WO2003059948A1

WO2003059948A1 - Dilated cardiomyopathy associated gene-2 (dcmag-2): a cytoplasmatic inducer of sarcomeric remodeling in cardiomyocytes

Info

Publication number: WO2003059948A1
Application number: PCT/EP2003/000363
Authority: WO
Inventors: Volker RÖNICKE; Birgit Reuner; Barbara Nave; Martin Funk; Stéphane LECLAIR; Kay Brinkmann; Thomas Henkel; Thomas Kirsch; Michael Becker
Original assignee: Medigene Ag
Priority date: 2002-01-15
Filing date: 2003-01-15
Publication date: 2003-07-24
Also published as: AU2003205611A1

Abstract

The present invention relates to a novel nucleic acid molecule and the protein of DCMAG-2, which is encoded by said molecule and relevant in congestive heart failure. Furthermore, the invention relates to a vector comprising said molecule, antibodies detecting said protein and transgenic non-human mammals overexpressing the corresponding gene product of DCMAG-2. Moreover, the present invention relates to a method of identifying of a compound for treating congestive heart failure which results from aberrant expression or regulation of the gene product of DCMAG-2.

Description

Dilated Cardiomyopathy Associated Gene-2 (DCMAG-2): A cytoplasmatic inducer of sarcomeric remodeling in cardiomyocytes

The present invention relates to a novel nucleic acid molecule and the protein of DCMAG-2, which is encoded by said molecule and relevant in congestive heart failure. Furthermore, the invention relates to a vector comprising said molecule, antibodies detecting said protein and transgenic non-human mammals overexpressing the corresponding gene product of DCMAG-2. Moreover, the present invention relates to a method of identifying a compound for treating congestive heart failure which results from aberrant expression or regulation of the gene product of DCMAG-2.

A variety of documents is cited throughout this specification. The disclosure content of said documents is herewith incorporated by reference.

The present invention is based on the identification and functional characterization of new target gene, which are causally involved in the initiation and progression of human heart insufficiency. In particular, a disease relevant activity of the target gene product was to be found. Finally, the present invention to the evaluation of the new genes in terms of therapeutic interventions in congestive heart failure.

The human body consists of several billion cells. These cells build up all tissues and in addition show a high degree of specialization in distinct organs, which sums up to more than two hundred different cell types in the mammalian organism. The basis for the heterogeneity of different cells in each individual is the variability in the protein content. While each cell of the organism contains an identical set of genomic DNA with only few exceptions like immune cells, the protein composition varies in quantity and quality. The main reason for this besides post-translational modification of proteins is the cell type specific gene expression. During early embryogenesis, which starts from a single cell (zygote), a distinct gene expression program defined by an increasingly complicated methylation pattern of the genomic DNA and maintained and propagated by master gene regulatory proteins sets the basis for an initial body plan. The plan is further modified and adapted by the determination and differentiation of an increasing number of highly specialized cell types, which clearly differ in structural and enzymatic properties of their proteins.

While this process of manifestation of distinct gene expression patterns in different morphological and functional properties is most obvious during embryogenesis, it is still present in further maturation to adulthood and in the adaptation of the adult organism to environmental demands. Furthermore, during pathological conditions there are clear alterations of the gene expression program detectable. Also the diseased heart, while progressing into steadily more severe stages of insufficiency, switches to a fetal gene expression program.

The systemic alterations in heart insufficiency or congestive heart failure (CHF) are reflected for instance in a dramatically reduced exercise tolerance and pulmonary edema. The macroscopic manifestation of the disease progression is the initial thickening of the left ventricular myocardium, which is followed by a severe dilatation of a thin walled left ventricular cavity. These alterations are reflected on the cellular level in distinct forms of cardiomyocyte hypertrophy. The early phase (adaptive) is characterized by thick cardiomyocytes with a parallel organization of sarcomeres while the later, decompensated stage shows elongated cells with a serial sarcomere organization.

Congestive heart failure is a life threatening disease. The prevalence and incidence in the US are 4.6 million and 550,000 cases respectively. There is no causal treatment available at the moment and the 50 per cent survival rate is less than five years.

An inevitable prerequisite for the development of new therapeutic principles in this indication is the identification and validation of new therapeutic targets. This can only be based on the observation of pathological relevant action of disease associated gene products.

This invention describes the identification and validation of a new gene product. Causal involvement in pathogenesis of the heart was manifested by a massive serial organization of sarcomere structures in cardiomyocytes due to the over expression of the induced gene. As the serial organization was not only found to be the cellular correlation to chamber dilatation in heart insufficiency (Gerdes et al. (1995) J Mol Cell Cardiol Mar; 27(3): 849-56), but was also found to be reversible (Zafeirides et al. (1998) Circulation Aug 18; 98(7): 656-62) and an active cellular reorganization driven by signaling cascades (Wollert et al. (1996), J Biol Chem Apr 19; 271 (16): 9535-45), any gene product involved in this remodeling would be a good target for a therapeutic intervention.

One striking advantage of this invention was the identification of the first cytoplasmatic protein which actively induces remodeling of cardiomyocytes. Until now only cytokines were known to induce an elongation of cardiomyocytes (Wollert et al., supra).

In order to identify the mechanisms which are responsible for these dramatic changes in cardiomyocyte morphology and function, we analysed the mRNA expression patterns of tissue samples from several diseased human hearts in comparison with healthy human hearts.

The analysis of gene expression patterns of tissue samples from diseased and healthy human hearts showed the significant up-regulation of a transcript homologous to an EST with unknown function (D80010: KIAA0188). The further comparison of the identified mRNA with the known EST revealed an insertion of 105 nucleotides. The analysis of the genomic organization led to the identification of a novel exon. This EST was named Dilated Cardiomyopathy Associated Gene 2 (DCMAG-2). This finding was unexpected and new, in particular, because the new exon was part of the coding region. However, there is a Mus musculus adult male testis cDNA (RIKEN full-length enriched library, AK019539), a Mus musculus 0 day neonate skin cDNA (RIKEN full-length enriched library, AK014526) and the Mus musculus sequence BAB31786 that show 83% homology within exon 4a on nucleotide level and 71% on protein level, respectively. The mouse clones AK019539, AK014526 and BAB31786 show an overall homology of 88% to DCMAG-2 on protein level. These mouse clones represent an isoform of mouse Lipinl . Therefore, DCMAG-2 might represent a new splice variant of human Lipinl .

DCMAG-2 shows homology to mouse Lipins1-3 and to human Lipin2 as shown below:

Table 1 : Homologies of DCMAG-2 to mouse Lipins1-3 and to human Lipin2

Furthermore, DCMAG-2 shows homology to human and mouse lipins as shown below in table 2:

Table 2: Homologies of DCMAG-2 to human and mouse lipins

The table lists homologies of DCMAG-2 nucleotide and protein sequence to public entrees identified using blast analysis. A detailed alignment of all sequences specified in the table is given in Fig. 12.

The proteins of NM 45693 and BC030537 are identical and published in 2002 to code for human lipin 1. Both transcripts together with D80010 do not contain the additional exon sequence found to be present in DCMAG-2. In contrast, the transcript of IMAGE:4819424 contains the exon sequence but is truncated at the C-terminus by 473 amino acids (aa). Therefore, this clone may code for a differentially polyadenylated isoform. Since it also contains a frameshift, it may be an artifact. In mice genes homologous to human lipins, this exon sequence is transcribed

Taken together, DCMAG-2 is a novel, human splice variant of Lipin 1. Since this variant may be heart specific, selective inhibitors will minimize side effects.

Highest sequence identity can be found from amino acid 1-109 and from amino acid 658 - 911 (also called amino-terminal and carboxy-terminal Lipin domains).

Mutations in mouse LPIN1 result in a phenotype of lipodystrophy. A LPIN3-mutant is a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertrigiyceridemia and insulin resistance (Peterfly et al. (2001) Nat. Genet. 27:121-124).

In order to identify a causal role of DCMAG-2 in the pathophysiology of congestive heart failure we recapitulated the up-regulation of the transcript in diseased heart samples by recombinant over-expression of DCMAG-2 in primary cardiomyocytes (pCMs) from neonatal rats. Unexpectedly, the consequence were morphological alterations in pCMs with many thin cell protrusions bearing ramifications. Astonishingly, we could identify sarcomeric structures in these tiny elongated cell protrusions, which clearly demonstrated the induction of a serial sarcomere organization due to the over-expression of DCMAG-2.

The massive induction of a decompensated hypertrophy (serial sarcomere organization), which is the most characteristic morphological alteration of cardiomyocytes in insufficient hearts, by DCMAG-2 over-expression pointed to a causal involvement of the newly identified protein in the pathogenesis of congestive heart failure. In particular, the observation, that a cytoplasmatic protein fulfills this function, was unexpected and new as until now only secreted proteins were found to be able to induce an elongation of cardiomyocytes (Wollert et al., supra).

In conclusion, based on an expression profiling approach we were able to identify a new transcript which was present in higher abundance in mRNA samples from diseased hearts than from normal hearts. The sequence analysis showed a homology to a known EST, but also revealed an insertion of 105 nucleotides. A comparison with genomic organization led to identification of a new coding exon, which alters the protein sequence.

The novel protein derived from the identified cDNA induced a massive serial organization of sarcomeres in cardiomyocytes after over expression. As this reflects the main morphological alteration of muscle cells in insufficient hearts, a causal role of the novel protein (DCMAG-2) in the pathogenesis of congestive heart failure was found. Further, the morphological alteration induced by DCMAG-2 could be the basis for the detailed analysis of cellular factors which are responsible for the sarcomeric remodeling in heart failure. It could be the read out for a drug screening assay to identify new chemical entities as remodeling inhibitors. In addition, DCMAG-2 could be used as a drug target for a new therapeutic intervention in congestive heart failure.

The invention creates the opportunity to analyze new intracellular signaling processes triggered by DCMAG-2 over expression, which are responsible for the sarcomeric rearrangement. These analyses can lead to the identification of new target proteins for a novel therapeutic intervention in congestive heart failure based on the inhibition or reversion of sarcomeric remodeling. In addition, DCMAG-2 by itself can be used as a target in a drug screening assay to identify new chemical entities with cardioprotective action.

The technical problem underlying the present invention was to provide tools useful in the diagnosis, prevention and treatment of heart-related diseases which are connected with the serial sarcomer organization in the heart.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims.

Thus, the present invention relates to a polynucleotide comprising a nucleotide sequence selected form the group consisting of: (a) a nucleotide sequence encoding the mature form of a protein comprising the amino acid sequence as given in SEQ ID NO: 2;

(b) a nucleotide sequence comprising or consisting of the DNA sequence as given in SEQ ID NO: 1 ;

(c) a nucleotide sequence hybridizing with the complementary strand of a nucleotide sequence as defined in (b) under stringent hybridization conditions;

(d) a nucleotide sequence encoding a protein derived from the protein encoded by a nucleotide sequence of (a) or (b) by way of substitution, deletion and/or addition of one or several amino acids of the amino acid sequence encoded by the nucleotide sequence of (a) or (b), whereby overexpression in heart tissue of the protein encoded by said nucleotide sequence results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer; (e) a nucleotide sequence encoding a protein having an amino acid sequence at least 60 % identical to the amino acid sequence encoded by the nucleotide sequence of (a) or (b), whereby overexpression in heart tissue of the protein encoded by said nucleotide sequence results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer;

(f) a nucleotide sequence encoding at least the domain of a polypeptide encoded by a nucleotide sequence as given in SEQ ID NO:6;

(g) a nucleotide sequence comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of (a) to (e);

(h) nucleotide sequences obtainable by screening an appropriate library under stringent conditions with a probe having at least 12 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 1;

(i) a nucleotide sequence encoding a fragment of at least 4 consecutive amino acids of a protein encoded by a nucleotide sequence of (a) or (b); and

(j) a nucleotide sequence which is degenerate as a result of the genetic code to a nucleotide sequence of any one of (a) to (h), whereby overexpression in heart tissue of the polypeptide encoded by said nucleotide sequence results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer; wherein said nucleotide sequence comprises at least 3 nucleotides of the DNA sequence as given in SEQ ID NO: 5.;

The term "mature form of the protein" defines in context with the present invention a protein translated from its corresponding mRNA and optional subsequently modified.

The term "hybridizing" as used herein refers to polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof, wherein said nucleotide sequence comprises at least 3 nucleotides of the DNA sequence as given in SEQ ID NO: 5. Therefore, said polynucleotides may be useful as probes in Northern or Southern Blot analysis of RNA or DNA preparations, respectively, or can be used as oligonucleotide primers in PCR analysis dependent on their respective size. Preferably, said hybridizing polynucleotides comprise at least 10, more preferably at least 15 nucleotides in length while a hybridizing polynucleotide of the present invention to be used as a probe preferably comprises at least 100, more preferably at least 200, or most preferably at least 500 nucleotides in length. It is well known in the art how to perform hybridization experiments with nucleic acid molecules, i.e. the person skilled in the art knows what hybridization conditions s/he has to use in accordance with the present invention. Such hybridization conditions are referred to in standard text books such as Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. "Stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C. Preferred in accordance with the present inventions are polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof, wherein said nucleotide sequence comprises at least 3 nucleotides of the DNA sequence as given in SEQ ID NO: 5, under stringent hybridization conditions, i.e. which do not cross hybridize to unrelated polynucleotides such as polynucleotides that not comprises at least 3 nucleotides of the DNA sequence as given in SEQ ID NO: 5. "Stringent hybridization conditions" refer, i.e. to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C. Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH₂PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50°C with 1 X SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

The term "altered cellular morphology" as used herein refers to a change in cell- size and shape, e.g. from a small, round basal cell to a broad, flat shape or an elongated shape with cell protrusions and/or a higher number of ramifications The term "serial sarcomer organization" as used herein refers to an elongation of cells characterized by serial, or end-to-end, elongation of contractile units of the cell; more sarcomeres added in cross direction than perpendicular to it. The term "altered amount and/or altered subcellular localization of at least one signaling molecule in the sarcomer" as used herein refers to increased or reduced quantity of protein (e.g. kinases, phosphatases, adapters, signal transduction molecules) in the cell or changes in the location of protein within the cell (e.g. from cytosolic diffuse distribution to association with the sarcomer resulting in a striated pattern distribution.

Preferably overexpression of the protein encoded by said nucleotide sequence/polynucleotide of the invention in heart tissue results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer.

A preferred embodiment of the invention relates to a nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with an above defined DNA sequence or with a complementary strand thereof.

An alternative embodiment of the invention relates to a vector comprising an above defined DNA sequence.

Many suitable vectors are known to those skilled in molecular biology, the choice of which would depend on the function desired and include plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells. As discussed in further details below, a cloning vector was used to isolate individual sequences of DNA. Relevant sequences can be transferred into expression vectors where expression of a particular polypeptide is required. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322 and pGBT9, Typical expression vectors include pTRE, pCAL-n-EK, pESP-1 , pOP13CAT.

Hence, in a preferred embodiment of the present invention the above-described a vector an expression vector wherein the DNA sequence or nucleic acid molecule is operatively linked to one or more control sequences allowing the transcription and optionally expression in prokaryotic and/or eukaryotic host cells.. The term "control sequence" refers to regulatory DNA sequences which are necessary to effect the expression of coding sequences to which they are Iigated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term "control sequence" is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.

The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is Iigated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is preferably used. Thus, the vector of the invention is preferably an expression vector. An "expression vector" is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic and/or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the Pι_, lac, trp or tac promoter in E. coli, and examples of regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDMδ, pRc/CMV, pcDNAI , pcDNA3 (In-vitrogene), pSPORTI (GIBCO BRL). An alternative expression system which could be used to express a cell cycle interacting protein is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The coding sequence of a nucleic acid molecule of the invention may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of said coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the protein of the invention is expressed (Smith, J. Virol. 46 (1983), 584; Engelhard, Proc. Nat. Acad. Sci. USA 91 (1994), 3224-3227). In plants, promoters commonly used are the polyubiquitin promoter, and the actin promoter for ubiquitous expression. The termination signals usually employed are from the Nopaline Synthase promoter or from the CAMV 35S promoter. A plant translational enhancer often used is the TMV omega sequences, the inclusion of an intron (lntron-1 from the Shrunken gene of maize, for example) has been shown to increase expression levels by up to 100-fold. (Mait, Transgenic Research 6 (1997), 143-156; Ni, Plant Journal 7 (1995), 661-676). Additional regulatory elements may include transcriptional as well as translational enhancers. Advantageously, the above-described vectors of the invention comprises a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed cells and, e.g., plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485). Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338). Useful scorable marker are also known to those skilled in the art and are commercially available. Advantageously, said marker is a gene encoding luciferase (Giacomin, PI. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a vector of the invention.

The present invention furthermore relates to host cells produced by introducing a nucleic acid molecule into the host cell which upon its presence in the cell mediates the expression of a gene encoding the polynucleotide of the invention or comprising a polynucleotide or a vector as described above or a polynucleotide according to the invention wherein the polynucleotides and/or nucleic acid molecule is foreign to the host cell.

By "foreign" it is meant that the polynucleotide or nucleic acid molecule is either heterologous with respect to the host cell, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host cell but located in a different genomic environment than the naturally occurring counterpart of said nucleic acid molecule. This means that, if the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural location in the genome of said host cell, in particular it is surrounded by different genes. In this case the polynucleotide may be either under the control of its own promoter or under the control of a heterologous promoter. The vector or nucleic acid molecule according to the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extrachromosomally. In this respect, it is also to be understood that the nucleic acid molecule of the invention can be used to restore or create a mutant gene via homologous recombination. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells.

The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of a protein of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is meant to include yeast, higher plant, insect and preferably mammalian cells. Depending upon the host employed in a recombinant production procedure, the protein encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated. A polynucleotide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Furthermore, methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

In a preferred embodiment the host cell is a human cell or human cell line.

Moreover, the present invention related to a method for the production of a protein or an immunologically active or functional fragment thereof comprising culturing an aforementioned host cell under conditions allowing the expression of the protein and recovering the produced protein from the culture. The term "immunologically active" as used herein refers to proteins or fragments thereof which are characterized by their capability to induce an immunological response in an immunized organism. Said response may be induced by the protein or fragment thereof either alone or in combination with a hapten, an adjuvant or other compounds known in the art to induce or elicit immunoresponses to a protein or fragment thereof.

The term "functional fragment" as used herein refers to a fragment of said protein having the same function as said protein. In particular, overexpression of said "functional fragment" results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer.

In a further preferred embodiment the invention relates to a protein or an immunologically active or functional fragment thereof encodeable by a DNA sequence as described herein above or obtainable by the aforementioned method.

Preferably said protein or fragment thereof is glycosylated, phosphorylated, and/or amidated.

Furthermore, the present invention relates to an antibody or an aptamer specifically recognizing the aforementioned protein or a fragment or epitope thereof. Said antibody may be a monoclonal or a polyclonal antibody.

A preferred embodiment of the invention relates to an antibody which is a monoclonal antibody.

The term "monoclonal" or "polyclonal antibody" (see Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988) also relates to derivatives of said antibodies which retains or essentially retains its binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region.

The term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (poly)peptides of similar structures. Cross-reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing binding of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, loc. cit.) to the polypeptide of interest as well as to a number of more or less (structurally and/or functionally) closely related polypeptides. Only those antibodies that bind to the polypeptide of interest but do not or do not essentially bind to any of the other (poly)peptides which are preferably expressed by the same tissue as the polypeptide of interest, i.e. heart, are considered specific for the polypeptide of interest and selected for further studies in accordance with the method of the invention,

In a further alternative embodiment the present invention relates to a transgenic non-human mammal whose somatic and germ cells comprise at least one gene encoding a functional polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution; said functional polypeptide has been modified, said modification being sufficient to increase the amount of said functional polypeptide expressed in the heart tissue of said transgenic non-human mammal, wherein said transgenic non-human mammal exhibits a disease of the heart.

A method for the production of a transgenic non-human animal, for example transgenic mouse, comprises introduction of the aforementioned polynucleotide or targeting vector into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. The non-human animal can be used in accordance with a screening method of the invention described herein. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe; see supra. A general method for making transgenic non-human animals is described in the art, see for example WO 94/24274. For making transgenic non-human organisms (which include homologously targeted non-human animals), embryonal stem cells (ES cells) are preferred. Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62:1073-1085 (1990)) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting. Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al., Nature 326:292-295 (1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87:27-45 (1985)), the CCE line (Robertson et al., Nature 323:445-448 (1986)), the AK-7 line (Zhuang et al., Cell 77:875-884 (1994)). The success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells (i. e., their ability, once injected into a host developing embryo, such as a blastocyst or morula, to participate in embryogenesis and contribute to the germ cells of the resulting animal). The blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant nonhuman females and are born as chimeric mice. The resultant transgenic mice are chimeric for cells having either the recombinase or reporter loci and are backcrossed and screened for the presence of the correctly targeted transgene (s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozygous for either the recombinase or reporter locus/loci. The transgenic non-human animals may, for example, be transgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, or cows. Preferably, said transgenic nonhuman animal is a mouse, a rabbit or a rat.

According to a preferred embodiment the transgenic non-human mammal of the invention is an animal, wherein said transgenic gene was introduced into the nonhuman mammal or an ancestor thereof, at an embryonic stage. In a further preferred embodiment of the transgenic non-human mammal of the invention the modification is activation or overexpression of said gene or leads to the enhancement of the synthesis of the corresponding protein. This embodiment allows for example the study of the interaction of various mutant forms of the aforementioned polypeptides on the onset of the clinical symptoms of a disease related to disorders in the heart. All the applications that have been herein before discussed with regard to a transgenic animal also apply to animals carrying two, three or more transgenes for example encoding different aforementioned nucleic acid molecules. It might be also desirable to inactivate or, more preferably, to enhance protein expression or function at a certain stage of development and/or life of the transgenic animal. This can be achieved by using, for example, tissue specific, developmental and/or cell regulated and/or inducible promoters which drive the expression of the transgen. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62) or the interferon-α/β induce expression of genes under control of the Mx-promoter (Dulat et al., Transplant Proc (2001 );33 (1-2):262-3). Similar, the expression of the mutant protein(s) may be controlled by such regulatory elements. As mentioned, the invention also relates to a transgenic non-human animal, preferably mammal and cells of such animals which cells contain (preferably stably integrated into their genome) at least one of the aforementioned nucleic acid molecule(s) or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to induction of the synthesis of (a) corresponding protein(s).

Techniques how to achieve this are well known to the person skilled in the art. However, it is also possible to use nucleic acid molecules which display a high degree of homology to endogenously occurring nucleic acid molecules encoding such a protein. In this case the homology is preferably higher than 60%, preferably at least 80%, especially at least 90%, advantageously at least 99%.

In cases where aforementioned gene is enhanced, optionally in combination with a modification of the function and/or expression of one or more further gene products, interrelationships of gene products in the onset or progression of the diseases of the heart may be assessed. In this regard, it is also of interest to cross transgenic non-human animals having different transgenes for assessing further interrelationships of gene products in the onset or progression of said disease. Consequently, the offspring of such crosses is also comprised by the scope of the present invention.

The present invention also relates in an alternative embodiment to a method for identifying a subject at risk for a disease of the heart, comprising the step of analyzing of at least one nucleic acid sequence or quantitating the amount of at least one RNA in the heart tissue of the subject, whereby

(a) said at least one nucleic acid sequence or RNA encodes an amino acid sequence: (aa) of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID

NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12; (ab) an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (aa); (ac) an amino acid sequence of (aa) with at least one conservative amino acid substitution;

(ad) an amino acid sequence that is an isoform of an amino acid sequence of any of (aa) to (ac); and

(ae) an amino acid that is encoded by a DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1% SDS at 65°C to the DNA molecule encoding an amino acid sequence of (aa), (ac) or (ad); and/or

(b) said at least one RNA is transcribed from the DNA sequence of SEQ ID NO: 1 , the DNA sequence of SEQ ID NO: 3, the DNA sequence of SEQ ID NO: 7, the DNA sequence of SEQ ID NO: 9 or the DNA sequence of SEQ ID

NO: 11 or a degenerate variant thereof. The term "disease of the heart" means, in accordance with the present invention, any disease that affects the normal function of the heart. This definition includes hereditary as well as acquired diseases such as diseases induced by a pathogen or diseases due to lack of exercise.

Several diseases of the heart are, for example, rheumatic fever/ rheumatic heart disease, hypertensive heart disease, hypertensive heart and renal disease, ischemic heart disease (coronary heart disease), diseases of pulmonary circulation (which include acute and chronic pulmonary heart disease), arrhythmias, congenital heart disease, angina and congestive heart failure. The term "analyzing of at least one nucleic acid sequence" is intended to comprise the analysis of nucleic acid sequence of the gene of DCMAG-2 in heart tissue of a subject and to compare said sequence with the sequence of a healthy subject. The term "healthy subject" in connection with the present invention means a subject without any indication for heart disease. Said analysis comprise the analysis of regulatory structures of the gene, e.g. promoter structures. The term "quantitating the amount of at least one RNA" is intended to mean the determination of the amount of mRNA in heart tissue as compared to a standard value such as an internal standard. The (internal) standard would advantageously be the amount of a corresponding RNA produced by a heart tissue not affected by a disease. Said (internal) standard would also include a mean value obtained from a variety of heart tissues not affected by a disease. A possible way to get samples of heart tissue is to take a biopsy (catheter) from the ventricular wall. Optionally, a standard would take into account the genetic background of the subject under investigation. Thus, quantitation of said subject's RNA is effected in comparison to the amount of RNA of one or a variety of samples of the same or a similar genetic background. A variable number of "non-failing" humans (humans that do not show an indication for any heart disease) are compared with a variable number of patients that suffer a distinct heart disease like dilated cardiomyopathy. The determination can be effected by any known technology of analyzing the amount of RNA produced in a sample such as a tissue sample. Techniques based on hybridization like Northern-Blot, dot-blot, subtractive hybridization, DNA-Chip analysis or techniques based on reverse transcription coupled to the polymerase chain reaction (RT-PCR) like differential display, suppression subtractive hybridization (SSH), fluorescence differential display (FDD), serial analysis of gene expression (SAGE) or representational difference analysis (see e.g. Kozian, D.H., Kirschbaum, B.J.; Comparative gene-expression analysis. (1999) 17:73-77. Generally, it is preferred that the assay is performed as a high throughput assay. This holds also true for the further methods described herein and in accordance with this invention. Samples of RNA may be prepared as described in the appended examples.

The term "isoform" means a derivative of a gene resulting from alternative splicing, alternative polyadenylation, alternative promoter usage or RNA editing. Isoforms can be detected by

(a) in silico analysis (e.g. by clustering analysis of any types of expressed sequences or the corresponding proteins, by alignment of expressed sequences with chromosomal DNA, by interspecies comparisons or by analysis of the coding as well as non-coding sequences like promoters or regulatory RNA processing sites for SNPs or known mutations causing a disease).

(b) any type of hybridisation techniques (e.g. Northern blots, nuclease protection assays, microarrays) starting from RNA (as described in Higgins, S.J., Hames, D. RNA Processing: A practical approach Oxford University Press (1994), Vol. 1 and 2; Sambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual. (1989) Cold Spring Harbor Laboratory Press).

(c) PCR-applications as well as hybridisation techniques starting from single strand or double strand cDNA obtained by reverse transcription, as described for example in Stoss, O., Stoilov, P., Hartmann, A.M., Nayler, O., Stamm, S. The in vivo minigene approach to analyse tissue-specific splicing. Brain Res. Brain Res. Protoc. (1999), 3:383-394.

Primers/probes for RT-PCR or hybridisation techniques are designed in a fashion that at least one of the primers/probes specifically recognizes one isoform. If differences in the molecular weight of isoforms are large enough to separate them by electrophoretic or chromatographic methods, it is also possible to detect multiple isoforms at once by employing primers/probes that flank the spliced regions. The isoforms are then sequenced and analysed as described in a). The term "DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1% SDS at 65°C to the DNA molecule encoding the amino acid sequence of (a), (c) or (d)" means that the two DNA molecules hybridize under these experimental conditions to each other. This term does not exclude that the two DNA sequences hybridize at higher stringency conditions such as 2xSSC, 0.1% SDS at 65°C nor does it exclude that lower stringency conditions such as 6xSSC, 0.1 % SDS at 60°C allow a hybridization of the two DNA sequences.

Stringent conditions for hybridization are well known by a person skilled in the art and described in more detail herein above. The invention is based upon the unexpected result that the DCMAG-2 gene coding for the protein sequences referred to above, preferred embodiments of which are given in all of the examples, is deregulated in the comparison of one or more failing heart samples to one or more non-failing heart samples and lead to an upregulation of the described polypeptides measured by their respective mRNAs or cDNAs. The significant changes in gene expression levels suggest a causative role in congestive heart failure.

However such a causative role for one specific indication of the heart leads to the assumption that a deregulation of the gene might play an important role in other diseases of the heart as well. Such involvement can easily be tested by methods well known in the art and described for example in example 1 of this invention by a comparison of the gene expression levels of such gene between a sample of a healthy mammal and of a mammal having the disease in question. Therefore the subject of this invention does not only relate to dilated cardiomyopathy but also to other diseases of the heart as specified throughout the specification. It is well accepted in the art that downregulation of gene expression of a upregulated target gene by means of a gene therapeutic intervention, compensatory molecules or specific inhibitors, for example of transcription or translation are potentially very promising therapeutic tools to treat a heart disease that is caused or promoted by the upregulation of such gene. Further, in accordance with the present invention it has surprisingly been found that the DCMAG-2 gene is overexpressed in diseases associated with the heart and in particular in patients suffering from congestive heart failure. By performing the method of the invention which may be in vivo, in vitro or in silico, the diagnosis of a disease of the heart established by a different methodology may be corroborated. Alternatively, it may be assessed whether a subject that is preferably throughout this specification a human displaying no sign of being affected by a disease of the heart is at risk of developing such a disease. This is possible in cases where the overexpression of the gene defined herein above is causative of the disease or is a member of a protein cascade wherein another gene/protein than the one identified herein above is causative for said disease. In this regard, the term "causative" is not limited to mean that the aberrant expression of one gene as identified above or which is a member of said protein cascade is the sole cause for the onset of the disease. Whereas this option is also within the scope of the invention, expression the invention also encompasses embodiments wherein said aberrant is one of a variety of causative events that lead to the onset of the disease. There is causal correlation between altered cellular function of cardiomyocytes and its protein composition. The latter is regulated by three main mechanisms: a. Gene expression b. Posttranscriptional modification (e.g. alternative splicing) c. Posttranslational modification

In a variation of the method of the invention quantitation of the above recited RNA is used to monitor the progress of a disease of the heart (said variation also applies to the method described herein below). This variation may be employed for assessing the efficacy of a medicament or to determine a time point when administration of a drug is no longer necessary or when the dose of a drug may be reduced and/or when the time interval between administrations of the medicament may be increased. This variation of the method of the invention may successfully be employed in cases where an aberrant expression of any of the aforementioned genes/genes as members of protein cascades is causative of the disease. It is also useful in cases where the aberrant expression of the gene/genes is the direct or indirect result of said disease.

In a preferred embodiment of the method of the invention the amount of the said RNA is quantitated using a nucleic acid probe which is a nucleic acid of: (a) the DNA sequence of SEQ ID NO: 1 , SEQ ID NO: 3, the DNA sequence of SEQ ID NO: 7, the DNA sequence of SEQ ID NO: 9 or the DNA sequence of SEQ ID NO: 11 or a degenerate variant thereof;

(b) a DNA sequence at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to a DNA sequence of (a);

(c) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12; each of said amino acid sequences having at least one conservative amino acid substitution;

(d) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (c);

(e) a nucleic acid sequence that encodes an amino acid sequence of (c) or (d) with at least one conservative amino acid substitution;

(f) a nucleic acid sequence that hybridizes in 4xSSC, 0.1% SDS at 65°C to the complementary strand of the DNA molecule encoding an amino acid sequence of (c), (d) or (e);

(g) a fragment of at least 15 nucleotides in length of (a) to (f); and (h) a nucleic acid probe comprising a sequence that specifically hybridizes under physiological conditions to the nucleotide sequence of: (i) the DNA sequence of the RNA transcribed from the DNA sequence of SEQ ID NO: 1 , the DNA sequence of SEQ ID NO: 3, the DNA sequence of SEQ ID NO: 7, the DNA sequence of SEQ ID NO: 9 or the DNA sequence of SEQ ID NO: 11 ; (ii) a DNA sequence at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to a DNA sequence of (i); (iii) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12 with at least one conservative amino acid substitution; (iv) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (iii); (v) a nucleic acid sequence that encodes an amino acid sequence of (iii) with at least one conservative amino acid substitution; (vi) a nucleic acid sequence that hybridizes in 2xSSC, 0.1% SDS at 65°C to the DNA molecule encoding an amino acid sequence of (iii), (iv) or (v); and (vii) a fragment of at least 15 nucleotides in length of (i) to (vi).

In a preferred embodiment of the method of the invention, said nucleic acid or RNA is obtained from heart tissue. A suitable way would be to take a biopsy (catheter) from the ventricular wall. The decision to do this is clearly affected by the severity of the disease and the general constitution of the patient. The cardiologist and the patient have to drive the final decision. In an additionally preferred embodiment of the method of the invention, said polypeptide is quantitated in heart tissue.

In another preferred embodiment, the method of the invention further comprises the step of normalizing the amount of RNA against a corresponding RNA from a healthy subject or cells derived from a healthy subject.

The term "normalizing the amount of RNA against a corresponding RNA from a healthy subject or cells derived from a healthy subject" means, in accordance with the present invention, that levels of mRNA from a comparative number of cells from the heart of said subject under investigation and from the heart of an individual not affected by a disease of the heart are compared. Alternatively, cells from the heart of the subject under investigation may be compared in terms of the indicated mRNA levels with cells derived from the heart of a healthy individual which are kept in cell culture and optionally form a cell line. Optionally, different sources of cells such as from different individuals and/or different cell lines may be used for the generation of the standard against which the mRNA level of the subject under investigation is compared. Using the Affymetrix Chip technology, there is also the possibility to use external standards (that are given separately to the hybridization cocktail) in order to normalize the values of different oligonucleotide-chips. In addition, the invention relates to a method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of a polypeptide in the heart tissue or the serum of the blood of the subject, the polypeptide selected of the group comprising: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of

SEQ ID NO: 12;

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least

99% identical to an amino acid sequence of (a); and

(c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution.

This embodiment of the invention makes use of the option that detection may not only be at the level of the mRNA but also at the level of the polypeptide translated from the mRNA. Whereas it is not excluded that the level of mRNA strictly correlates with the level of polypeptide translated from the mRNA, this may not always be the case. Accordingly, it may be assessed whether the mRNA or the protein level, if different, is more appropriate to establish if the heart of a subject is prone to develop a disease of the heart. Factors that contribute to differences in the expression levels of mRNA and protein are well-known in the art and include differential mRNA-export to the protein-synthesis machinery as well as differences in the translation efficacy of different mRNA species. Other considerations influencing the choice of the detection level (in RNA or protein) include the availability of an appropriate screening tool, instrumentation of the lab, experience of the lab personnel and others.

In a preferred embodiment of the method of the invention, the amount of the said polypeptide is quantitated using an aptamer or an antibody or an antigen-binding portion of said aptamer or antibody that specifically binds a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12;

99% identical to an amino acid sequence of (a); and

The antibody used in accordance with the invention may be a monoclonal or a polyclonal antibody (see Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988) or a derivative of said antibody which retains or essentially retains its binding specificity. Said antibody may correspond to an antibody described herein above. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region. The term "specifically binds" in connection with the antibody used in accordance with the present invention has been defined herein above.

In a particularly preferred embodiment of the method of the invention, said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.

The term "humanized antibody" means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity. Optionally, the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO90/07861.

In an also particularly preferred embodiment of the method of the invention the antibody is the antibody particularly described herein above or a derivative thereof.

In an additionally preferred embodiment of the method of the invention, said derivative of said antibody is an scFv fragment. The term "scFv fragment" (single-chain Fv fragment) is well understood in the art and preferred due to its small size and the possibility to recombinantly produce such fragments.

The specifically binding antibody etc. may be detected by using, for example, a labeled secondary antibody specifically recognizing the constant region of the first antibody. However, in a further particularly preferred embodiment of the method of the invention, the aptamer or the antibody or derivative of said aptamer or antibody or derivative thereof itself is detectably labeled at the binding portion.

Detectable labels include a variety of established labels such as radioactive (¹²⁵l, for example) or fluorescent labels (see, e.g. Harlow and Lane, loc. cit.). Binding may be detected after removing unspecific labels by appropriate washing conditions (see, e.g. Harlow and Lane, loc. cit.).

According to a preferred embodiment of the method of the invention said polypeptide is quantitated in heart tissue.

Methods for the determination of the amount of a particular protein in a specified tissue are known by a person skilled in the art.

In yet another preferred embodiment, the method of the invention further comprises the step of normalizing the amount of polypeptide against a corresponding polypeptide from a healthy subject or cells derived from a healthy subject.

The same considerations as developed for the previous embodiment on the mRNA level apply here to the normalization of protein levels.

Additionally, the invention relates to a method for identifying a compound that decreases the level in heart tissue of a polypeptide selected from the group consisting of:

(a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12;

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (a); and

(c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of:

(i) contacting a DNA encoding said polypeptide under conditions that would permit the translation of said polypeptide with a test compound; and (ii) detecting a decreased level of the polypeptide relative to the level of translation obtained in the absence of the test compound.

The term "compound" in accordance with the present invention shall mean any biologically active substance that has an effect on heart tissue or a single heart cell, whereas such compound has a positive or negative influence upon such heart tissue or heart cell. Preferred compounds are nucleic acids, preferably coding for a peptide, polypeptide, antisense RNA or a ribozyme or nucleic acids that act independently of their transcription respective their translation as for example an antisense RNA or ribozyme; natural or synthetic peptides, preferably with a relative molecular mass of about 1.000, especially of about 500, peptide analogs polypeptides or compositions of polypeptides, proteins, protein complexes, fusion proteins, preferably antibodies, especially murine, human or humanized antibodies, single chain antibodies, F_ab fragments or any other antigen binding portion or derivative of an antibody, including modifications of such molecules as for example glycosylation, acetylation, phosphorylation, famesylation, hydroxylation, methylation or esterification, hormones, organic or inorganic molecules or compositions, preferably small molecules with a relative molecular mass of about 1.000, especially of about 500. The term "under conditions that would permit the translation of said polypeptide" denotes any conditions that allow the in vitro or in vivo translation of the polypeptide of interest. As regards in vitro conditions, translation may be effected in a cell-free system, as described, for example in Stoss, Schwaiger, Cooper and Stamm (1999). J. Biol. Chem. 274: 10951-10962, using the TNT-coupled reticulocyte lysate system (Promega). With respect to in vivo conditions, physiological conditions such as conditions naturally occurring inside a cell are preferred.

Based on the finding that expression of genes encoding the above recited polypeptides is aberrant, the method of the invention allows the convenient identification or isolation of compounds that counteract such aberrant expression such that normal expression levels are restored or essentially restored. In the case that the method of the invention is carried out in vitro, for example, in a cell-free system, then introduction into a cell would not be necessary. Rather, the test compound would be admixed to the in vitro expression system and the effect of said admixture observed.

The effect of the contact of the DNA of interest with the test compound on the protein level may be assessed by any technology that measures changes in the quantitative protein level. Such technologies include Western blots, ELISAs, RIAs and other techniques referred to herein above. The change in protein level, if any, as a result of the contact of said DNA and said test compound is compared against a standard. This standard is measured applying the same test system but omits the step of contacting the compound with the DNA. The standard may consist of the expression level of the polypeptide after no compound has been added. Alternatively, the DNA may be contacted with a compound that has previously been demonstrated to have an influence on the expression level.

Compounds tested positive for being capable of reducing the amount of polypeptide produced are prime candidates for the direct use as a medicament or as lead compounds for the development of a medicament. Naturally, the toxicity of the compound identified and other well-known factors crucial for the applicability of the compound as a medicament will have to be tested. Methods for developing a suitable active ingredient of a pharmaceutical composition on the basis of the compound identified as a lead compound are described elsewhere in this specification.

Additionally, the invention relates to a method for identifying a compound that specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12, said method comprising the steps of (i) providing said polypeptide; (ii) contacting one or a plurality of compounds with said polypeptide; and

(iii) identifying one or a plurality of compounds that is capable of binding said polypeptide.

In a preferred embodiment of the method of the invention said binding results in inactivation of said polypeptide. Said inactivation may be, for example, an inhibition of enzymatic activity (as described herein below) and/or the initiation or inhibition of a signal cascade.

Based on the function of this protein in dilated cardiomyopathy (DCM) development a cell based assay can be developed to identify potential inhibitors. The protein under investigation is expressed in cardiomyocytes (e. g. by infection with recombinant adenovirus). The expression of said protein leads to characteristic morphological alterations. Reversal or reduction of these morphological alterations can be used e.g. in an HTS assay to identify compounds which act as activators of these proteins. The system can be automated by use of digital image analysis systems.

Another possibility is to identify first proteins which are binding partners of the described proteins. This is especially important for structural proteins or adaptor proteins in signal transduction pathways. Methods to identify compounds capable of binding include affinity chromatography with immobilized target protein and subsequent elution of bound proteins (e. g. by acid pH), co-immunoprecipitation and chemical crosslinking with subsequent analysis on SDS-PAGE. The influence of compounds on these protein-protein interactions can be monitored by techniques like optical spectroscopy (e. g. fluorescence or surface plasmon resonance), calorimetry (isothermal titration microcalorimetry) and NMR. In the case of optical spectroscopy either the intrinsic protein fluorescence may change (in intensity and/or wavelength of emission maximum) upon complex formation with the binding compound or the fluorescence of a covalently attached fluorophore may change upon complex formation. The claimed protein or its identified binding partner may be labelled on e. g. cysteine or lysine residues with a fluorophore (for a collection of fluorophores see catalogues of Molecular Probes or Pierce Chemical Company) which changes its optical properties upon binding. These changes in the intrinsic or extrinsic fluorescence may be applied for use in a HTS assay to identify compounds capable of inhibiting or activating the mentioned protein-protein interaction. If the protein referred to above exhibits enzymatic activity (e. g. Kinase, Protease, Phosphatase) the activation of this activity may be monitored by using labelled (fluorescently, radioactively or immunologically) derivates of the substrate. This activity assay which is based on labelled substrates can be used for development of a HTS assay to identifiy compounds acting as inhibitor.

Moreover, the invention relates to a method for identifying a compound that decreases the level in heart tissue of an mRNA encoding a polypeptide selected from the group consisting of:

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (a); and (c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of (i) contacting a DNA giving rise to said mRNA under conditions that would permit transcription of said mRNA with a test compound; and (ii) detecting a decreased level of the mRNA relative to the level of transcription obtained in the absence of the test compound. This embodiment of the invention is very similar to the previously discussed one with the exception that here mRNA levels are detected whereas in the previous embodiment protein levels are detected. Methods of assessing RNA levels which also apply to this embodiment have been described herein above.

An alternative embodiment of the invention relates to a method of identifying a compound that decreases the expression of a polypeptide in heart tissue, the polypeptide being selected from the group consisting of:

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (a); and (c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of:

(i) contacting a transgenic non-human mammal according to any one of claims 15 to 17 with a test compound, and (ii) detecting a decreased level of expression of said polypeptide relative to the expression in the absence of said test compound.

In a preferred embodiment of the method of the invention the test compound prevents or ameliorates a disease of the heart in said transgenic non-human mammal.

In this embodiment, the effect of the test compound may be assessed by observing the disease state of the transgenic animal. Thus, if the animal suffers from a disease of the heart prior to the administration of the test compound and the administration of the test compound results in an amelioration of the disease, then it can be concluded that this test compound is a prime candidate for the development of a medicament useful also in humans. In addition the compound could also inhibit disease establishment by treatment in advance.

A further embodiment of the invention is a method for identifying one or a plurality of isogenes of a gene coding for a polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12, whereby overexpression in heart tissue of the protein encoded by said identified isogen(s) results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer; said method comprising the steps of: (i) providing a first nucleic acid molecule of at least 12 nucleotides coding for at least a part of said polypeptide; and (ii) identifying a second nucleic acid molecule that (a) has a homology of 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% or (b) hybridizes in 4xSSC, 0.1 SDS at 45°C to the complementary strand of the nucleic acid molecule encoding said amino acid sequences.

The term "isogenes" is used herein to describe genes that are considered to be generated by gene duplication. They can be identified by comparing the homology of the DNA-, RNA-, or protein-sequence of interest with other DNA, RNA or protein-sequences of the same species. There might be strong differences in the degree of homology between isogenes of the same species. This may be dependent on the time-point, when the gene duplication event took place in evolution and the degree of conservation during evolution. Isogenes can be identified and cloned by RT-PCR as has been demonstrated by Screaton et al. (1995) EMBO J. 14:4336-4349 or Huang et al. (1998) Gene 211 : 49-55. Isogenes can also be identified and cloned by colony hybridization or plaque hybridization as described in Sambrook, Fritsch, Maniatis (1989), Molecular Cloning, Cold Spring Harbor Laboratory Press. In a first step, either a genomic or a cDNA library e.g. in bacteria or phages is generated. In order to identify isogenes, colony hybridization or plaque hybridization is slightly modified in a way that cross- hybridizations are detectable under conditions of lower stringency. This can be achieved by lowering the calculated temperature for hybridization and washing and/or by lowering the salt concentration of the washing solutions (Sam brook, Fritsch, Maniatis (1989) Cold Spring Harbor Laboratory Press). For example, a low-stringency washing condition may include 2 washing steps at a temperature between 45°C and 65°C with 4xSSC, 0,1 % SDS for 30 min (50 ml) and finally two washing steps with 50 mi of a solution containing 2xSSC, 0.1 % SDS for 30 min. After detection, signal intensity of colonies containing an isogene is dependent on the homology of a gene and its isogene(s).

Furthermore, the invention relates to a method for identifying one or a plurality of genes the expression of which in heart tissue is modulated by inhibiting or decreasing the expression of a polypeptide selected from the group consisting of:

(c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of:

(i) contacting a plurality of heart tissue cells with a compound that inhibits or decreases the expression of said polypeptide under conditions that permit the expression of said polypeptide in the absence of a test compound, and

(ii) comparing a gene expression profile of said heart cell in the presence and in the absence of said compound. The term "gene expression profile" shall mean all expressed genes of a cell or a tissue. Such profile can be assessed using methods well known in the art, for example isolation of total RNA, isolation of poly(A) RNA from total RNA, suppression subtractive hybridization, differential display, preparation of cDNA libraries or quantitative dot blot analysis, as for example described in Example 1 of this specification.

This embodiment of the method of the invention is particularly suitable for identifying further genes the expression level of which is directly affected by the aberrant expression of any of the aforementioned genes. In other words, this embodiment of the method of the invention allows the identification of genes involved in the same protein cascade as the aberrantly expressed gene. Typically, the method of the invention will be a method performed in cell culture. The method of the invention allows for the design of further medicaments that use other targets than the aberrantly expressed gene. For example, if a potential target downstream of the aberrantly expressed gene is indeed targeted by a medicament, the negative effect of the aberrantly expressed gene may be efficiently counterbalanced. Compounds modulating other genes in the cascade may have to be refined or further developed prior to administration as a medicament as described elsewhere in this specification.

Additionally, the invention relates to a method for identifying one or a plurality of genes whose expression in heart tissue is modulated by the inhibition or decrease of the expression of a polypeptide selected from the group consisting of:

(a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID

NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12;

(c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of: (i) providing expression profiles of: (1) a plurality of heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and (2) a plurality of heart tissue cells from or derived from a subject not suffering from a disease of the heart; and (ii) comparing the expression profiles (1) and (2).

In variation to the method described herein above, this embodiment of the method of the invention compares the expression profiles of cells from a healthy subject and a subject suffering from a heart disease. In this regard, the term "cells derived from a heart" includes cells that are held in cell culture or even cell lines that autonomously grow in cell culture and that were originally derived from heart tissue. By comparing the two expression profiles, differences in expression levels of genes involved in the disease of the heart may be identified. As with the preceding embodiment, these genes may be part of a cascade involving the aberrantly expressed gene. Examples of such cascades are signaling cascades, Once genes are identified that are expressed at a different level in a diseased heart, they may be tested up-regulation or down-regulation by bringing them into contact with suitable test compounds. Again, these test compounds may then, with or without further development, be formulated into pharmaceutical compositions.

In a preferred embodiment, the method of the invention further comprises the steps of

(iii) determining at least one gene that is expressed at a lower or higher level in the presence of said compound; and

(iv) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

This preferred embodiment of the invention requires that one of the genes the expression of which may directly or indirectly be lowered or increased by the expression of the aberrant gene is identified. Then, a further panel of test compounds may be tested for the capacity to increase or decrease the expression of said further gene. Compounds that are successfully tested would be prime candidates for the development of medicaments for the prevention or treatment of a disease of the heart.

In another preferred embodiment, the method of the invention further comprises the steps of

(iii) determining at least one gene that is expressed at a lower or higher level in said heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and (iv) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

In variation of the previously discussed embodiment, this embodiment requires that at least one gene is identified by comparing the expression profiles of tissue or cells derived from a healthy subject and from a subject suffering from a disease of the heart. Subsequently, at least one compound is identified that is capable of increasing or decreasing the expression of said gene.

Additionally, the invention relates to a method for identifying a protein or a plurality of proteins in heart tissue whose activity is modulated by a polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12; said method comprising the steps of (i) providing said polypeptide; and (ii) identifying a further protein that is capable of interacting with said polypeptide.

One possible method to identify protein-protein interactions is the Yeast two-hybrid screen described by Golemis & Khazak (1997), Methods Mol Biol. 63:197-218. Other well established methods in order to identify protein-protein interactions are co-immunoprecipitations or in vitro protein interaction assays like GST-pulldown assays (such as described in Stoss, Schwaiger, Cooper and Stamm (1999). J. Biol. Chem. 274: 10951-10962). In a further preferred embodiment of the method of the invention said compound is a small molecule or a peptide derived from an at least partially randomized peptide library.

Additionally, the invention relates in a preferred embodiment to a method of refining a compound identified by the method as described herein above, said method comprising the steps of: .

(1) identification of .the binding sites of the compound and the DNA or mRNA molecule by site-directed mutagenesis or chimeric protein-studies;

(2) molecular modeling of both the binding site of the compound and the binding site of the DNA or mRNA molecule; and

(3) modification of the compound to improve its binding specificity for the DNA or mRNA.

All techniques employed in the various steps of the method of the invention are conventional or can be derived by the person skilled in the art from conventional techniques without further ado. Thus, biological assays based on the herein identified nature of the polypeptides may be employed to assess the specificity or potency of the drugs wherein the increase of one or more activities of the polypeptides may be used to monitor said specificity or potency. Steps (1) and (2) can be carried out according to conventional protocols. A protocol for site directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochem. 254: 157-178. The use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships is reviewed in Szklarz and Halpert (1997) Life Sci. 61 :2507-2520. Chimeric proteins are generated by ligation of the corresponding DNA fragments via a unique restriction site using the conventional cloning techniques described in Sambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual. (1989) Cold Spring Harbor Laboratory Press. A fusion of two DNA fragments that results in a chimeric DNA fragment encoding a chimeric protein can also be generated using the gateway-system (Life technologies), a system that is based on DNA fusion by recombination. A prominent example of molecular modeling is the structure-based design of compounds binding to HIV reverse transcriptase that is reviewed in Mao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol. 60: 1251-1265. For example, identification of the binding site of said drug by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the (poly)peptide primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug. As regards step (2), the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (1)) and computer simulations of the structure of the binding site provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other residues in the polypeptide of interest.

Finally, in step (3) the drug can be modified to improve its binding affinity or ist potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of the polypeptide of interest and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction.

Identification of binding sites may be assisted by computer programs. Thus, appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptide by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Modifications of the drug can be produced, for example, by peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220- 234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three- dimensional and/or crystallographic structure of activators of the expression of the polypeptide of the invention can be used for the design of peptidomimetic activators, e.g., in combination with the (poly)peptide of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

In accordance with the above, in a preferred embodiment of the method of the invention said compound is further refined by peptidomimetics.

The invention furthermore relates in a further preferred embodiment to a method of modifying a compound identified or refined by the method as described herein above as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophylic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof. The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).

The invention additionally relates to a method for inducing a disease of the heart in a non-human mammal, said disease is connected with the disordered expression of a polypeptide, comprising the step of contacting the heart tissue of said mammal with a compound that inhibits or decreases the expression of said polypeptide selected from the group consisting of:

NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least

99% identical to an amino acid sequence of (a); and (c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution. This embodiment of the invention is particularly useful for mimicking factors/developments leading to the onset of the disease. The fact, that differences in the expression of a protein contributes to heart failure has been shown for phospholamban, for example. Mice over-expressing phospholamban develop heart failure. This effect is thought to be due to the inhibition of Serca. (Minamisawa et al. (1999) Cell, 99:313-322).

In a preferred embodiment of the method of the invention said compound that inhibits or decreases is a small molecule, an antibody or an aptamer that specifically binds said polypeptide. The terms "small molecule" as well as "antibody" have been described herein above and bear the same meaning in connection with this embodiment. The invention moreover relates in a further preferred embodiment to a method further comprising producing a pharmaceutical composition comprising formulating the compound identified, refined or modified by the method of any of the preceding claims with a pharmaceutically active carrier or diluent. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 1012 copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.

The invention also relates to a method for preventing or treating a disease of the heart in a subject in need of such treatment, comprising the step of decreasing the level of a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12;

99% identical to an amino acid sequence of (a); and

Further, the invention relates to a method of preventing or treating a disease of the heart in a subject in need of such treatment comprising the step of decreasing the level of mRNA encoding a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of:

The invention in a preferred embodiment relates to a method wherein such decrease is effected by administering the pharmaceutical composition obtained by the method of the invention.

In a further preferred embodiment of the method of the invention such a decrease is effected by introducing the nucleic acid sequence recited herein above into the germ line or into somatic cells of a subject in need thereof.

In a most preferred embodiment of the method of the invention said disease of the heart is congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, specific heart muscle disease, rhythm and conduction disorders, syncope and sudden death, coronary heart disease, systemic arterial hypertension, pulmonary hypertension and pulmonary heart disease, valvular heart disease, congenital heart disease, pericardial disease or endocarditis.

In a further embodiment the invention relates to the use of a compound, an aptamer or an antibody identified, refined or modified by the method as described herein above for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of heart diseases, especially congestive heart failure. The figures show:

Fig. 1 : Hybridization pattern of DCMAG-2 on 19 HG-U95A oligo arrays (Affymetrix). Individual relative signal intensities of DCMAG-2, average values and standard deviations for NF (non-failing control) and DCM (Dilated Cardiomyopathy) samples are given.

Fig. 2: Hybridization pattern of DCMAG-2 on HG-U95A oligo arrays (Affymetrix) in different tissues. Individual relative signal intensities of DCMAG-2 as well as ratios between tissues are given.

Fig. 3: cDNA sequence of the EST fragment of DCMAG-2 identified by suppression subtractive hybridization (SSH).

Fig. 4: Sequence of DCMAG2 transcript as cloned from dilated cardiomyopathy patient RNA. Start and stop codons are underlined. 105 nucleotides corresponding to the additional exon 4a are marked in italic letters.

Fig. 5: Predicted protein sequence of DCMAG2. 35 amino acids resulting from the additional coding exon 4a are marked in italic letters; the nuclear localization signal is underlined.

Fig. 6: Detailed view on the genomic region around exon 4a which is inserted in DCMAG2 by alternative splicing as shown in the dotted lines but not present in D80010 (KIAA0188, shown in the broken line).

Figure 7: Morphometric analysis of stimulated and CFP expressing cardiomyocytes: Cardiomyocytes were stimulated, infected with a recombinant adenovirus (AV 151 , Flag-CFP) and analysed 48 hrs later. PE: Phenylephrine (100 μM); LIF: Leukemia inhibitory factor (1 ng/ml); ET-1: Endothelin-1 (10 nM); ISO: Isoprenaline (10 μM) Figure 8: Morphometric analysis of stimulated and Flag-DCMAG-2-CFP expressing cardiomyocytes:

Cardiomyocytes were stimulated, infected with a recombinant adenovirus (AV 223, Flag-DCMAG-2-CFP) and analysed 48 hrs later. PE: Phenylephrine (100 μM); LIF: Leukemia inhibitory factor (1 ng/ml); ET-1 : Endothelin-1 (10 nM); ISO: Isoprenaline (10 μM)

Figure 9: Morphological alterations of pCMs induced by over-expression of DCMAG-2:

Cardiomyocytes were stimulated, infected with a recombinant adenovirus (AV 223, Flag-DCMAG-2-CFP) and analysed 48 hrs later. LIF: Leukemia inhibitory factor (1 ng/ml); ET-1 : Endothelin-1 (10 nM); ISO: Isoprenaline (10 μM)

Figure 10: Immunofluorescence analysis of DCMAG-2 over-expressing cardiomyocyte:

Cardiomyocytes were stimulated with PE, infected with a recombinant adenovirus (AV 223, Flag-DCMAG-2-CFP) and analysed 48 hrs later by immunostaining with an anti -actinin antibody and a Cy3-conjugated secondary antibody. PE: Phenylephrine (100 μM).

Figure 11 : Hybridization pattern of 212276_at on HG-U133A oligonucleotide arrays (Affymetrix) in different tissues:

Individual relative signal intensities as calculated from MAS5 software (Affymetrix) and ratios between tissues are given.

Figure 12: Multiple sequence alignment with hierarchical clustering of DCMAG-2 and homologous sequences listed in Table 2.

Alignment was performed using Multalin version 5.4.1 (Copyright I.N.R.A. France 1996) according to Corpet (1988). The following parameters were applied: symbol comparison table: blosum62, gap weight: 12, gap length weight: 2, consensus levels: high=90% low=50%. Identities of all eight sequences are given in red. ! is anyone of IV, $ is anyone of LM, % is anyone of FY, # is anyone of NDQEBZ.

Figure 13: Detection of DCMAG-2 in different cell fractions by Western Blot. Heart tissue was fractionated by differential centrifugation and corresponding samples were analyzed by 12 % SDS-PAGE. Protein was blotted onto nitrocellulose membrane (Amersham) and DCMAG-2 was detected with a primary polyclonal rabbit antibody raised against a peptide comprising residues 118-135 of DCMAG-2 (Biogenes, Berlin) and anti-rabbit Ig-HRP conjugate (Amersham) as secondary antibody. PeNu: pellet of nuclear fraction, Nu: nuclear fraction, Wnu: wash of nuclear fraction, Pa: organelle containing fraction, Cyt: cytoplasmatic fraction. The molecular mass of Rainbow marker (Amersham) bands in kilodaltons (kDa) is indicated on the left of the gel. The band corresponding to full-length DCMAG-2 is indicated by the arrow and runs slightly above the 105 kDa marker band.

Figure 14: Influence of DCMAG-2 overexpression on cardiomyocyte contractility. Heart rings were infected with adenovirus containing cyan fluorescent protein (CFP, control) or DCMAG-2 fused to CFP. Maximal contractile forces of constantly paced heart rings were measured by a stepwise increase of ring tension (pre-load). Compiled data were statistically analysed using Sigmastat version 2.0 software (SPSS Inc., Chicago). Means, 25/75% quartiles and max/min values for heart cardiomyocytes overexpressing control virus and DCMAG-2, respectively, are depicted in boxplots. There is a statistically significant difference between the input groups (p<0.001).

Figure 15: Ultrasonic examination of paced rabbit after myocardial injection of AV223 (DCMAG-2-CFP).

Rabbit hearts were analysed by ultrasonic examination after two weeks of pacing and one week after gene transfer. Ten measurements were performed for each animal (AV 157: n=5; AV 223: n=8) after one week and after two weeks of pacing.

Fractional shortening was calculated based on enddiastolic and endsystolic diameter. Values depicted are percent alteration of fractional shortening after two weeks in relation to the one week measurement. AV 157 (yellow fluorescent protein, YFP), AV 223 (DCMAG-2 fused to cyan fluorescent protein, CFP). There is a statistically significant difference between the input groups (p<0.001).

Figure 16: Analysis of DCMAG-2 phosphorylation by PKB/Akt. YFP-tagged DCMAG-2 and native PKB proteins were co-precipitated using specific antibodies (Roche and Ceil Signaling respectively). The band corresponding to phosphorylated and radioactively labelled DCMAG-2 is indicated by the arrow. This band is visible only if PKB and DCMAG-2 were co-precipitated. When either DCMAG-2 or PKB were precipitated alone, no phosphorylation could be detected. The band in lane 4 from the right is due to spill-over from Lane 5.

Figure 17: Influence of DCMAG-2 overexpression on the phosphorylation of PKCβ. Control cells were compared with DCMAG-2 expressing cells by phosphoblot analysis.

Figure 18: Influence of DCMAG-2 overexpression on the activation of raf. Control cells were compared with DCMAG-2 expressing cells using a raf-1 immunoprecipitation kinase cascade assay kit (Upstate).

Fig. 19: Morphological alterations of pCMs induced by over-expression of DCMAG- 2 in the presence or absence of the MEK-1/2 specific inhibitor PD98059.

Figure 20: Influence of DCMAG-2 overexpression on the phosphorylation of MEK. Control cells were compared with DCMAG-2 expressing cells by phosphoblot analysis

Figure 21 : Influence of DCMAG-2 overexpression on the phosphorylation of Erk1 and Erk2.

Control cells were compared with DCMAG-2 expressing cells by phosphoblot analysis Figure 22: Analysis of tyrosine phosphorylation.

DCMAG-2 was precipitated with the anti-GFP antibody (Roche) from DCMAG-2 over-expressing cells (lane 5-9) and control cells (lane 1-4). Cells of lane 3-4 and 7-8 were incubated with fetal calf serum (10%). Precipitates were loaded onto an 8% gel. Tyrosine phosphorylation was detected using the anti phospho-tyrosine antibody (Calbiochem). As a control a sample was also blotted for DCMAG-2 protein using the anti-GFP antibody(Roche, lane 9). DCMAG-2 is indicated by the arrow.

Examples

The following examples illustrate the invention. These examples should not be construed as limiting: the examples are included for purposes of illustration and the present invention is limited only by the claims.

Example 1: Isolation of total RNA from heart tissue

RNA was isolated from different human heart samples of non-failing patients (NF) and patients suffering from dilated cardiomyopathy (DCM) (see table 3). Suppression Subtractive Hybridization (SSH) was used to enrich for transcripts that are over-represented in the DCM patient group (data not shown). Changes in the expression profile were confirmed by means of Affymetrix GeneChip technology. Total RNA was isolated from tissue of explanted hearts of left ventricle of human NF and DCM patients, respectively, according to the protocol of Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159) with some minor modifications. Approximately 500 mg tissue were disrupted using a mortar and pestle and grinded under liquid nitrogen. The suspension of tissue powder and liquid nitrogen was decanted into a cooled 50 ml polypropylene tube and nitrogen allowed to evaporate completely without thawing the sample. After addition of 1 ml Trizol (GibcoBRL) per 100 mg tissue the sample was homogenized immediately using a rotor-stator homogenizer (Ultra-Turrax T8, IKA Labortechnik) for 60 s at maximum speed. The sample was incubated for 5 min at room temperature (RT) and mixed with 200 μl chloroform per 1 ml Trizol. After vigorous shaking for 15 s the sample was incubated for 2-3 min at RT and centrifuged at 12000g for 15 min at 4 °C. The aqueous phase was transferred to a fresh 50 ml polypropylene tube and centrifuged again for 15 min. The supernatants was mixed with 0.5 ml isopropanol per 1 m Trizol and precipitated at RT for 10 min. After centrifugation at 12000g for 10 min at 4 °C the RNA pellet was washed with 1 ml 75% EtOH per 1 ml Trizol and dried at RT for 5-10 min. To completely dissolve RNA 50 μl DEPC-treated water per 1 ml Trizol was added and the sample was incubated at 60 °C for 10 min, final storage was at -80 °C. An aliquot was used for quantification by A₂6o measurement and separation on a formaldehyde agarose gel (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) to check integrity and size distribution.

Table 3: Patient data of human heart samples used for RNA isolation.

Example 2: Affymetrix array technology

50 μg total RNA was purified using RNeasy Mini columns (Qiagen) as described by the manufacturer. 5 μg of purified total RNA was used for synthesis of first and second strand cDNA and double stranded cDNA then used to synthesize the biotinylated cRNA probe as recommended by Affymetrix. 15 μg of fragmented labeled cRNA was hybridized to the human genome U95A array and stained by streptavidin-phycoerythrin (SAPE). Signal intensities were amplified using a biotinylated anti-streptavidin antibody and a second SAPE staining step. Data were analyzed by means of Microarray Suite and Data Mining Tool software provided by Affymetrix. Fold changes in gene expression were analyzed comparing average intensity values of 11 DCM heart samples with that of 8 normal controls (see Fig.

1). Disease-related regulation of DCMAG-2 was first identified by SSH and quantitative dot blot analysis (data not shown). By means of Affymetrix GeneChip analysis it . could be confirmed that the relative expression level of DCMAG-2 is significantly induced by a factor of 1 ,4 upon DCM. The probability of type 1 error is less than 5% as determined in a Mest and Mann-Whitney test. Using Affymetrix Gene Chip technology it was shown that the expression of

DCMAG2 is increased in heart and skeletal muscle as compared to kidney and liver (see Fig. 2).

To determine tissue-specificity, the expression of DCMAG-2 was analyzed in 12 different human tissues on human genome U133 microarrays. Applying MAS5 software, the probability of its signal intensity values had to be over 95% (p<0.05). The gene is represented by the target identifier 212276_at on the HG-U133A microarray. Oligonucleotide probes of 212276_at on the HG-U133A do not allow to distinguish between splice variants DCMAG-2 and DCMAG-2b, since they are directed to the 3'-end of the transcript.

As shown in Fig. 11 , the transcript was detected to be expressed mainly in heart and skeletal muscle. The level in ten other human tissues used for RNA profiling was detected to be nearly the same and very low. Therefore, we do not expect side effects on these tissues upon DCMAG-2 inhibition. Since the expression level in skeletal muscle was detected to be high, an inhibiting substance has to be specific for the heart form of DCMAG-2.

Example 3: Cloning, sequencing and bioinformatic analysis DCMAG-2 was amplified from human RNA by One step RT-PCR (Qiagen) according to the manufacturers protocols. The cDNA fragment identified by SSH (shown in Fig. 3) was found to be a part of the EST D80010:KIAA0188. For further cloning into selected vectors the upstream primer [gcgaattcatgaattacgtggggcag, the first ATG of the coding region is indicated in bold] and downstream primer [ccgctcgagcgctgaggcagaatgaat, the last codon is indicated in bold] were selected to contain restriction sites for EcoRI and Xhol, respectively. The EcoRI/Xhol restriction fragment was cloned into pBluescriptll vector (Sambrook et al., supra). The insert of 2775 nt was sequenced (MediGenomix) and analyzed by bioinformatic tools available from DoubleTwist at https://www.doubletwist.com. The full-length cDNA-sequence of DCMAG-2 is given in Fig. 4. The predicted protein consists of 925 amino acids (the amino acid sequence is given in Fig. 5) and displays a calculated molecular weight of 102.2 kDa. Figure 6 shows the detailed view on the genomic region around exon 4a.

We also cloned the DCMAG-2 isoform without exon 4a (DCMAG-2 beta). This clone was full-length sequenced and is identical to KIAA0188.

Example 4: Isolation of primary cardiomyocytes from neonatal rats Neonatal rats (P2-P7) were sacrificed by cervical dislocation. The ventricles of the beating hearts were removed and cardiomyocytes were isolated with the "Neonatal Cardiomyocyte Isolation System" (Worthington Biochemicals Corporation, Lakewood, New Jersey) according to the protocol. Briefly, the ventricles were washed twice with ice cold Hank's Balanced Salt Solution without Potassium and Magnesium (CMF-HBBS) and minced with a scalpel to an average volume of one cubic millimeter . The heart tissue was further digested over night with trypsin at 10°C. Next morning trypsin inhibitor and collagenase were added. After an incubation at 37^CC and mild agitation for 45 minutes the cells were dispersed by pipetting. The solution was further purified by 70 μm mesh (Cell Strainer) and centrifuged twice for 5 minutes at 60 x g. The cell pellet was resuspended in plating medium and counted. Cells were seeded with a density of 2 x 10⁴/cm² on gelatine (Sigma, Deisenhofen) coated dishes. The next morning cells were washed twice with DMEM and maintenance medium was added.

Plating medium: DMEM/M-199 (4/1); 10% Horse serum, 5% Fetal calf serum;

1 mM Sodiumpyruvate; Antibiotics and antimycotics Maintenance medium: DMEM/M-199 (4/1); 1 mM Sodiumpyruvate

Example 5: Stimulation of isolated cardiomyocytes from neonatal rats

Stimulation of primary cardiomyocytes (as described in the figure legends in detail) from neonatal rats (pCMs) was started two to six hours after medium was changed to maintenance medium. Different stimuli or combinations of stimuli were added to the cells. Used stimuli were:

Phenylephrine (PE) at 100 μM (Sigma)

Leukemia inhibitory factor (LIF) at 1 ng/ml (Roche Diagnostics)

Endothelin-1 (ET-1) at 10 nM (Roche Diagnostics) Isoprenaline (ISO) an 10 μM (Sigma)

Directly after stimulation pCMs were infected with recombinant adenoviruses to express the green fluorescent protein or variants thereof at a MOI of five. Cells were incubated for 48 hours at humidified atmosphere at 37°C and 5% C0₂ followed by an analysis of morphological alterations.

Example 6: Generation of recombinant adenoviruses

Recombinant adenoviruses were produced according to the simplified system developed by He et al. (He TC, Zhou S, da Costa LT, Yu J, Kinzler KW and Vogelstein B (1998): A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA. 95: 2509-2514). To generate a recombinant adenovirus genome in order to express the green-fluorescent-protein (GFP), the pAdTrack vector was combined with the pAdEasy-1 plasmid by homologous recombination. Briefly, 5 μg of the pAdTrack plasmid were linearized with the restriction enzyme Pme I and gel-purified. Approximately 100 ng of the linearized vector were combined with 100 ng of the pAdEasy-1 plasmid and aqua bidest. was added to a final volume of 7 μl. This solution was combined with 20 μl of electro- competent bacteria (BJ5183) and transferred to an electroporation cuvette (2.0 mm). The electroporation was performed using the Bio-Rad Gene Pulser (2.500 V, 200 Ohms, 25 μFD). Then 500 μl LB-medium were added. The bacterial culture was incubated at 37°C for 20 minutes in a bacterial shaker and afterwards plated on two LB-agar plates (1/10, 9/10) containing 50 μg/mi Kanamycin. After overnight incubation at 37°C twelve of the smallest colonies were picked and grown for at least 12 hours in 2 ml LB-medium (50 μg Kanamycin) at 37°C in a bacterial shaker. Plasmid DNA from these overnight cultures was purified by alkaline lysis and digested with the restriction enzyme Pac I. One of the positive clones which showed two fragments after cleavage (30 kb and 3.0 or 4.5 kb) was transformed into the bacterial strain DH5α by electroporation (2.500 V, 200 Ohms, 25 μFD). A single colony was picked and transferred into 500 ml LB-medium (50 μg/ml Kanamycin) and grown for 12 to 16 hours in a bacterial shaker. Plasmid DNA was prepared by the Tip-500 column (Qiagen, Hilden, Germany) according to the manufacturers instructions. Then 10 μg DNA were digested with Pac I, ethanol precipitated and resuspended in 40 μl H₂0 (cell culture grade).

The packaging was performed in HEK 293 cells (ATCC: ) by lipofection. The day before transfection cells were seeded into two T-25 flask (2 x 10⁶ cells each) in DMEM (10% fetal calf serum). For each flask 20 μl of the Pac I digested adenovirus genome was mixed with 20 μl of Lipofectamine (GIBCO BRL) in 500 μl OptiMem I medium and incubated for 15 minutes at room temperature. Meanwhile cells were washed twice with 4 ml serum-free DMEM. Then 2.5 ml OptiMem I was added to each flask followed by the DNA Lipofectamine solution. After incubation for 6 hrs at 37°C and 5% C0₂ medium was changed to DMEM (10% fetal calf serum). Packaging was monitored by GFP expression of transfected cells. Cells were harvested after 7 to 14 days, depending on the efficiency of the packaging. To harvest the cells they were detached by pipetting. Cells were sedimented by centrifugation at 100 x g for 5 minutes and the pellet was resuspended in 1 ml of (20 mM Tris pH 8.0, 2 mM MgCI₂, 140 mM NaCl, 3 mM KCI). After three freeze- thaw cycles in liquid nitrogen and a 37°C waterbath the cell lysate was centrifuged at 150 x g. For the amplification of the recombinant adenovirus 80% of the supernatant were used. The rest was stored at - 80°C after adding glycerol to a final concentration of 25%.

The first amplification was performed in one T-25 flask with HEK 293 cells at a density of 70 to 80%. Cells were harvested and lysed as described above. The virus titer was determined after two to four rounds of further amplification in T-75 flasks.

The titer of infectious particles was determined by end-point-dilution with HEK 293 cells based on the TCID 50 method (Mahy and Kangro, Virology Methods Manual, Academic Press. p37). Another series of adenoviruses for the monocistronic expression of recombinant proteins were generated based on the pShuttle vector. In the first step the pShuttle plasmid was cut with Sal I and Kpn I, gel purified, blunted with T4-polymerase and religated to eliminate the multiple cloning site. The vector was digested with EcoR I, blunted with T4-polymerase and religated to get rid of the single EcoR I site in the vector backbone. Next the vector was linearized with Bgl II and desphosphorylated. A whole expression cassette for a Flag-CFP (cyano variant of GFP) fusion protein derived from a modified pCI vector was inserted into the Bgl II site. The new construct was named plasmid #151.

The pCI vector (Promega) was modified in the following way. It was cut with BsrG I, blunted with Klenow-fragment in the presence of dNTPs and religated to eliminate the BsrG I site. The new vector was cut with Nhe I and Not I and gel purified. A PCR fragment containing the coding region for the CFP and the following restriction sites was inserted into the Nhe I and Not I sites:

Spe l-Xba l-EcoR l-Xho l-CFP coding region(2. codon until end)-STOP-Not I The pECFP-C1 plasmid (Ciontech) served as a template for the PCR amplification. The new vector was cut with Xba I and EcoR I and gel purified. The coding region for the Flag epitope was constructed by oligo annealing and inserted into the Xba I and EcoR I sites. Flag oligo 5': CTA GAT CCA CCA TGG ATT ACA AGG ATG ACG ACG ATA AGG Flag oligo 3': AAT TCC TTA TCG TCG TCA TCC TTG TAA TCC ATG GTG GAT

(the first ATG of the coding region is indicated in bold) Next the whole expression cassette was isolated by digestion of this vector with Bgl II and BamH I and gel purification of the smaller band. The resulting fragment was inserted into the Bgl II site of the vector described above to get plasmid #151. Plasmid #151 was linearized with Pme I and gel purified. The resulting recombinant adenovirus AV 151 for the expression of a Flag-CFP fusion protein was generated as described above. To get the recombinant adenovirus AV 223, which led to the expression of a Flag- DCMAG-2-CFP fusion protein, plasmid #151 was cut with EcoR I and Xho I and gel purified. A PCR fragment containing the entire coding region of DCMAG-2 without stop codon was inserted into the EcoR I and Xho I sites. To do this the EcoR I site was added in frame to the 5' end of the coding region and the Xho I site in frame to the 3' end by the PCR primers. Again a human heart cDNA library served as template for the PCR amplification. The resulting plasmid was cut with Pme I and gel purified. Recombinant adenoviruses were generated as described above.

Example 7: Morphometric analysis of stimulated cardiomyocytes

Morphometric analyses of cardiomyocytes were performed 48 hrs after stimulation with hormones, hormone analoga or cytokines as described in DE 19962154. Said stimulation leads to modifications in the localization of specific signaling molecules, especially DCMAG-1 , in the sarcomer of a heart tissue cell. This effect results in morphological alteration of the cell, indicating the state of hypertrophy. The PE stimulation led to larger cells with an increased width whereas the LIF stimulation resulted in an elongation of pCMs. The PE/ET-1 stimulation gave similar results as the PE stimulation, while ET-1 stimulated cells were only slightly larger than unstimulated cells. The ET-1/ISO/LIF stimulation in comparison with LIF stimulated cells in contrast led to a slight increase in width and length (see DE 19962154). The same pattern of morphological alteration was seen in control virus infected cells after stimulation (Figure 7). In particular the flat morphology without cell extensions of PE stimulated cells was very characteristic. The recombinant over-expression of a Flag-DCMAG-2-CFP fusion protein in pCMs led to vast morphological alterations of pCMs under all stimulation conditions (Figure 8). The very characteristic feature were thin elongated cellular protrusion with additional ramifications.

While cell protrusions were detectable under all stimulation conditions, the most spectacular manifestation of these morphological alterations was seen after treatment with LIF containing stimulation cocktails (Figure 9).

Example 8: Immunofluorescence analysis of DCMAG-2 over-expressing cardiomyocytes

Cells were prepared and treated as described in Example 4 and Example 5, but plated on glass coverslips in a density of 6.5 x 10⁴ cells/cm². Two days after stimulation and infection cells were washed twice with cold (4°C) phosphate buffered saline (PBS) incubated with cold (-20°C) Methanol/Aceton (7/3 vol/vol) for 20 minutes at -20°C, and washed three times with PBS (room temperature (RT) for 5 minutes each. Fixed cells were incubated with 10 % fetal calf serum in PBS for one hour in a humidified chamber at RT and incubated with first antibody for 1 hour at 37°C followed by three washing steps with PBS, 5 minutes each. The incubation with the secondary antibody was performed in the same way. After three washed with PBS samples were dipped into aqua bidest. and mounted with Histoclear (Linaris). Examination of fluorescence staining was performed on a confocal laser scanning microscope (TCS SP2, Leica). primary antibody: monoclonal anti- -actinin (A7811 , Sigma); 1 :800 in 0.5 % Tween-20, 0.5 % bovine serum albumine in PBS secondary antibody: Cy3-conjugated F(ab')2 fragment goat anti mouse

IgG (Jackson ImmunoResearch Laboratories, Inc.); 1:200 in 0.5 % Tween-20, 0.5 % bovine serum albumine in PBS pCMs were stimulated with PE which leads to protrusions induced by the overexpression of DCMAG-2. The subcellular analysis of protrusions with an antibody specific for sarcomeric -actinin revealed a striated appearance (Figure 10). The identification of α-actinin, the mean component of the sarcomeric Z-band, in a striated appearance in those protrusions revealed an extensive serial sarcomere organization induced by DCMAG-2.

Example 9: Cellular localization of DCMAG-2

200 mg frozen human heart tissue (DCM84) was powdered in liquid nitrogen. After evaporation of the liquid nitrogen, 1 ml 20 mM Tris/HCI, pH 7.4, 250 mM sucrose, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and protease inhibitors (1 tablet CompleteMini, Roche, for 10 ml buffer) were added. Cells were broken up for 3 min with a douncer using a loose pistil. The cell lysate was centrifuged with 3000g for 5 min at 4 °C. The supernatant was centrifuged with 13000g for 25 min at 4 °C. The supernatant was the cytoplasmic fraction and the pellet contained cell organelles (e.g. microsomes). The pellet of the first centrifugation step was resuspended in 500 μl 20 mM Tris/HCI, pH 7.4, 1 M sucrose, 1 mM EDTA, 10 mM NaF, 10 mM KCI and protease inhibitors (1 tablet CompleteMini, Roche, for 10 ml buffer). The suspension was centrifuged with 10000g for 30 min at 4 °C. The pellet of this centrifugation step was the nuclear fraction, whereas the supernatant was the wash fraction of nuclei.

The nuclear fraction was resuspended in 500 μl 20 mM Tris/HCI, pH 7.4, 400 mM KCI, 1 mM EDTA, 1 mM DTT, 0.1 % (v/v) Triton X100, 10 % (v/v) glycerol and protease inhibitors (1 tablet CompleteMini, Roche, for 10 ml buffer) and sonicated 3 times for 1 min with 5 W. The suspension was centrifuged with 13000g for 2 min at 4 °C. The pellet of this centrifugation step was the pellet of the nuclear fraction. Reducing SDS-PAGE sample buffer was added to each fraction, and samples were prepared by 5 min 98 °C treatment. After SDS-PAGE on 12 % polyacrylamide gels (Biorad), protein was blotted onto nitrocellulose membrane (Hybond-C extra, 45 micron, Amersham). The membrane was blocked with 5 % (w/v) skim milk solution. A polyclonal rabbit anti-DCMAG-2 antibody directed against a peptide comprising residues 118-135 of DCMAG-2 and purified by peptide-affinity chromatography (Biogenes, Berlin) was used at 73 μg/ml for dectection of DCMAG-2. Bound primary antibody was detected by a anti-rabbit Ig-HRP conjugate (Amersham) using LumiGlo as substrate (Cell Signaling Technology). As shown in Fig. 13, the Western blot revealed that the majority of DCMAG-2 was found in the organelle fraction; a significantly lower amount was found in the nuclear fraction and the pellet of the nuclear fraction. Therefore an association of DCMAG-2 with organelles like microsomes was assumed. The Immunofluorescence analysis was done as described omitting the antibody- staining steps. Instead, the YFP-tag DCMAG-2 was visualized directly using the Zeiss Axiovert 100 fluorescence microscope. Some cells received 50 μM PD98059 for 48 h (once daily application), while control wells only received the solvent DMSO. As shown in Figure 19, addition of MEK inhibitior PD98059 showed that the inhibition of the MAPK pathway blocks the elongation of cells caused by the overexpression of DCMAG-2.

Example 10: Effect of DCMAG-2 on the contractility of cardiomyocytes cultured in the 3D heart model Heart rings were prepared from neonatal rat cardiac myocytes (NCM) by a standard protocol. Primary cardiomyocytes were isolated as described in Example 4. Cells were washed once in 50 ml Ring Medium (DMEM from Biochrom, 4.5 g/l glucose, 10% horse serum, 1 mM Na-pyruvate, 2% Chick Embryo Extract (CEE) from Gibco, 1% Pen/Strep/Actinomycin). The cell pellet was then resuspended in a mixture of 0.945 mg Rat Tail Collagen Type I, 2x DMEM containing 5% CEE, 20% horse serum, 2.5% antibiotics, the appropriate amount of NaOH to buffer the acidic collagen solution, 10% MatriGel from Tebu and 2.5x10⁶ cells per ring. Heart rings were casted as described (Eschenhagen et al., 1997; Zimmermann et al., 2000) and maintained in Ring Medium. 48 h after casting rings were infected with 10-20 MOIs (multiplicity of infection) of control virus (cyan fluorescent protein, CFP) or of recombinant adenovirus containing a CFP-fusion of the target gene. 8 days after casting NCMs formed in an extracellular matrix mixture a compact, heart tissue-like ring, in which cells upon microscopic observation showed re-established cell-cell contacts in form of a three- dimensional network. Heart rings were trained by constant mechanical stretching for additional 7 days to strengthen the engineered heart tissue (Fink et al., 2000) before force measurements were performed.

To analyse contractility trained heart rings were suspended on a force transducer (Ingenieurbϋro Jackel) reaching into an organ bath and incubated at 37 °C in Tyrode's Salt solution, 1 g/l Na-bicarbonate and 0₂. Maximal contractile force was determined by stepwise increasing the tension on the heart ring (Frank Starling mechanism) and monitoring ring contraction with the BEMON software (Ingenieurbϋro Jackel). Heart rings were paced with 120 mA and 120 bpm throughout the length of the measurement. Recorded force parameters were analysed using AMON software (ingenieurbϋro Jackel). To analyse the new potential target DCMAG-2 in terms of its causative role in DCM two batches of heart rings were prepared and infected with recombinant target (n=14) or control adenovirus (n=11) containing a DCMAG-2-CFP-fusion or CFP, respectively. Resulting values are listed in Table 5 and 6, boxplot data have been calculated and are shown in Fig. 14. Upon force measurements heart rings overexpressing DCMAG-2 were characterised by a reduction of contractility to 28,6% in comparison to control virus-infected rings. This indicates a strongly negative impact of DCMAG-2 overexpression on the contractile behaviour of cardiomyocytes.

Since DCMAG-2 overexpression significantly (p<0.001) reduces contractility in the 3-D heart model, we expect a therapeutic intervention increasing expression and/or function of DCMAG-2 to result in an increased contractility and performance of the insufficient heart.

Table 5: Contraction force of independent heart rings infected with control virus measured in mN.

Table 6: Contraction force of independent heart rings infected with target virus containing DCMAG-2 measured in mN.

Example 11:Target Validation in Rabbit CHF Model

In vivo validation of CHF targets was performed in a tachycardia-induced heart insufficiency model. Right ventricular pacing over a two week period was used to provoke a massive dilatation of the left ventricle in rabbits. Adenovirally mediated target gene transfer was performed after one week of pacing, in order to assess the effect of the target protein during the second week of pacing. The following experimental schedule was performed: Day 0: first blood sample taken

Ultrasonic examination of heart geometry / function Implantation of pacemaker

Day 3: start of pacing at 320 beats per minute

Day 10: second blood sample taken

Ultrasonic examination of heart geometry / function

Myocardial injection of adenovirai suspension Omission of pacing for one day

Day 11 : restart of pacing at 360 beats per minute Day 18: third blood sample taken

Ultrasonic examination of heart geometry / function

Tip-catheter measurement of heart function during β-adrenergic stimulation

Explantation and storage of heart samples for further analysis

Pathological examination of whole animal Experiments were performed with New Zealand White (NZW) rabbits (Harlan Winkelmann, Borchen). Body weight was between 2.5 and 3.5 kg. Two permanent cannulae (Venflon Pro, 22 GA) were positioned into the Venae auriculars laterals for further pharmacological intervention.

Blood samples, 3 ml each, were taken from the Arteria auriculars media. Sedation was performed with Propofol (1 %, Fresenius Kabi). Initially at 5 - 10 mg / kg body weight (i. v.) until effect was observed and then continued with 1.3 - 2.0 mg / kg body weight. Animals were shave ventrally at neck, thorax and abdomen. Artificial respiration (Kindertubus, Rϋsch 2.5 mm inner diameter; Hallowell EMC, Vόlker) was performed at 30 breaths per minute at a volume of 6 - 8 ml / kg bodyweight with 100 % oxygen. Artificial respiration pressure was approximately 10 cm water column. For continued sedation animals received Disoprivan (2%, Astra Zeneca) at 80 - 110 mg / kg body weight per hour. Ultrasonic Examination

Ultrasonic examination of the heart was performed two-dimensionally (M-mode, Hewlett Packard Sonos 1000, 5 MHz Sektorschallkopf). Measurement focused on left ventricular endsystolic and enddiastolic diameter at level of the papillary muscle. The average of ten measurements were taken to calculate fractional shortening.

Pacemaker Implantation

Surgery was controlled by continuous ECG (Medtronic 9790 Programmer Vitatron).

Fentanyl (0.02 - 0.03 mg / kg body weight) was used as a sedativum.

The lead (2 F) was forwarded via the Vena jugularis externa in a catheter guide way into the right ventricle. The whole process was controlled by ECG and X-ray of the thorax. Pacemaker (Vita DDD, Vitatron) was inserted subcutaneously caudal to the costal arch. Carprofen was given as a post-surgerial analgeticum for four days at 4 mg / kg body weight per 24 hours. Wound healing was examined every day during this period. Gene Transfer

Transfer of target genes was performed by delivery of recombinant adenovirus directly into the myocardial wall (free wall of left ventricle). Sedation was performed in the same way as for the pacemaker implantation. Access to the heart was made via the third intercostal space. Direct myocardial injection of the viral suspension was performed after opening of the pericardium. In total 0.5 - 0.7 ml of virus suspension with a titre of infectious particles of 5 x 10⁹ per ml were injected at different point at the left ventricular free wall. Carprofen was given as a post-surgerial analgeticum for four days at 4 mg / kg body weight per 24 hours in combination with Buprenorphin at 0.01 - 0.05 mg / kg bodyweight. Wound healing was examined every day during this period.

Results depicted in Fig. 15 show that overexpression of DCMAG-2 significantly reduces fractional shortening. Therefore, we expect a therapeutic intervention increasing expression and/or function of DCMAG-2 to result in an increased performance of the insufficient heart.

Example 12:Analysis of phosphorylation of MEK, PKC-b and Erk Primary cardiomyocytes (pCMs) were prepared as described in Example 4 and plated in six-well plates at a density of 1x10⁶ cells/well. Cells were stimulated as described in example 5 for 48 h with Leucocyte Inhibitor Factor (Lif; 10 ng/ml), Isoproterenol (100 μM) and Endothelin-1 (ET-1 ; 200 nM). Cells were harvested by washing once in ice-cold PBS and scraping into 100 μl/well 1x Western Loading Dye (10% glycerol, 2% SDS, 150 mM DTT in 300 mM Tris, pH 6.8) and transferring to Eppendorf tubes. The tubes were vortexed to shear DNA and boiled for 5 min. 40 μl were loaded per lane (corresponds to 4x10⁵ cells per lane) onto a 10% SDS-polyacylamide gel and proteins separated by electrophoresis. Proteins were transferred onto a nitrocellulose membrane and incubated with antibodies (antibodies from NEB) recognizing phosphorylated PKC-β, MEK and Erk, respectively. The Western blot procedure was as follows: nitrocellulose membranes were blocked in 1% BSA (Sigma, Fraction V) for 1 h. Membranes were then incubated with the phospho-specific antibody for MEK, PKC-β or Erk (dilution 1 :1000) in 1% BSA-0.1 %Tween in Tris-buffered saline solution (TBS-T) overnight at 4 °C. Membranes were washed 4-5 times in TBS-T and then incubated with anti- rabbit-HRP conjugated secondary antibody (Amersham, dilution 1 :10000) for 2 h at RT. Membranes were washed again 4-5- times in TBS-T and then incubated with LumiGlo (NEB), the ECL developing agent as per instructions. Bands were visualized using the digital Fuji LAS1000 system and quantified using the AIDA software. To check for total protein amount the same Western blot membrane was stripped by incubation for 30 min at 50 °C in SDS (0.7% β-Mercaptoethanol, 20% SDS, 60 mM Tris pH 6.8), washed extensively in Tris-buffered saline and reblotted as per protocol using antibodies against the non-phosphorylated form of the respective protein (from NEB at a 1 :1000 dilution). Measurements were repeated at least six times.

As shown in figure 17, no differences in PKCβ phosphorylation was detected in control cells compared to DCMAG-2 expressing cells.

MEK phosphorylation was increased in DCMAG-2 expressing cells compared to control cells (fig. 20).

In addition, Erk1 and Erk2 phosphorylation was increased in DCMAG-2 expressing cells (fig. 21).

Example 13: Analysis of tyrosine phosphorylation of DCMAG-2

To detect tyrosine phosphorylation of DCMAG-2 the Western blot protocol was followed as described above. The only exception was the use of anti-phospho- tyrosine antibody in a 1 :1000 dilution (0.1 μg/ml) from Calbiochem as primary antibody.

Figure 22 shows that DCMAG-2 is tyrosine-phosphorylated. This phosphorylation is independent of serum stimulation.

Example 14: Measurement of Raf-kinase activity

Raf activity was measured using the raf-1 immunoprecipitation kinase cascade assay kit from Upstate (Cat no 17-173) according to manufacturers protocol. Briefly, 2 μg of anti.-raf antibody was added to a microcentrifuge tube. 100 μl of a 50% protein G agarose slurry that has been washed and resuspended in PBS was added. 300 μl of ice-cold PBS was added and incubated for 30 min on a rotator at 4 °C, after which the Protein G was pelleted at 14000 rpm for 15 s. The supernatant was removed and washed twice with ice-cold buffer A: 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3Vo4, 0.1% β-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.1 mM PMSF, and one Boehringer Complete Mini protease inhibitor tablet. The pellet was then resuspended in 200 μl Buffer A and 200 μl of cell culture cells (expressing DCMAG-2, plated and treated as described in Examples 3 and 4) lysed in Buffer A were added. The mixture was incubated for 2 h on a rotating wheel to immunoprecipitate raf-1. The mixture was centrifuged and washed twice with 500 μl Buffer A and once with ice-cold ADB (provided in the kit). The supernatant was removed and the pellet placed on ice. 20 μl ADB, 10 μl magnesium/ATP cocktail, 0.4 μg MEK1 , 1 μg ERK1/2 were added to each tube and the mixture incubated for 30 min in a shaking incubator. The reaction was stopped by the addition of 3x Western loading dye and the samples boiled for 5 min and then loaded onto a 10% SDS-PAGE gel. Phosphorylated Erk and MEK were detected using the Western Blot protocol mentioned earlier. Measurements were repeated at least three times.

As shown in figure 18, no differences in raf activation was detected in control cells compared to DCMAG-2 expressing cells.

Example 15: Analysis of phosphorylation of DCMAG-2 by PKB/Akt

Inspection of the DCMAG-2 sequence revealed a putative Akt-1 phosphorylation site RKRDKRS (residues 418-424) where S424 should be phosphorylated. A further analysis of the sequence displayed a second site RGRNTTI (residues 593- 599) which is a possible substrate for Akt-1. The second site complies the consensus sequence RXιRX₂X₃T/SX , published by Alessi et al. (1996), where Xi is any amino acid, X₂ and X₃ are small residues other than glycine and X is a bulky hydrophobic residue. Both of these sites are also present (in slightly modified form) in Lipin-1 , but not in Lipin-2 and Lipin-3. To confirm DCMAG-2 phosphorylation by PKB/Akt, cells were plated and transfected with DCMAG-2 virus or control virus as described. Cells were lysed in raf-1 kinase assay buffer as described above. After a short spin (10000 rpm 5 min 4 °C), the supernatant was incubated with anti-GFP antibody (1 :500) from Roche, that also recognizes YFP and CFP, and anti-mouse antibody coupled to agarose. In some tubes, 20 μl of immobilized AKT antibody was added ( Cell Signaling) as well.

The complexes were immunoprecipitated on a rotating wheel for 2 h or overnight at 4 °C and then the pellet washed twice in the lysis buffer and once in PKB assay buffer: 100 mM Tris, pH 7.4, 20 mM MgCI2. Radioactive labeled ATP was added (0.1 μCi/tube) and the reaction allowed to proceed for 30 min at 30 °C. It was then stopped using 5x Western loading dye, the sample boiled for 5 min and loaded onto an 8% gel. The gel was fixed in 20% ethanol, 10% acetic acid for 30 min at RT and then dried on a gel-drier for 2 h at 80 °C. Radioactively labeled phosphorylated DCMAG-2 was detected by means of a STORM phosphorimager (Molecular Dynamics). Measurements were repeated at least three times. As shown in Fig. 16, phosphorylated DCMAG-2 could only be detected, when DCMAG-2 and PKB/Akt proteins were co-precipitated using specific antibodies. Therefore we concluded, that DCMAG-2 is phosphorylated by PKB/Akt.

Example 16: Identification of adaptor proteins that bind to DCMAG-2

Comparison of the DCMAG-2 sequence with sequence motifs, to which adaptor proteins bind, revealed, that residues 234-240 (REWSPTP) and - more likely - residues 890-897 (RSHSSDFP) could be phosphorylation-dependent binding sites for 14-3-3 proteins. The minimal binding motif for 14-3-3ζ is RSXpSXP where pS designates a phosphoserine residue (Muslin et al., 1996). For BCR the corresponding sequence is RSQSQN, which might indicate, that the proline residue is not absolutely necessary. As Akt-1 contains a similar sequence RSGSPS (residues 121-126), 14-3-3 proteins could function as adaptors which bridge Akt-1 with DCMAG-2. In addition, the 14- 3-3 binding site could couple DCMAG-2 to other effector proteins containing a cognate motif. The proposed interaction of DCMAG-2 with 14-3-3 protein could be confirmed experimentally by a yeast two-hybrid assay described below. Cloning of 14-3-3 genes and DCMAG-2 deletion mutant. The 14-3-3 genes were cloned using RT-PCR (Qiagen). A heart cDNA library prepared from heart samples KN4, KN6, KN7, DCM93, DCM100, DCMN102, DHZM2, DHZM5 was used as template. The primers are listed in Table 7.The PCR products were recombined in the pDNOR vector (Invitrogen) according to manufacturers protocol. The clones were checked by PCR and confirmed by sequencing. 14-3-3epsilon5 I GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGCCATCATGgatgatcgagaggat

14-3-3epsilon3 GGGGACCACTTTGTACAAGAAAGCTGGGTGtggtttctcttgttggc

14-3-3tau5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGCCATCATGgagaagactgagctgatc

14-3-3tau3 GGGGACCACTTTGTACAAGAAAGCTGGGTGgttttcagccccttctg

14-3-3zeta5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGCCATCATGgataaaaatgagctggtt

14-3-3zeta3 GGGGACCACTTTGTACAAGAAAGCTGGGTGattttCCCCtccttctcctgct

14-3-3beta5 |GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGCCATCATGacaatggataaaag

Table 7: Primers for cloning proteins 14-3-3 epsilon, 14-3-3 tau, 14-3-3 zeta and 14-3-3 beta.

DCMAG-2 deletion mutants were generated by PCR using the DCMAG-2 full- length construct (Example 3) as template. The primers used are listed in Table 8. The PCR product was recombined in the Donor vector pDNOR201 (Invitrogen) via an in vitro BP reaction as recommended by the manufacturer. The recombination event was checked by PCR and confirmed by sequencing. Since this clone codes for a protein starting at amino acid 100 and ending with amino acid 658, this clone was named: DCMAG-2-N100C658

EST5-100-attR1-5 G GGG ACA AGT TTG TAC AAA AAA GCA GGC Tct atg cac ctg gee ace tec ccc ate c

EST5-dC658-3 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTa get gac at agg cag aag ag

Table 8: Primers used for cloning of DCMAG-2 deletion mutant.

Construction of the expression clones.

The expression clones were obtained via a LR reaction following manufacturers protocol (Invitrogen). Genes of interest were swapped from the pDONR vectors to the yeast-two-hybrid vectors of interest by LR recombination reaction.

Recombination events were checked by PCR.

Creation of a yeast two-hybrid mini matrix

Yeast methods were performed according to Golemis and Khazak (1997). The yeast two-hybrid bait vectors 413MetLexN0.att, 413MetLecC0.att and one containing the Gateway cassette, prey vectors 424GBN0.att, 424GBC0.att and again one containing the Gateway cassette were used. Yeast strains EGY48LacZ- GFP (ιvra3::6^*LexOp-lacZ, /ys2::6*LexOpCYC1GFP, his3, trpl , 6*LexAOp-LEU2, matα) and EGY199UL (ιvra3::6*LexOp-lacZ, his3, trpl , 6*LexAOp-LEU2, mat a) were used. Yeast cells were grown in YPD or selective minimal medium (Sherman, 1986). Transformations were done using the high-efficiency method of Gietz et al. (1992). The bait plasmids were first introduced in yeast strain EGY48LacZ-GFP resulting in the strain EGY48LacZ-GFP-bait. Self activation of the bait was checked by plating the yeast on minimal glucose medium X-Gal (5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside) with or without histidine. Protein expression was verified by Western blot analysis using a polyclonal rabbit anti LexA serum. The same transformation procedure was used to introduce the preys in the EGY199UL strain. The resulting prey was named EGY199UL-prey. A strain carrying the bait and the prey were mated (Golemis and Khazak, 1997) so that each EGY48LacZ- GFP-bait was challenged against each EGY199UL-prey for interaction. The interactions were identified by plating the colonies on medium A (contains glucose and X-gal, deficient in histidine, tryptophane and uracil) and medium B: (contains raffinose, galactose, X-gal, deficient in histidine, tryptophane and uracil). Protein protein interactions were considered to be positive when colonies were growing and becoming blue on medium B but not on medium A. Interaction between 14-3-3 and DCMAG-2-N100C658. The yeast two-hybrid mini-matrix allowed us to identify an interaction between DCMAG-2-N100C658 and the 14-3-3 proteins (Beta, Epsilon, Tau and Zeta). This interaction was obtained when 14-3-3 proteins were used as bait and DCMAG-2- N100C658 as prey. Moreover, positive interactions were found only when the 14-3- 3 sequences were fused to the C-terminus of the Lex-A DNA binding domain but not to the N-terminus. This finding is a hint that the interactions takes place between the C-terminal part of the 14-3-3 proteins and the DCMAG-2 protein. The interactions between the 14-3-3 proteins and the DCMAG-2 deletion mutant N100C658 likely takes place at that the first 14-3-3 docking site located between the amino acid 230 and 240 of DCMAG2. However others 14-3-3 docking site on the DCMAG2 can not be rule out.

Taken together, yeast to hybrid experiments have confirmed the interaction of 14- 3-3 proteins with the DCMAG-2 protein. Since DCMAG-2 is phosphorylated by PKB/Akt (Example 14, Fig. 16), 14-3-3 proteins could function as adaptors which bridge Akt-1 with DCMAG-2.

Abbreviations aa amino acids

ACEI inhibitors of angiotensin converting enzyme

Amp ampicillin

BPB bromphenol blue cDNA copy deoxyribonucleic acid

CHF congestive heart failure

DCM dilative cardiomyopathy ddH₂0 double-distilled water

DEPC diethyl pyrocarbonate EtOH ethanol f female

FDD fluorescence differential display

GAPDH glyceraldeyde-3-phosphate dehydrogenase

HCM hypertrophic cardiomyopathy ICM ischemic cardiomyopathy

ID identity m male mRNA messenger ribonucleic acid

NaOAc sodium acetate NF non-failing/healthy patient

RT room temperature

SSH suppression subtractive hybridization

U units vol volume XC xylene cyanol References

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Claims

A polynucleotide comprising a nucleotide sequence selected form the group consisting of: (a) a nucleotide sequence encoding the mature form of a protein comprising the amino acid sequence as given in SEQ ID NO: 2;

(d) a nucleotide sequence encoding a protein derived from the protein encoded by a nucleotide sequence of (a) or (b) by way of substitution, deletion and/or addition of one or several amino acids of the amino acid sequence encoded by the nucleotide sequence of (a) or (b), whereby overexpression in heart tissue of the protein encoded by said nucleotide sequence results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer;

(e) a nucleotide sequence encoding a protein having an amino acid sequence at least 60 % identical to the amino acid sequence encoded by the nucleotide sequence of (a) or (b), whereby overexpression in heart tissue of the protein encoded by said nucleotide sequence results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer;

(f) a nucleotide sequence encoding at least the domain of a polypeptide encoded by a nucleotide sequence as given in SEQ ID NO:6; (g) a nucleotide sequence comprising at least 15 consecutive nucleotides of a nucleotide sequence of any one of (a) to (e);

(h) nucleotide sequences obtainable by screening an appropriate library under stringent conditions with a probe having at least 12 consecutive nucleotides of a nucleotide sequence of any one of SEQ ID NO: 1 ;

(j) a nucleotide sequence which is degenerate as a result of the genetic code to a nucleotide sequence of any one of (a) to (h), whereby overexpression in heart tissue of the polypeptide encoded by said nucleotide sequence results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer; wherein said nucleotide sequence comprises at least 3 nucleotides of the

DNA sequence as given in SEQ ID NO: 5.;

The polynucleotide of claim 1 , whereby overexpression in heart tissue of the protein encoded by said polynucleotide results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer.

A nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with a DNA sequence of claim 1 or 2 or with a complementary strand thereof.

A vector comprising a DNA sequence of claim 1 or 2.

A vector comprising a nucleic acid molecule of claim 3. The vector of claim 4 or 5 which is an expression vector wherein the DNA sequence or nucleic acid molecule is operatively linked to one or more control sequences allowing the transcription and optionally expression in prokaryotic and/or eukaryotic host cells.

A host cell containing a vector of any one of claims 4 to 6 or a DNA sequence of claim 1 or 2.

The host cell of claim 7 which is a bacterial, insect, fungal, plant or animal cell.

The host cell of claim 7 which is a human cell or human cell line.

A method for the production of a protein or an immunologically active or functional fragment thereof comprising culturing a host cell of any of claims

7 to 9 under conditions allowing the expression of the protein and recovering the produced protein from the culture.

A protein or an immunologically active or functional fragment thereof encodeable by a DNA sequence of claim 1 or 2 or obtainable by the method of claim 10.

The protein of claim 11 which is glycosylated, phosphorylated, and/or amidated.

An antibody or an aptamer specifically recognizing the protein of claim 11 or 12 or a fragment or epitope thereof.

The antibody of claim 13, which is a monoclonal antibody.

A transgenic non-human mammal whose somatic and germ cells comprise at least one gene encoding a functional polypeptide selected from the group consisting of:

(a) the polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to the amino acid sequence of (a); and

(c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution; said functional polypeptide has been modified, said modification being sufficient to increase the amount of said functional polypeptide expressed in the heart tissue of said transgenic non-human mammal, wherein said transgenic non-human mammal exhibits a disease of the heart.

The transgenic non-human mammal according to claim 16, wherein said transgenic gene was introduced into the non-human mammal or an ancestor thereof, at an embryonic stage.

A transgenic non-human mammal according to claim 15 or 16, wherein the modification is activation or overexpression of said gene or leads to the enhancement of the synthesis of the corresponding protein.

A method for identifying a subject at risk for a disease of the heart, comprising the step of analyzing of at least one nucleic acid sequence or quantitating the amount of at least one RNA in the heart tissue of the subject, whereby

(a) said at least one nucleic acid sequence or RNA encodes an amino acid sequence: (aa) of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12; (ab) an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (aa); (ac) an amino acid sequence of (aa) with at least one conservative amino acid substitution;

(ad) an amino acid sequence that is an isoform of the amino acid sequence of any of (aa) to (ac); and (ae) an amino acid that is encoded by a DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1% SDS at 65°C to the DNA molecule encoding an amino acid sequence of (aa), (ac) or (ad); and/or (b) said at least one RNA is transcribed from the DNA sequence of SEQ ID NO: 1 , the DNA sequence of SEQ ID NO: 3, the DNA sequence of

SEQ ID NO: 7, the DNA sequence of SEQ ID NO: 9 or the DNA sequence of SEQ ID NO: 11 or a degenerate variant thereof.

The method according to claim 18, wherein the amount of the said RNA is quantitated using a nucleic acid probe which is a nucleic acid of:

(a) the DNA sequence of SEQ ID NO: 1 , SEQ ID NO: 3, the DNA sequence of SEQ ID NO: 7, the DNA sequence of SEQ ID NO: 9 or the DNA sequence of SEQ ID NO: 11 or a degenerate variant thereof;

(d) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of

(c);

(e) a nucleic acid sequence that encodes an amino acid sequence of (c) or (d) with at least one conservative amino acid substitution; (f) a nucleic acid sequence that hybridizes in 4xSSC, 0.1% SDS at 65°C to the complementary strand of the DNA molecule encoding an amino acid sequence of (c), (d) or (e); (g) a fragment of at least 15 nucleotides in length of (a) to (f); and

(h) a nucleic acid probe comprising a sequence that specifically hybridizes under physiological conditions to the nucleotide sequence of:

(i) the DNA sequence of the RNA transcribed from the DNA sequence of SEQ ID NO: 1, the DNA sequence of SEQ ID NO:

3, the DNA sequence of SEQ ID NO: 7, the DNA sequence of SEQ ID NO: 9 or the DNA sequence of SEQ ID NO: 11; (ii) a DNA sequence at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to a DNA sequence of (i);

(iii) a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12 with at least one conservative amino acid substitution; (iv) a nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (iii);

(v) a nucleic acid sequence that encodes an amino acid sequence of (iii) with at least one conservative amino acid substitution; (vi) a nucleic acid sequence that hybridizes in 2xSSC, 0.1% SDS at 65°C to the DNA molecule encoding an amino acid sequence of (iii), (iv) or (v); and

(vii) a fragment of at least 15 nucleotides in length of (i) to (vi). The method of claim 18 or 19, wherein said nucleic acid or RNA is obtained from heart tissue.

The method of any one of claims 18 to 20 further comprising the step of normalizing the amount of RNA against a corresponding RNA from a healthy subject or cells derived from a healthy subject.

A method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of a polypeptide in the heart tissue or the serum of the blood of the subject, the polypeptide selected of the group comprising:

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12;

The method according to claim 22, wherein the amount of the said polypeptide is quantitated using an aptamer or an antibody or an antigen- binding portion of said aptamer or antibody that specifically binds a polypeptide selected from the group consisting of:

(a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 12; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (a); and (c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution.

24. The method according to claim 23, wherein said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.

25. The method according to claim 23 or 24, wherein the antibody is the antibody of claim 13 or 14 or a derivative thereof.

26. The method of claim 25, wherein said derivative of said antibody is an scFv fragment.

27. The method of any of claims 23 to 26, wherein the aptamer or the antibody or derivative of said aptamer or antibody is detectably labeled at the binding portion.

28. The method of any one of claims 23 to 27 wherein said polypeptide is quantitated in heart tissue.

29. The method of any one of claims 23 to 28 further comprising the step of normalizing the amount of polypeptide against a corresponding polypeptide from a healthy subject or cells derived from a healthy subject.

30. A method for identifying a compound that decreases the level in heart tissue of a polypeptide selected from the group consisting of:

(b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (a); and (c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of: (i) contacting a DNA encoding said polypeptide under conditions that would permit the translation of said polypeptide with a test compound; and (ii) detecting a decreased level of the polypeptide relative to the level of translation obtained in the absence of the test compound.

31. A method for identifying a compound that specifically binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, said method comprising the steps of (i) providing said polypeptide;

(ii) contacting one or a plurality of compounds with said polypeptide; and (iii) identifying one or a plurality of compounds that is capable of binding said polypeptide.

32. The method of claim 31 , wherein said binding results in inactivation of said polypeptide.

33. A method for identifying a compound that decreases the level in heart tissue of an mRNA encoding a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 2,

SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% identical to an amino acid sequence of (a); and (d) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of (i) contacting a DNA giving rise to said mRNA under conditions that would permit transcription of said mRNA with a test compound; and

(ii) detecting a decreased level of the mRNA relative to the level of transcription obtained in the absence of the test compound.

A method for identifying a compound that decreases the expression of a polypeptide in heart tissue, the polypeptide being selected from the group consisting of:

(a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12;

The method according to claim 34, wherein the test compound prevents or ameliorates a disease of the heart in a transgenic non-human mammal according any of claims 15 to 17.

A method for identifying one or a plurality of isogenes of a gene coding for a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, whereby overexpression in heart tissue of the protein encoded by said identified isogen(s) results in an altered cellular morphology, in a serial sarcomer organization, in an altered amount, and/or an altered subcellular localization and/or in posttranslational modification of at least one signaling molecule in the sarcomer; said method comprising the steps of

(i) providing a first nucleic acid molecule of at least 12 nucleotides coding for at least a part of said polypeptide; and

(ii) identifying a second nucleic acid molecule that (a) has a homology of 60%, preferably at least 80%, especially at least 90%, advantageously at least 99% or (b) hybridizes in 4xSSC, 0.1 SDS at 45°C to the complementary strand of the nucleic acid molecule encoding said amino acid sequences.

37. A method for identifying one or a plurality of genes the expression of which in heart tissue is modulated by inhibiting or decreasing the expression of a polypeptide selected from the group consisting of:

(i) contacting a plurality of heart tissue cells with a compound that inhibits or decreases the expression of said polypeptide under conditions that permit the expression of said polypeptide in the absence of a test compound, and (ii) comparing a gene expression profile of said heart cell in the presence and in the absence of said compound.

38. A method for identifying one or a plurality of genes whose expression in heart tissue is modulated by the inhibition or decrease of the expression of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12;

(c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution, or of an mRNA encoding said polypeptide, said modulation being indicative of a disease of the heart, said method comprising the steps of: (i) providing expression profiles of

(1) a plurality of heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and

(2) a plurality of heart tissue cells from or derived from a subject not suffering from a disease of the heart; and

(ii) comparing the expression profiles (1) and (2).

The method of claim 37 further comprising the steps of

The method of claim 38 further comprising the steps of (iii) determining at least one gene that is expressed at a lower or higher level in said heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and (iv) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.

A method for identifying a protein or a plurality of proteins in heart tissue whose activity is modulated by a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12; said method comprising the steps of (i) providing said polypeptide; and (ii) identifying a further protein that is capable of interacting with said polypeptide.

The method of any one of claims 30 to 35, 37, 39 and 40, wherein said compound is a small molecule or a peptide derived from an at least partially randomized peptide library.

The method of any one of 30 to 35, 37, 39, 40, or 42 further comprising refining the compound identified, said method comprising the steps of:

(1) identification of the binding sites of the compound and the DNA or mRNA molecule by site-directed mutagenesis or chimeric protein studies;

The method of any one of claims 30 to 35, 37, 39, 40, 42 and 43, wherein said compound is further refined by peptidomimetics. The method of any one of claims 30 to 35, 37, 39, 40, 42 to 44 further comprising modifying the compound identified or refined as a lead compound to achieve:

(i) modified site of action, spectrum of activity, organ specificity, and/or

(ii) improved potency, and/or

(iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or

(v) modified onset of therapeutic action, duration of effect, and/or

(vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or

(vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or

(viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by

(i) esterification of carboxyl groups, or

(ii) esterification of hydroxyl groups with carbon acids, or

(iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or

(iv) formation of pharmaceutically acceptable salts, or

(v) formation of pharmaceutically acceptable complexes, or

(vi) synthesis of pharmacologically active polymers, or

(vii) introduction of hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or

(ix) modification by introduction of isosteric or bioisosteric moieties, or

(x) synthesis of homologous compounds, or

(xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or

(xiii) derivatisation of hydroxyl group to ketales, acetales, or

(xiv) N-acetylation to amides, phenylcarbamates, or

(xv) synthesis of Mannich bases, imines, or

(xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof.

A method for inducing a disease of the heart in a non-human mammal, said disease is connected with the disordered expression of a polypeptide, comprising the step of contacting the heart tissue of said mammal with a compound that inhibits or decreases the expression of said polypeptide selected from the group consisting of: 0363

91

(c) a polypeptide having an amino acid sequence of (a) with at least one conservative amino acid substitution^'.

The method according to claim 46, wherein said compound that inhibits or decreases is a small molecule, an antibody or an aptamer that specifically binds said polypeptide. The method of any of the preceding claims further comprising producing a pharmaceutical composition comprising formulating the compound identified, refined or modified with a pharmaceutically active carrier or diluent.

A method for preventing or treating a disease of the heart in a subject in need of such treatment, comprising the step of decreasing the level of a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of:

A method of preventing or treating a disease of the heart in a subject in need of such treatment comprising the step of decreasing the level of mRNA encoding a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12;

51. The method of claims 49 or 50, wherein such decrease is effected by administering the pharmaceutical composition obtained by the method of claim 45.

52. The method of claim 49 or 50, wherein such a decrease is effected by introducing the nucleic acid sequence recited in claim 19 into the germ line or into somatic cells of a subject in need thereof.

53. The method of any of claims 18 to 52, wherein said disease of the heart is congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, specific heart muscle disease, rhythm and conduction disorders, syncope and sudden death, coronary heart disease, systemic arterial hypertension, pulmonary hypertension and pulmonary heart disease, valvular heart disease, congenital heart disease, pericardial disease or endocarditis.

54. Use of a compound of any one of the 30 to 35, 37, 39, 40, 42 a refined or modified compound of any one of the claims 43 to 45, or an aptamer or an antibody of any of claims 13, 14 or 23 to 27 for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of heart diseases, especially congestive heart failure.