WO2008110356A2

WO2008110356A2 - Protein pi 16 secreted from the heart and uses thereof

Info

Publication number: WO2008110356A2
Application number: PCT/EP2008/001975
Authority: WO
Inventors: Robert Frost; Stefan Engelhardt
Original assignee: Robert Frost; Stefan Engelhardt
Priority date: 2007-03-12
Filing date: 2008-03-12
Publication date: 2008-09-18
Also published as: WO2008110356A3

Abstract

The present invention relates to the use of (a) polypeptide(s) the expression of which is indicative for hypertrophy or a heart disease or (a) nucleic acid molecule(s) encoding such (a) polypeptide(s), for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease. Moreover, the present invention relates to the use of such (a) polypeptide(s) or (a) nucleic acid molecule(s) for the preparation of a diagnostic composition for detecting hypertrophy or a heart disease, as well as to methods for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease. Additionally, the present invention relates to fragments and splice variants of (a) polypeptide(s) the expression of which is indicative for hypertrophy or a heart disease or (a) nucleic acid molecule(s) encoding such (a) polypeptide(s),as well as to corresponding vectors, host cells and pharmaceutical or diagnostic compositions comprising said (a) polypeptide(s), (a) nucleic acid molecule(s), vectors or host cells. Moreover, the present invention relates to a non-human transgenic animal specifically expressing in the heart a nucleic acid molecule encoding a polypeptide the expression of which is indicative for hypertrophy or a heart disease and to a corresponding nucleic acid expression construct, as well as to a corresponding vector and host cell. The present invention also relates to the use of said transgenic animal for the screening and/or validation of medicaments.

Description

Proteins secreted from the heart and uses thereof

Cardiomyocyte hypertrophy and fibrosis are of central importance for the development of congestive heart failure. Heart failure remains one of the most frequent causes of death in industrialized countries despite significant progress in pharmacological treatment¹. Remodeling of the myocardium, characterized by hypertrophy of cardiomyocytes and interstitial fibrosis, is of central importance for the development and progressive clinical course of heart failure. Communication between cardiac cells via secreted factors may contribute to myocardial remodeling. For example, conditioned medium from cultured fibroblasts induces hypertrophy of cardiomyocytes, and conditioned medium from cardiomyocytes promotes proliferation of fibroblasts ². Furthermore, stem cell therapy after myocardial infarction may prevent cardiac remodeling through paracrine factors ³'⁴. Paracrine factors that are known to contribute to cardiomyocyte hypertrophy and myocardial remodeling include angiotensin II, transforming growth factor-β, endothelin, catecholamines and insulin-like growth factor-1 ^{5 6}. The number of identified proteins secreted from the heart is small, possibly due to their low expression level. In contrast, bioinformatic analyses imply very high numbers of secreted proteins, comprising up to 2000 proteins for the mouse secretome ⁷'⁸. These approaches assume that the presence of a secretion signal sequence per se is sufficient to mediate effective secretion of a particular protein. However, there is no strictly defined consensus sequence for a functionally active signal sequence. Signal sequences are typically characterized by hydrophobic amino acids followed by a signal peptidase cleavage site. However, not all sequences with these characteristics function as signal sequences, e.g. because the signal sequence is not accessible after folding of the tertiary structure. Also identification of the genes itself in genomic data is still difficult and the estimates of the total number of human genes vary widely⁹. ESTs on the other hand are often truncated and miss the N-terminal signal sequence. Furthermore, tissue-specific expression data for the majority of the identified proteins are insufficient, making the identification of tissue specific secretomes difficult. Lastly, many of the putative secreted genes were identified by homology screening but homologues do not need to have the same subcellular localization. Taken together, computational screens may miss secreted proteins and may yield a high rate of false positive results.

In general, secreted proteins play a major role in intercellular communication. Several secreted peptides and proteins have been described to be involved in cardiac remodeling ⁶. However, many proteins known to be secreted in the heart like extracellular matrix proteins, proteinases and autocrine / paracrine factors like TGF β, TNF α, FGF, PDGF and IL-6 have been shown to be secreted by activated cardiac fibroblasts ⁶. Little is known about the secretome of cardiomyocytes. Cardiomyocyte specific overexpression of the βradrenergic receptor for example is accompanied by an activation of fibroblasts and interstitial fibrosis although the initial stimulus for remodeling was set in cardiomyocytes ¹⁹, however, the underlying mechanism is largely unknown.

The technical problem underlying the present invention is the provision of means and methods for the diagnosis, treatment or prevention of hypertrophy or a heart disease.

The technical problem is solved by provision of the embodiments characterized in the claims.

In a first aspect, provided is a method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need a polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6 and 8;

(b) a polypeptide comprising a fragment of the amino acid sequence of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment has PI16 function;

(c) a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440-459 of SEQ ID NO: 4, or any one of corresponding fragments (by comparison of homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having Pl 16 function; and

(d) a polypeptide comprising an amino acid sequence at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identical to the polypeptide of (a), (b) or (c), optionally having Pl 16 function, and, optionally, at least one further polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or a variant of said further polypeptide.

The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that proteinase inhibitor 16 (Pl 16), a newly identified protein secreted by the heart, is strongly upregulated in the heart early during the development of heart failure and cardiac hypertrophy and inhibits cardiomyocyte hypertrophy in vivo and in vitro. Although there are putative PI16-homologues in several species, a function of Pl 16 was largely unknown. Bioinformatical analysis revealed that Pl 16 contains a SCP- domain. This evolutionary highly conserved protein domain can be found in more than 600 mainly extracellular eukaryotic proteins of many different species including bacteria ²³. It was recently shown that PSP94 binding protein (PSPBP), the human homolog of Pl 16, may be partially bound in the serum to the prostate secretory protein of 94 amino acids (PSP94) ²⁴ (see also WO 03/093474). PSP94 is a protein that is upregulated in prostate cancer and may have growth regulating properties, in this disease ²⁵, but the signaling pathway or a receptor are unknown. In the gene ontology classification ²⁶ Pl 16 was inferred as protease by electronic annotation. The sequence analysis (ProtFun 2.2 ²⁷) performed in context of the present invention supports an enzymatic function (probability 0.48; odds 1.67) and negates a function as structural protein (probability 0.008; odds 0.28). Proteases have several important functions in cardiac disease; for example, matrix metalloproteinases and their inhibitors (TIMPS, tissue inhibitors of matrix metalloproteinases) play a crucial role in extracellular matrix modification in heart failure ²⁸.

Particularly, secreted and transmembrane proteins are of interest as therapeutic targets or possibly even as therapeutic agents. One advantage of the means and methods of the present invention is based on the use of such proteins, since they are accessible to various drug delivery mechanisms, because they are presented on the cell surface or within the extracellular space. A secreted protein or an extracellular receptor domain might even be utilized directly as a therapeutic agent, or it might be targeted for example by specific antibodies or small molecules.

A genetic yeast secretion trap screen in order to systematically search for genes encoding proteins that are secreted by the heart was performed in context of the present invention. Based on a murine cardiac cDNA-library, 54 cardiac proteins containing a secretion signal were identified. Many of these proteins have not been previously studied or even discovered in the heart, and the function of several is completely unknown. Among the latter is Pl 16, a protein that was found to be secreted by cardiomyocytes and that inhibits cardiomyocyte hypertrophy both in vitro and in vivo.

In contrast thereto, whether proteins secreted from the myocardium itself contribute to hypertrophy and fibrosis was largely unknown from the prior art.

It was further found out in context of the present invention that Pl 16, a 489 amino acid protein with so far unknown function, also displayed enhanced expression on the protein level after serum stimulation of primary cardiomyocytes as well as in failing myocardium. Additionally, it was found Pl 16 to be rapidly secreted by primary cardiomyocytes into the culture medium, where it inhibited cardiomyocyte growth.

Transgenic mice overexpressing Pl 16 in a cardiomyocyte-specific manner showed normal cardiac function but had smaller hearts with hypotrophic cardiomyocytes.

In view of the above, it is evident that PI16 represents a potential novel therapeutic and diagnostic target in heart failure and hypertrophy, like, for example cardiac hypertrophy.

In a second aspect, the present invention relates to a method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need one or more polypeptide(s), the expression of which is indicative for hypertrophy or a heart disease, or (a) variant(s) of said polypeptide(s).

In a third aspect, the present invention relates to a method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need a nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NO: 1 , 3, 5 and 7;

(b) a nucleic acid encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6 and 8;

(c) a nucleic acid encoding a polypeptide comprising a fragment of the amino acid sequence of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment has Pl 16 function;

(d) a nucleic acid encoding a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440- homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having Pl 16 function;

(e) a nucleic acid of at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identity to the nucleic acid of (a), (b), (c) or (d), wherein, optionally, said nucleic acid encodes a polypeptide having Pl 16 function; and

(f) a nucleic acid encoding a polypeptide as defined and provided herein, and, optionally, at least one further nucleic acid molecule encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or a variant of said further nucleic acid molecule.

In a fourth aspect, the present invention relates to a method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need one ore more nucleic acid molecule(s) encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or (a) variant(s) of said nucleic acid molecule(s).

In another aspect, the present invention relates to the use of a polypeptide selected from the group consisting of:

(d) a polypeptide comprising an amino acid sequence at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identical to the polypeptide of (a), (b) or (c), optionally having Pl 16 function, and, optionally, at least one further polypeptide, the expression of which is indicative for hypertrophy or a heart disease, for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

In a further aspect, the present invention relates to the use of one or more polypeptide(s), the expression of which is indicative for hypertrophy or a heart disease, for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

In an additional aspect, the present invention relates to the use of a nucleic acid molecule selected from the group consisting of:

(c) a nucleic acid encoding a polypeptide comprising a fragment of the amino acid sequence of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment has PI16 function;

(d) a nucleic acid encoding a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440- 459 of SEQ ID NO: 4, or any one of corresponding fragments (by comparison of homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having PI16 function;

(f) a nucleic acid encoding a polypeptide as defined and provided herein, and, optionally, at least one further nucleic acid molecule encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

Furthermore, the present invention relates to the use of one ore more nucleic acid molecule(s) encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

The nucleic acid/nucleic acid molecule as well as the nucleic acid construct as defined in or according to the present invention may be comprised by a vector, preferably by a viral vector, more preferably by an adenoviral vector.

The term "vector" as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In a preferred embodiment, the vectors of the invention are suitable for the transformation of cells, like fungal cells, cells of microorganisms such as yeast or bacterial cells or animal cells. In a particularly preferred embodiment such vectors are suitable for use in gene therapy.

In one aspect of the invention, the vector as provided is suitable for stable transformation of an organism, and hence is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, for example a promoter as defined herein, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a nucleotide sequence desired to be expressed. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the selected host organism. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, New York, (1982), 462-481 ; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), IpI , rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.

Examples of vectors suitable to comprise the nucleotide sequences/nucleic acid molecules of the present invention to form the vector of the present invention are known in the art.

For example, such vectors may be suitable for gene therapy, i.e. the vector of the present invention may also be a gene transfer and/or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes or nucleic acid constructs into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; Schaper, Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389 (1997), 239-242 and references cited therein.

Nucleic acid molecules or nucleic acid costructs encoding the herein disclosed Pl 16 (poly)peptides/proteines or the vectors as disclosed and described herein comprising said nucleic acid molecules or nucleic acid costructs may therefore be particularly designed for gene therapy approaches. Said compounds may also be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, baculoviral systems or systems based on vaccinia virus or Semliki Forest Virus can be used as eukaryotic expression system for said compounds disclosed in the context of the invention. For gene therapy, various viral vectors which can be utilized are, for example, adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can also incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the inserted polynucleotide sequence.

Moreover, the present invention relates to the use of (a) polypeptide(s) or (a) nucleic acid molecule(s) as defined or provided herein, for the preparation of a diagnostic composition for detecting hypertrophy or a heart disease.

Additionally, the present invention relates to a method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the amount of expression or activity of a polypeptide as defined or provided herein, and, optionally, of at least one further polypeptide, the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and

(b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein an increased (or decreased) amount of expression or activity of said polypeptide, and, optionally, an increased or decreased amount of expression or activity of said at least one further polypeptide, is indicative for said hypertrophy or heart disease. In another aspect, the present invention relates to a method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the amount of expression or activity of one ore more polypeptide(s), the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and

(b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein an increased amount of expression or activity of said one ore more polypeptide(s) is indicative for said hypertrophy or heart disease.

In a further aspect, the present invention relates to a method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the presence or amount of expression of a nucleic acid molecule as defined or provided herein, and, optionally, the presence or amount of expression of at least one further nucleic acid molecule encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and

(b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein the presence (or absence) or an increased (or decreased) amount of expression of said nucleic acid molecule, and, optionally, the presence or absence or an increased or decreased amount of expression of said at least one further nucleic acid molecule, is indicative for said hypertrophy or heart disease.

Moreover, the present invention relates to a method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the presence or amount of expression of one or more nucleic acid molecule(s) encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and (b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein the presence or an increased amount of expression of said one or more nucleic acid molecule(s) is indicative for said hypertrophy or heart disease.

In a preferred embodiment of the present invention, said further polypeptide or one or more polypeptide(s) may be or comprise CD14 (human GenelD according to Entrez Gene (NCBI): 929) or a variant thereof or CxcM4 (GenelD: 9547) or a variant thereof. In this preferred embodiment, it is preferred that an increased amount of expression or activity of said further polypeptide or said one or more polypeptide(s) is indicative for said hypertrophy or heart disease.

In another preferred embodiment of the present invention, said further polypeptide or one ore more polypeptide(s) may be or comprise CD 164 (GenelD: 8763) or a variant thereof or Psap (GenelD: 5660) or a variant thereof. In this preferred embodiment, it is preferred that a decreased amount of expression or activity of said further polypeptide or said one ore more polypeptide(s) is indicative for said hypertrophy or heart disease.

In another preferred embodiment of the present invention, said further nucleic acid molecule or one or more nucleic acid molecule(s) may be or comprises a nucleic acid encoding CD14 or a variant thereof or a nucleic acid encoding Cxcl14 or a variant thereof. In this preferred embodiment, it is preferred that the presence or an increased amount of expression of said further nucleic acid molecule or said one or more nucleic acid molecule(s) is indicative for said hypertrophy or heart disease.

In another preferred embodiment of the present invention, said further nucleic acid molecule or one or more nucleic acid molecule(s) may be or comprise a nucleic acid encoding CD164 or a variant thereof or a nucleic acid encoding Psap or a variant thereof. In this preferred embodiment, it is preferred that the absence or an decreased amount of expression of said further nucleic acid molecule or said one or more nucleic acid molecule(s) is indicative for said hypertrophy or heart disease.

In another specific embodiment of this invention, it is also envisaged that the absence or a decreased amount of (a) nucleic acid molecule(s) encoding PI16 or the corresponding PI16 polypeptide(s) is indicative for hypertrophy or a heart disease. However, it is of note that it is preferred in context of the present invention that the presence or an increased amount of (a) nucleic acid molecule(s) encoding Pl 16 or the corresponding Pl 16 polypeptide(s) is indicative for hypertrophy or a heart disease

The term "indicative for hypertrophy or a heart disease" as used herein refers to (a) nucleic acid molecule(s) or (a) polypeptide(s) the presence/absence, amount of expression and/or activity of which is different at a condition of hypertrophy or a heart disease compared to a condition without said hypertrophy or a heart disease. In this context, "different" can mean increased/higher as well as decreased/lower. Particularly, in this context, "different" can mean at least 0.25, 0.5, 0,75, 1 , 2, 3, 4, 5, 6, 10, 20, 50 or even 100 fold increased/higher or decreased/lower than compared to a condition without said hypertrophy or a heart disease.

The term "PI16 function" as used herein, inter alia refers to the capability to bind a binding partner of Pl 16, like, for example, PSP94 (²⁴, WO 03/093474), to inhibit protease activity, to act as an antihypertrophic agent and/or to be secreted (for example by (hypertrophied) cardiomyocytes). A "functional part of PI16" or "a functional fragment of the sequences of SEQ ID NO: 2, 4, 6 and/or 8 may be capable to fulfill at least one of the functions of the full length protein. A functional fragment may be or may comprise the SCP domain.

Particularly, a "hypertrophy" to be diagnosed, prevented or treated in context of the present invention may be selected from the group consisting of:

(a) cardiac hypertrophy or cardiomyocyte hypertrophy;

(b) hypertrophy of blood vessel cells; and

(c) vascular disorders.

Moreover, a "hypertrophy" to be diagnosed, prevented or treated in context of the present invention may be a hypertrophy in context of a skin disease or a hypertrophy of adipose tissue.

A "heart disease" to be diagnosed, prevented or treated in context of the present invention may particularly be selected from the group consisting of: (a) cardiac hypertrophy or cardiomyocyte hypertrophy; (b) heart failure;

(c) (heart) fibrosis;

(d) atherosclerosis; and

(e) remodeling post-myocardial infarction.

Beneath the above mentioned disorders or diseases, also (any other kind of) remodeling is intended to be diagnosed, prevented or treated in a further aspect of the present invention.

The term "remodeling" as used in this context particularly refers to tissue remodeling, i.e. this term refers to a change in the structure of a tissue, like, for example, by an alteration of the cell size (hypertrophy, hypotrophy), the cell number (apoptosis, proliferation) or the composition or extent of the extracellular matrix. Particularly, the term "remodeling" or "tissue remodeling" as used throughout this invention refers to a remodeling process of the heart, the skin, the vessels or the adipose tissue, preferably to a remodeling process of the heart.

Cardiac remodeling, for example in the course of cardiac failure, is often characterized by cardiomyocyte hypertrophy, cardiomyocyte apoptosis and interstitial fibrosis. It is shown that a pathological stimulus in one cell type can have an impact on other cell types and influence tissue remodeling in general. For example, cardiomyocyte specific overexpression of the beta_radrenergic receptor in transgenic mice, results not only in a hypertrophy and apoptosis of cardiomyocyes, but also in an activation of fibroblasts with consecutive interstitial fibrosis ¹⁹. Since Pl 16 is per se even a secreted protein, it is highly possibly that upregulation of Pl 16 as seen during cardiac disease or hypertrophy has an impact on other cells as cardiac fibroblasts, blood cells or endothelial cells and that Pl 16 might influence cardiac remodeling in a more general way. An influence in blood cells or endothelial cells might also have an impact on blood clotting Since Pl 16 is also strongly expressed in skin, vessels and adipose tissue, Pl 16 might have also an impact on remodeling in these tissues.

In a particular embodiment of the present invention, it is envisaged that the term "diagnosing" as used herein refers to the first identification of a hypertrophy or a heart disease as well as to the follow up of these conditions. In a further aspect, the present invention relates to a polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8;

(b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6 and 8, wherein said fragment has PI16 function;

(c) a polypeptide comprising a fragment of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment corresponds (by comparison of homology) to any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440-459 of SEQ ID NO: 4, and, optionally, wherein said fragment has Pl 16 function; and

(d) a polypeptide comprising an amino acid sequence at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identical to the polypeptide of (a), (b) or (c), optionally having PI16 function.

In another aspect, the present invention relates to a nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 5 or 7;

(b) a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8;

(c) a nucleic acid encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6 or 8, wherein said fragment has PU 6 function;

(d) a nucleic acid encoding a polypeptide comprising a fragment of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment corresponds (by comparison of homology) to any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369^39 and 440-459 of SEQ ID NO: 4, and, optionally, wherein said fragment has Pl 16 function;

(e) a nucleic acid of at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identity to the nucleic acid of (a), (b), (c) or (d), wherein, optionally, said nucleic acid encodes a polypeptide having PI16 function; and

(f) a nucleic acid encoding a polypeptide as provided herein and described above. The meanings of the terms "polypeptide" and "nucleic acid molecule'V'nucleotide sequence" are well known in the art, and are, if not otherwise defined herein, used accordingly in the context of the present invention. For example, "nucleotide sequence" as used herein refers to all forms of naturally occurring or recombinantly generated types of nucleic acids and/or nucleotide sequences as well as to chemically synthesized nucleic acids/nucleotide sequences. This term also encompasses nucleic acid analogs and nucleic acid derivatives such as, e. g., locked DNA, PNA, oligonucleotide tiophosphates and substituted ribo-oligonucleotides. Furthermore, the term "nucleotide sequence" also refers to any molecule that comprises nucleotides or nucleotide analogs.

Preferably, the term "nucleotide sequence" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The "nucleotide sequence" in the context of the present invention may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded, "nucleic acid molecule'V'nucleotide sequence" also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.

Furthermore, the term "nucleic acid molecule'V'nucleotide sequence" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., US 5525711 , US 4711955, US 5792608 or EP 302175 for examples of modifications). The nucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and, if not otherwise defined, without any size limitation. For instance, the nucleotide sequence may be genomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding such RNAs or chimeroplasts (Colestrauss, Science, 1996, 1386-1389). Said nucleotide sequence may be in the form of a plasmid or of viral DNA or RNA. "Nucleic acid molecule'V'nucleotide sequence" may also refer to (an) oligonucleotide(s), wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included. The meaning of the term "homologous" and "homology", respectively, particularly with respect to two Nucleic acid molecule or amino acid sequences/polypeptides to be compared, is also known in the art. These terms are used herein accordingly. For example, the tern "homology'V'homologous" is used herein in the context of a nucleotide sequence or amino acid sequence/polypeptide which has a homology, that is to say a sequence identity, of at least 40%, of at least 50%, of at least 60%, preferably of at least 70%, more preferably of at least 80%, even more preferably of at least 90% and particularly preferred of at least 95%, especially preferred of at least 98% and even more preferred of at least 99% to another, preferably entire, nucleotide sequence or amino acid sequence/polypeptide.

Methods for sequence comparison, particularly nucleotide or amino acid sequence comparison, and hence, determination of homology/sequence identity are well known in the art. For example, the degree of homology can be determined conventionally using known computer programs such as the DNASTAR program with the ClustalW analysis. This program can be obtained from DNASTAR, Inc., 1228 South Park Street, Madison, Wl 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 OAS UK (support@dnastar.com) and is accessible at the server of the EMBL outstation.

When using the Clustal analysis method to determine whether a particular sequence is, for instance, 90% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

Preferably, the degree of homology/identity of two nucleotide sequences, like two hybridizing nucleotide sequences, is calculated over their complete length, preferably of their coding sequences.

If the two nucleotide sequences to be compared by sequence comparisons differ in length, the degree of homology refers to the shorter sequence and that part of the longer sequence that matches the shorter sequence. In other words, when the sequences which are compared do not have the same length, the degree of homology preferably either refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence. In this context, the skilled person is readily in the position to determine that part of a longer sequence that "matches" the shorter sequence.

Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding nucleotide sequences or polypeptides (e.g. encoded thereby). Nucleotide/amino acid sequences which are homologous to the herein- described particular nucleotide/amino acid sequences and represent derivatives/variants of these sequences are normally variations of these sequences which, preferably, represent modifications having the same biological function. They may be either naturally occurring variations, for instance sequences from other ecotypes, varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. Allelic variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques. Deviations from the above-described sequences may have been produced, e.g., by deletion, substitution, insertion and/or recombination. The polypeptides encoded by the different variants of the nucleotide sequences of the invention or the variants of the polypeptides of the present invention preferably exhibit certain characteristics they have in common. These include for instance biological activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc. The biological activity of a Pl 16 (poly)peptide as described herein, for example its protease inhibiting activity can be tested in corresponding testing assays, for example by using Pl 16 or a suitable modified form thereof. Such assays can easily be established by the skilled person based on his common general knowledge and the teaching provided herein. Moreover, the present invention relates to a host cell comprising the nucleic acid/nucleic acid molecule, the polypeptide, the nucleic acid construct or the vector as defined in or according to the present invention. Said Host cell may be an embryonic stem cell, like, for example, a mouse embryonic stem cell.

Generally, the host cell of the present invention may be a prokaryotic or eukaryotic cell, comprising the nucleotide sequence/nucleic acid molecule, the nucleic acid construct, the vector and/or the polypeptide of the invention or a cell derived from such a cell and containing the nucleotide sequence/nucleic acid molecule, the nucleic acid construct, the vector and/or the polypeptide of the invention. In a preferred embodiment, the host cell comprises, for example due to genetic engineering, the nucleotide sequence or the vector of the invention in such a way that it contains the nucleotide sequences of the present invention integrated into the genome. For example, such host cell of the invention, but also the host cell of the invention in general, may be a bacterial, yeast, fungus, plant, animal or human cell. In one particular aspect, the host cell of the present invention is capable to express or expresses the nucleotide sequence/nucleic acid molecule of this invention. An overview of examples of different expression systems to be used for generating the host cell of the present invention, for example the above-described particular one, is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456- 463), Griffiths, (Methods in Molecular Biology 75 (1997), 427-440).

The transformation or genetically engineering of the host cell with a nucleotide sequence/nucleic acid molecule or the vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. Moreover, the host cell of the present invention is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc.

Additionally, the present invention relates to a pharmaceutical or diagnostic composition comprising the nucleic acid/nucleic acid molecule, the polypeptide, the vector or the host cell as defined in or according to the present invention.

For Example, when employed as or in a pharmaceutical composition according to this invention, Pl 16 may be administered in form of an biologically active peptide or in form of the full-length Protein. It is preferred that PI16 is applied in purified form when employed as or in a pharmaceutical composition according to this invention.

The compositions of the invention may be in solid or liquid form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Furthermore, it is envisaged that the medicament/pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.

Administration of the suitable (pharmaceutical) compositions may be effected by different ways, e.g., by parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral administrations include intraperitoneal, intramuscular, intradermal, subcutaneous intravenous or intraarterial administration. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like a specifically effected organ.

Examples of suitable pharmaceutical carriers, excipients and/or diluents, the compositions of this invention may optionally administered with, are well known in the art and include NaCI solutions, 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. Suitable carriers may comprise any material which, when combined with the (biologically active) nucleic acid/nucleic acid molecule, polypeptide, nucleic acid construct or vector as defined in or according to the present invention, retains the biological activity of said compounds (see Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed). Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions). The buffers, solvents and/or excipients as employed in context of the pharmaceutical composition are preferably "physiological" as defined herein-below. 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 may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present including, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.

The pharmaceutical compositions as disclosed and described herein are intended to be administered to a subject at a suitable dose. 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 depend 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. The person skilled in the art is readily in the position to determine the dosage regimen suitable for the particular compositions of the present invention.

Furthermore, it is envisaged that the pharmaceutical composition of the invention might comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. These further biologically active agents may be e.g. antibodies, antibody fragments, hormones, growth factors, enzymes, binding molecules, cytokines, chemokines, nucleic acid molecules and drugs. It is of note that the present invention is not limited to pharmaceutical compositions. Also compositions to be used in research or as diagnostic(s) are envisaged. It is, for example, envisaged that the biologically active peptides/proteins as disclosed herein are used in a diagnostic setting. For such a purpose, the inventive biologically active peptide/protein of this invention may be detectably labelled. Such labels comprise, but are not limited to radioactive labels (like [³H]hydrogen [¹²⁵l]iodide or [¹²³l]iodide), fluorescent labels (including but nor limiting fluorescent proteins, like green fluorescent protein (GFP) or fluorophores, like fluorescein isothiocyanate (FITC)) or NMR labels (like gadolinium chelates). The here defined labels or markers are in no way limiting and merely represent illustrative examples. The diagnostic compositions of this invention are particularly useful in a diagnostic medical setting.

The pharmaceutical compositions as disclosed and described herein are, in one aspect of this invention, intended to be administered under physiological conditions. Several buffers, are considered to represent "physiological solutionsTphysiological conditions" in context of this invention. Examples of such buffers are, e.g. phosphate- buffered saline (PBS: 115 mM NaCI, 4 mM KH₂PO₄, 16 mM Na₂HPO₄ pH 7.4), Tris buffers, acetate buffers such as citrate buffers or similar buffers such as those used in the appended examples. Generally, the pH of a buffer representing "physiological solution conditions" should lie in a range from 6.5 to 8.5, preferably in a range from 7.0 to 8.0, most preferably in a range from 7.2 to 7.7 and the osmolarity should lie in a range from 10 to 1000 mmol/kg H₂O, more preferably in a range from 50 to 500 mmol/kg H₂O and most preferably in a range from 200 to 350 mmol/kg H₂O. Optionally, the protein content of a buffer representing physiological solution conditions may lie in a range from 0 to 100 g/l, neglecting the protein with biological activity itself, whereby typical stabilizing proteins may be used, for example human or bovine serum albumin.

As also mentioned below, for example, repeated intravenously, subcutaneously, intranasally or per inhalation application of Pl 16 protein or parts of it or of Pl 16 proteinanaloga or of specific small molecules might reduce, stop or even reverse cardiomyocte hypertrophy during cardiac disease or even inhibit development of cardiac hypertrophy in patients with high risk to develop cardiac disease like patients with familiar forms of heart failure. In a further aspect, the present invention relates to a non-human transgenic animal specifically expressing in the heart a nucleic acid/nucleic acid molecule as defined in or according to the present invention or a nucleic acid/nucleic acid molecule encoding a polypeptide as defined in or according to the present invention. Thereby, for example, said nucleic acid molecule may be expressed under the control of a heart-specific promoter. Said promoter may be a cardiomyocyte specific promoter, like, for example the α-myosin heavy chain (αMHC) promoter.

In a particular aspect, the present invention relates to a non-human transgenic animal comprising the nucleic acid construct as described or provided herein, like, for example, the acid construct as depicted in Fig. 12.

In a further aspect, the present invention relates to a nucleic acid construct comprising a nucleic acid molecule/nucleic acid as described or provided herein or a nucleic acid molecule/nucleic acid encoding a polypeptide as described or provided herein and a promoter as defined or described herein. Particularly, said nucleic acid construct is envisaged to be nucleic acid construct as depicted in Fig. 12. Generally, the nucleic acid construct of the present invention is capable to express the nucleic acid molecule/nucleic acid as described or provided herein or a nucleic acid molecule/nucleic acid encoding a polypeptide as described or provided herein in a heart specific manner, like, for example in a cardiomyocyte specific manner.

In a further aspect, the present invention relates to the use of the transgenic animal as described and provided herein for the screening and/or validation of drugs or medicaments. For example, these drugs or medicaments may comprise (an) antagonist(s), preferably (an) agonist(s), of Pl 16 or of other proteins described or disclosed herein. Furthermore, these drugs or medicaments may have either an direct (e.g. an agonist or an antagonist) or an indirect impact on Pl 16 or the Pl 16 signalling pathway or on hypertrophy (e.g. of cardiomyocytes) or a heart disease. Such drugs or medicaments are particularly intended to be drugs or medicaments for the treatment or prevention of a hypertrophy or a heart disease, like a hypertrophy or a heart disease as defined herein, but could be also to screen for cardiac side effects as a hypertrophy or heart disease of other drugs. Detailed, non limiting, description of the findings made in context of the present invention:

Identification of proteins secreted in the heart by a secretion trap screen.

A murine cardiac cDNA-library with a complexity of 2 x 10⁷ independent clones, which means an average gene transcript was represented more than a 100 times, was generated. The cDNA library also contained also transcripts from weakly expressed genes, as tested by PCR amplification of a variety of genes (data not shown). Then, 1.7 x 10⁷ yeast transformants (about once the cDNA-library) were successively plated and 1900 clones that grew on selection media containing 2% sucrose were isolated. Only yeast clones carrying cDNA-fragments encoding for secreted proteins grew under selection conditions. By using an iterative cross hybridization strategy, the number of clones that were sequenced could be reduced to 347, and thereby, 54 non-redundant cardiac cDNAs encoding for putatively secreted proteins could be identified (table 2). Currently the function of approximately one third of these genes is unknown. Then, the mRNA expression of 25 of the identified genes was determined in a well-characterized murine heart failure model (βi-adrenergic receptor transgenic mice ¹⁹, figure 1). Compared to healthy wild-type littermates, upregulation of Protease inhibitor 16 (Pl 16; GenelD 74116; +420±130%, p<0.01), CD14 (GenelD 12475; +100±26%, p<0.01) and Cxcl14 (GenelD 57266; +68±30%, p<0.05) and downregulation of CD164 (GenelD 53599; -67±4%, p<0.001) and PSAP (GenelD 19156; -55+6%, p<0.001) were found. Pl 16 showed the strongest regulation in heart failure besides ANP. Therefore, one particular aim of the present invention was to further characterize Pl 16.

Cloning of PI16 and identification of a splice variant.

The murine Pl 16 gene comprises 7 exons (exon 7 encodes for the 3^'-UTR) spanning approximately 10,200 bp and resides on chromosome 17 ²⁰. Murine Pl 16 was cloned using primers at the 5^" and 3^'-ends of the putative coding sequence and confirmed the predicted sequence by sequencing (1470 bp). In the heart, thereby a more weakly expressed so far unknown splice variant comprising 684 bp that lacks exon 5 was identified (figure 2). The coding sequence of the human full-length homolog is shorter (1392 bp), but due to a smaller exon 5 is the splice variant is slightly longer (714 bp). Bioinformatic analysis with SMART ²¹ revealed that Pl 16 contains a SCP (sperm-coating glycoprotein)-domain. There are homologues to Pl 16 in other mammals like human (ENSP00000362778, Ensemble Peptide ID) and opossum (ENSMODP00000016841 ; aa identity 55.9%) but also in chicken (ENSGALP00000000778; aa identity 47.0%) (figure 3). The evolutionarily most distant homolog that was identified was in fish (ENSDARP00000067664; aa identity 32.9%), which suggests that Pl 16 evolved in vertebrates.

Protein expression of PM 6; Organ specific expression of PM 6.

The open reading frame of full length Pl 16 encodes a protein of 489 amino acids. A polyclonal antibody directed specifically against the full-length Pl 16 protein was generated (Pl 16-FL antibody). To examine the organ expression pattern of Pl 16, diverse organs from three months old wild-type FVB mice were isolated and the expression of Pl 16 was determined by Western blotting. Pl 16 protein expression was strongest in aorta and skin and weaker in adipose tissue (figure 4a). In healthy hearts, Pl 16 protein expression was low. Suprisingly, the polyclonal Pl 16 FL antibody detected three specific Pl 16 bands in tissue lysates both under reducing and non- reducing conditions. One band migrates at 74 kDa and two bands close to 100 kDa, i.e. 100 kDa and 108 kDa).

Two additional antibodies directed against exon 5 (excluding the SCP domain, which is localized in exons 1 to 4) and a small peptide in exon 2 of Pl 16, respectively, were generated. Both the original antibody directed against full-length PM 6 as well as the two new antibodies detected a virtually identical pattern of Pl 16 bands, when employed for Western blotting analysis in tissue lysates (Figure 4b). Bioinformatic analysis indicated potential glycosylation sites. Therefore, myocardial lysates from PI16-transgenic mice were deglycosylated using PNGaseF. Enzymatic deglycosylation lead to a significant shift of the Pl 16 bands to lower molecular weights suggesting glycosylation of the PI16 protein (Figure 4c). However, a different glycosylation pattern cannot explain the existence of more than one Pl 16 band. The theoretical molecular weight of PI16 is 52.7 kDa, however recombinant PI16 from E. coli fused to a GST-tag (27 kDa) and lacking its signal sequence (2 kDa) runs at about 95 kDa suggesting a molecular weight of unmodified Pl 16 of about 70 kDa (data not shown). This is in line with the deglycosylation experiment. The higher molecular weight bands of Pl 16 may represent a further modification or a covalent linkage to another protein.

PH 6 is a secreted protein.

The identification of Pl 16 in the secretion trap screen and the presence of a bioinformatically predicted N-terminal signal peptide both suggest secretion of Pl 16.

To determine whether Pl 16 is secreted from mammalian cells, neonatal rat cardiomyocytes (NRCM) were transfected with a PI16 expressing adenovirus (Adv- Pl 16) and COS7 cells were transfected with an eucaryotic expression vector (pTRex). In a first analysis, predominantly the lower Pl 16 band (74 kDa) was detected in cells transfected with Pl 16 (figure 5a), and a 100 kDa band predominated in the culture medium. This indicates that the N-terminal signal sequence of Pl 16 is functional and that Pl 16 is secreted from cardiomyocytes and putatively also other cells. In addition, upon secretion PI16 is further modified or covalently linked to another protein as suggested by the size increase detected by SDS polyacrylamid gel electrophoresis.

In a refined analysis with a possibly slightly stronger overexpression of Pl 16, interestingly, additionally to the 74 kDa band also the 108 kDa band was detected in lysates from cells transfected with PI16 (Figure 5b). Again, the 100 kDa band predominated in the culture medium. This confirms the indication that the N-terminal signal sequence of Pl 16 is functional and that Pl 16 is secreted from cardiomyocytes. Moreover, this indicates that the two cellular Pl 16 bands and the soluble extracellular Pl 16 together represent the three bands detected in tissue lysates that are a mixture of cells and extracellular material (Figure 4b).

Classically secreted proteins pass the endoplasmic reticulum and the Golgi apparatus and are ultimately transported to the plasma membrane in secretory vesicles. To elucidate the intracellular localization of PM 6, neonatal rat cardiomyocytes were transfected with a recombinant adenovirus expressing murine Pl 16 (MOI 0.1). lmmunofluorescent staining with the affinity-purified antibody directed against full-length Pl 16 revealed a distribution of Pl 16 in small cytoplasmatic vesicles as it is typical for secreted proteins (data not shown). Endogenous Pl 16 protein in non-transfected cardiomyocytes was only scarcely detected. This could be due to the low Pl 16 expression in non-stimulated cardiomyocytes or due to a rapid and effective secretion of Pl 16.

Therefore, cryosections of murine wild-type hearts were made and Pl 16 was stained by immunofluorescence (figure 6). Again, only small amounts of Pl 16 were visible intracellular^; however, Pl 16 was readily detectable in the intercellular space suggesting extracellular accumulation and possibly membrane association of Pl 16 in the heart after rapid secretion from cardiomyocytes.

Expression of PI16 is upregulated in heart failure.

In βi-adrenergic receptor transgenic mice, a well-characterized heart failure model using cardiac specific overexpression of the βi-adrenergic receptor, a profound upregulation (up to +470±55%, p<0.01) of Pl 16 full-length mRNA was found (figure 7). Pl 16 upregulation in this model starts in very young (two months old) animals and precedes the macroscopically visible myocardial remodeling, suggesting a possible role in the pathogenesis of heart failure. Messenger RNA expression of Pl 16 was also strongly upregulated (+200±70%, p<0.01) in mice after pressure overload of the left ventricle by thoracic aortic banding (figure 7). Furthermore, Pl 16 mRNA appeared to be upregulated (+323±160%, p=0.11) in human heart failure (figure 7). In accordance with these findings, also Pl 16 protein expression was found to be markedly upregulated in heart failure (figure 8). As in the adult murine heart there is only weak Pl 16 expression in isolated neonatal rat cardiomyocytes under normal conditions. However, Pl 16 expression is strongly induced in cardiomyocytes after serum stimulation (figure 9). In line with the previous experiments the lower Pl 16 band predominates in isolated cells.

Pl 16 inhibits hypertrophy of cardiomyocytes.

To determine the effect of PI16 on cardiomyocyte growth, neonatal rat cardiomyocytes were transfected with the PI16-expressing adenovirus. While Pl 16 overexpression had no effect on cardiomyocyte growth under normal conditions (data not shown), it inhibited isoproterenol/phenylephrine induced cardiomyocyte hypertrophy as assessed by isoleucin incorporation (figure 10). Interestingly, immunofluorescent staining demonstrated that the average cardiomyocytes size was already strongly reduced by overexpressing Pl 16 in only about 10% (MOI 0.1) of the cells, consistent with secretion of Pl 16 (lacZ-Adv, + 59% vs. PI16-Adv, +27%, p<0.01 ; figure 11). The amount of cells expressing Pl 16 was determined by co- staining with an antibody directed against PM 6 (data not shown). At MOI 0.4 about 30% of the cells expressed PI16, and at MOI 1.6 about 90%. Use of higher MOIs resulted in accumulation of Pl 16 in the endoplasmic reticulum and Golgi apparatus probably due to time-consuming posttranslational modifications. Next, the effect of Pl 16 expression on changes in the gene expression program typically associated with cardiomyocyte hypertrophy was analyzed. Treatment of NRCM with isoproterenol/phenylephrine led to a marked induction of ANP and BNP- mRNA expression, which was blunted in Adv-PI16 expressing cells (Figure 11 b). Then, it was sought to determine, whether endogenous Pl 16 is required for cardiomyocyte growth control. As to this, synthetic RNAi directed against Pl 16 was transfected into NRCM. This led to a marked suppression of Pl 16 protein levels (Figure 11c). Subsequent analysis of NRCM cell size by immunofluorescent staining (data not shown) revealed a marked increase in cardiomyocyte cell size and reorganization of the sarcomeres. These characteristics of cardiomyocyte hypertrophy were accompanied by a significant increase of cardiomyocyte protein synthesis through RNAi-mediated suppression of PI16 (Figure 11c). To determine the role of Pl 16 in the intact mammalian heart, transgenic mice that overexpress Pl 16 in a cardiomyocyte-specific manner under the control of the alphaMHC-promoter (figure 12) were generated. A first estimation revealed that these mice show about a 40-fold overexpression of Pl 16 in the heart. To assess the exact expression of the transgenic protein relative to endogenous Pl 16 levels, stepwise dilutions of myocardial lysates from Pl 16 transgenic mice with lysates from wild-type littermates were then performed (data not shown). Thereby, the level of transgene expression was determined to be about 20-fold higher than endogenous PI16 expression in nonfailing myocardium. These PI16-transgenic mice (Pl 16-Tg) developed normally, had a normal cardiac function (figure 13-15) and showed a normal myocardial structure without any signs of interstitial fibrosis (sirius red staining, data not shown). However, Pl 16-Tg mice had significantly smaller hearts than wild-type mice (figure 16). In line with these findings, individual cardiomyocytes from Pl 16-Tg mice were significantly smaller (- 24%, p<0.01 ; figure 17-18).

Without being bound by theory, the following conclusions can be drawn from the findings provided herein:

As already mentioned herein-above, this study describes the first systematical search for secreted proteins in the heart using a biological screen. 54 cardiac cDNAs comprising a secretion signal have been identified by a secretion trap screen in yeast. Among them are well-known genes like the atrial natriuretic peptide, but also genes with unknown cardiac expression and genes with so far completely unknown function and expression. 30 of the identified proteins are putatively secreted. 15 further proteins have secretion signals and one membrane domain but might be secreted after shedding from the plasma membrane²².

To avoid enrichment of mRNAs encoding for secreted proteins from non- cardiomyocytes, a cardiac cDNA library from young and healthy wild-type mice that showed no signs of fibroblast activation were generated.

The regulation of the identified genes during the development of heart failure was then studied. The mRNA-expression of Pl 16, a so far largely uncharacterized protein, was strongly upregulated early in heart failure and cardiac hypertrophy, like, for example, in murine and human heart failure. It is demonstrated herein that Pl 16 is expressed and secreted in the mammalian heart (e.g. from cardiomyocytes) and subsequently accumulates extracellularly in the heart. Furthermore, Pl 16 is detectable in the serum, e.g. by specific binding molecules, like, for example, antibodies, specifically directed against Pl 16 (data not shown). Cardiomyocyte specific overexpression of Pl 16 in transgenic mice increased serum levels of Pl 16 indicating that serum Pl 16 originates (at least partially) from the heart. Therefore, serum levels of PI16 altered during cardiac disease may be detected and, accordingly, Pl 16 is appropriable as a serum biomarker for diagnosis and course of cardiac and other disease. Also, the Pl 16 splice variant lacking exon 5 is upregulated in heart failure (data not shown). Therefore, also the splice variant might be usable as a serum biomarker for cardiac and other disease. Similar, the other proteins identified by the secretion trap screen to be secreted from the heart might be also detectable in the blood. Specifically, the proteins with altered expression in heart failure (PI16, CD14, CD164, Cxcl14, PSAP) might be usable alone, in combination or in combination with further serum biomarkers for diagnosis, following up, prevention and course of cardiac diseases.

Expression of Pl 16 in primary cardiomyocytes revealed a potent function of Pl 16 in the regulation of cardiomyocyte growth. Interestingly, this effect already occurred at very low MOIs, i.e. when only a small fraction of cardiomyocytes was transfected with a PI16-expressing adenovirus. Again, this supports a paracrine function of Pl 16. When expressed in the hearts of transgenic mice, Pl 16 was found to cause a profound inhibition of cardiac growth, in the absence of any impairment of cardiac structure or function. In line with the experiments conducted in vitro on isolated cardiomyocytes, this hypotrophic effect was traced back to a suppression of cardiomyocyte growth. The rapid and strong induction of Pl 16 and its growth- inhibitory effect under conditions of cardiac stress is reminiscent of the natriuretic peptides ANP and BNP ^{29 30} and the cytokine GDF15 ³¹. All three secreted proteins are induced in cardiac disease and serve as endogenous feedback mechanisms to control excessive growth promoting stimuli. Secretion of Pl 16 may serve a similar role as an endogenous cardioprotective signalling pathway and therefore can be used as therapeutic strategy in disease states such as cardiac failure and hypertrophic cardiomyopathy.

As already mentioned above, Cardiac remodeling, for example in the course of cardiac failure, is often characterized by cardiomyocyte hypertrophy, cardiomyocyte apoptosis and interstitial fibrosis. It is shown that a pathological stimulus in one cell type can have an impact on other cell types and influence tissue remodeling in general. For example, cardiomyocyte specific overexpression of the beta-adrenergic receptor in transgenic mice, results not only in a hypertrophy and apoptosis of cardiomyocyes, but also in an activation of fibroblasts with consecutive interstitial fibrosis ¹⁹. Since PI16 is per se even a secreted protein, it is highly possibly that upregulation of Pl 16 as seen during cardiac disease or hypertrophy has an impact on other cells as cardiac fibroblasts, blood cells or endothelial cells and that Pl 16 might influence cardiac remodeling in a more general way. An influence in blood cells or endothelial cells might also have an impact on blood clotting Since Pl 16 is also strongly expressed in skin, vessels and adipose tissue, Pl 16 might have also an impact on remodeling in these tissues.

Therefore, for example, repeated intravenously, subcutaneously, intranasally or per inhalation application of PI16 protein or (a)functional part(s) of it or of PI16 proteinanaloga or of specific small molecules might reduce, stop or even reverse cardiomyocte hypertrophy during cardiac disease or even inhibit development of cardiac hypertrophy in patients with high risk to develop cardiac disease like patients with familiar forms of heart failure. Additionally, application of Pl 16 as said above might have a favorable impact on pathological remodeling of the skin, vessels or adipose tissue.

Furthermore, cardiomyocyte-specific overexpression of Pl 16 in mice strongly reduces cardiomyocyte size. Therefore, also a gene therapy aiming in expressing Pl 16 in the heart e.g. by virus or direct cardiac application might reduce, inhibit or prevent (cardiomyocyte) hypertrophy or another heart disease. Since Pl 16 is a secreted protein that is found in the serum, it might be even possible to overexpress Pl 16 elsewhere in the body e.g. by a virus or a expression plasmid to achieve a Pl 16 effect in the heart.

Furthermore, numerous studies have demonstrated that the signaling pathways that control cardiomyocyte hypertrophy are also involved in the proliferation of foetal and neonatal cardiomyocyte and even in hypertrophy and proliferation of other muscle cells and of nonmuscle cells (Brooks, Cardiovasc Res. 1998; 39:301-11). For example, the RNA helicase CHAMP inhibits not only hypertrophy in post-mitotic adult cardiomyocytes, but also proliferation of foetal and neonatal cardiomyocytes (Liu, Proc Natl Acad Sci USA 2002; 99:2043-8). Therefore, Pl 16 might also has an impact on tissue remodeling like on hypertrophy or proliferation of cells also in other tissues where Pl 16 protein is found to be present in high concentrations as especially in skin, vessels and adipose tissue. Application, overexpression or inhibition of Pl 16 might have a favorable effect on disease stages of these tissues.

As mentioned above, proteins other than Pl 16, identified in context of the present invention to be secreted from the heart, might (also) be involved in (cardiac) hypertrophy and heart disease. For example, Toll like receptors (TLR) are important receptors of the innate immune system. They recognize general patterns of pathogens like for example lipopolysaccaride (LPS) from gram-negative bacteria. For the full activation of at least TLR4, LPS has to be presented by CD14 that is typically secreted from macrophages. Recently TLR4 deficient mice have been generated. These mice showed that TLR4 signaling is involved in cardiomyocyte hypertrophy during cardiac stress (Ha, Cardiovasc Res. 2005;68:224-34). TLR4 activation is likely to be independent of LPS in this setting suggesting the existence of an endogenous, so far not identified ligand for the TLR4. CD14 mRNA was identified in the present study to be expressed also in adult cardiomyocytes (data not shown) and to be upregulated in the failing heart. Therefore, without being bound by theory, CD14 secreted from cardiomyocytes during cardiac stress might present the so far unknown endogenous ligand to TLR4 and thereby promote cardiomyocyte hypertrophy. Inhibition of CD14 by e.g. specific antibodies, small molecules or other specific binding molecules might prevent cardiomyocyte hypertrophy during cardiac failure.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1 : Identification of proteins secreted from the heart by a secretion trap screen

Differential mRNA expression of genes identified by the yeast secretion trap screen in a murine heart failure model. RNA was isolated from the left ventricular myocardium of 6 months old β_radrenergic receptor-transgenic mice, gene expression levels were determined by real-time PCR and compared to wild-type littermates. Expression of protease inhibitor 16 (Pl 16) was nearly as strongly upregulated as ANP, a typical marker gene in heart failure. Furthermore, CD14 (CD14 antigen) and Cxcl14-mRNA (chemokine (C-X-X motif) ligand 14) were upregulated and mRNA-expression of CD164 and Psap (prosaposin) was downregulated in failing compared to non-failing myocardium. RIKEN cDNAs are abbreviated with the corresponding cDNA number.

Figure 2: Identification of a new splice variant of PM 6

Agarose gel depicting the PCR-products of a PCR with primers at the 3^Λ- and 5^'-end of Pl 16. The lower band represents a new splice variant of murine and human Pl 16 that lacks exon 5.

Figure 3: Identification of PM 6 homologues

Partial alignment of the amino acid sequences of homologues of Pl 16 by CLUSTAL W ³². The evolutionary oldest Pl 16 homolog is found in fish. Black labeling of residues indicates identity within the homologues, grey labeling indicates conservative replacement.

Figure 4: Protein expression of PM 6

a: Organ specific expression of Pl 16:

Detection of Pl 16 in diverse tissues of wild-type FVB mice by Western blotting. Strong Pl 16 expression was found in aorta, skin and to a lower extent in adipose tissue, but Pl 16 expression in the heart of healthy wild-type animals was low. The antibody directed specifically against Pl 16 detected three specific Pl 16 bands in eukaryotic tissues, one migrating at 74kDa and two at approx. 10OkDa.

b: Characterisation of three different polyclonal antibodies raised against PI16: (upper panel) Localization of a peptide (aa 84-94 of mouse PI16) and the protein fragment comprising exon 5 used to raise anti-PI16 polyclonal antibodies. All three antibodies were affinity-purified using the respective antigen.

c: Pl 16 is a glycosylated protein: After treatment of myocardial tissue of Pl 16 transgenic mice with PNGaseF, Pl 16 was detected by Western blotting.

Figure 5: PM 6 is a secreted protein a: first analysis:

Detection of Pl 16 by Western blotting in lysates from neonatal rat cardiomyocytes (NRCM) and in conditioned medium from neonatal rat cardiomyocytes 48 hours after transfection with a PI16-expressing or a control adenovirus (left panel). Predominantly the lower Pl 16 band was detected within the cells and a 10OkDa band in the culture medium. A similar result was found after transfection of COS7 cells with a Pl 16 expressing vector (right panel).

b: refined analysis:

Detection of Pl 16 by Western blotting in lysates from NRCMs and in conditioned medium from neonatal rat cardiomyocytes 48 hours after transfection with a Pl 16- expressing or a lacZ-expressing control adenovirus (Adv, MOI 0.1 , upper panel). Predominantly the 74 kDa band and the 108 kDa band are detected in lysates from cells transfected with Pl 16. In contrast, the 100 kDa band predominates in the culture medium. Detection of β-actin was used to monitor for potential contamination of the culture medium with cellular material derived from lysed cells.

Figure 6: PM 6 accumulates extracellularly in the heart lmmunofluorescent detection of Pl 16 in cryosections of a wild-type mouse heart. Pl 16 (bright signal) accumulates extracellulary around cardiomyocytes.

Figure 7. mRNA Expression of PM 6 is strongly upregulated early in heart failure

Determination of Pl 16 mRNA expression in cardiomyopathic myocardium by realtime PCR. Total RNA was isolated from left ventricular myocardium (n>4 each) of a mouse model of cardiac failure (β-i-adrenergic receptor transgenic mice), of mice with pressure overload-induced cardiac hypertrophy (after transverse aortic constriction, TAC) and from human failing myocardium. Figure 8: Strong upregulation of PU 6 protein in heart failure

Western blotting of heart lysates from left ventricular myocardium from wild-type and βi-adrenergic receptor transgenic mice. Mice were studied at 12 months of age.

Figure 9: Induction of Pl expression in vitro after serum stimulation

Induction of PI16 in neonatal rat cardiomyocytes after serum stimulation (5%, 24 hours). The Western blot shown is representative of three independent experiments.

Figure 10: PI16 inhibits hypertrophy of cardiomyocytes in vitro

Quantitative analysis of cardiomyocyte protein synthesis through determination of [³H]-leucine, namely [³H]-isoleucine incorporation. Cells transfected with lacZ adenovirus or PM 6 adenovirus were stimulated with isoproterenol (5 μM)/phenylephrine (50 μM) or left untreated.

Figure 11 : PI16 regulates cardiomyocyte size in vitro

a: PI16 inhibits hypertrophy of cardiomyocytes in vitro:

Morphometric analysis of cardiomyocyte surface areas after immunofluorescent staining. Cells transfected with lacZ adenovirus or Pl 16 adenovirus were stimulated with isoproterenol (5 μM)/phenylephrine (50 μM) or left untreated. More than 50 individual cells per group were analyzed by digitizing the images and computerized pixel counting.

b: Pl 16 inhibits expression of marker genes for cardiac hypertrophy: Expression of ANP- and BNP-mRNA as indicators of a prohypertrophic gene expression program. Real-time PCR was carried out on total mRNA preparations from NRCM treated with isoproterenol/phenylephrine (5/50 μM). Data are means of three independent experiments with three replicates each. Adv-PI16 or Adv-lacZ were used at an MOI of 1.6. ^**P<0.01 for Adv-PI16 vs. Adv-lacZ Iso/PE-stimulated cells, t-test.

c: Suppression of Pl 16 by RNAi induces cardiomyocyte growth: (left panel) Western blotting of PI16 in lysates derived from PM6-RNAi and control- RNAi-treated (scr-RNAi) NRCM infected with Adv-PI16 (MOI 0.1). (right panel) Determination of [³H]-isoleucine incorporation as a marker of protein synthesis in NRCM after 40 h starving without serum. NRCM were either transfected with Scrambled-RNAi (scr-RNAi), PM 6-RNAi (40 nM) or just exposed to the transfection reagent (control). Data are means from four independent experiments, each comprising three replicates per treatment condition. P<0.01 for Pl 16-RNAi vs. scr- RNAi by ANOVA, ^* P<0.05 for PI16-RNAi vs. scr-RNAi transfected, unstimulated cells, by Bonferroni's post test.

Figure 12: Generation of PU 6 transgenic mice

Transgenic mice overexpressing Pl 16 (Pl 16-Tg) cardiomyocyte specific under the control of the α-myosin heavy chain (αMHC) promoter were generated.

Figure 13-15: PI16 transgenic mice have a normal heart function

In vivo cardiac hemodynamics (heart rate, left ventricular pressure and dP/dt max) were analyzed in anesthetized mice by left ventricular catheterization. Dobutamine was administered via the left jugular vein (n=5-9).

Figure 16: PI16 transgenic mice have smaller hearts

Ventricular weight to body weight ratio of wild-type and Pl 16-Tg mice. Age, 3 months, n=7.

Figure 17: Inhibition of cardiomyocyte growth in PI16 transgenic mice.

Histological analysis of left ventricular myocardium from 3 months old wild-type and Pl 16-Tg mice. 5 μm sections were cut from paraffin-embedded tissues and were stained with hematoxylin eosin.

Figure 18: Inhibition of cardiomyocyte growth in PI16 transgenic mice.

Morphomethc analysis of myocyte cross-sectional areas. 40-60 individual cells per animal were determined by digitizing the images and computerized pixel counting (5- 6 animals per group). The Examples illustrate the invention.

Example 1 : Material and Methods

Secretion Trap Screen.

The secretion trap screen was performed as described ¹⁰'¹¹ with some modifications. Briefly, the host yeast strain O66-2 (kindly provided by C. Weitz, Department of Neurobiology, Harvard Medical School, Boston) lacks invertase, an enzyme that must be secreted for the strain to grow on sucrose. A cDNA library was prepared (Superscript Plasmid System, Invitrogen) from left heart ventricles from 4-5 months old wild-type FVB mice using random hexamer primers. The cDNA library was then cloned into a yeast expression vector encoding a mutant invertase (pSuc2^dMSP provided by C. Weitz), which lacked the start codon for methionine and the signal sequence necessary for secretion. After transformation of the host strain with the library, colonies could form on sucrose only in those cases in which a clone from the cDNA library provided both a start codon and a functional signal sequence in-frame with the invertase. cDNA inserts of positive clones were amplified by PCR with flanking primers, sequenced and analyzed by a BLAST search. To avoid sequencing of redundant clones, an iterative cross hybridization of the PCR-products of newly identified clones was performed with ³²P-labeled probes directed against repeatedly found cDNAs.

Prediction of signal peptides of the identified genes was performed by Signal P 3.0 ¹² and prediction of non-classical secretion was carried out by SecretomeP 1.0 ¹³. Transmembrane helices have hydrophobic properties similar to signal peptides. To detect membrane proteins among the identified genes, transmembrane helices were predicted by TMHMM Server 2.0 ¹⁴.

RNA isolation and real-time quantitative PCR.

Total RNA was extracted from frozen tissue using the RNeasy Mini Kit (Qiagen, Hilden, Germany) including DNase-digestion according to the manufacturer^'s instructions. Real-time PCR was performed with Sybr Green as fluorescent dye and data were calculated with the 2-ΔΔCT method ¹⁵. Please refer to the Supplementary Methods for a detailed description. Human heart tissue.

Human heart specimens were analyzed from stored samples that had originally been obtained from patients undergoing heart transplantation ¹⁶. After removal of the hearts, samples had been snap frozen in liquid nitrogen and stored at -8O⁰C. Non- failing donor hearts that had not been transplanted for technical reasons were used as controls. Echocardiography and past medical history of the donors had shown no indications of heart disease. The present study was approved by the Ethics Committee of the Medical Faculty of the University of Wuerzburg.

Generation of a PI16-specifιc antibody and Western blot analysis.

Polyclonal antibodies directed against Pl 16 were generated and affinity purified as detailed in the Supplementary Methods section.

Generation of PM 6 adenovirus.

Mouse full-length Pl 16 was cloned under the control of the CMV promotor into the vector pAD/CMV/Dest according to the manufacturer^'s protocol (Invitrogen). HEK293A cells were then transfected with the Pl 16 vector and cultured until they rounded off as a sign of high adenovirus load. After three rounds of virus amplification with increasing amounts of HEK293A cells the virus titer was determined with a plaque assay in agarose covered HEK293A cells.

Isolation of primary cardiac cells.

Neonatal rat cardiomyocytes were isolated as described ^{16 ir}.

lmmunofluorescent staining.

Cells or cryosections were fixed for 10 min using 4% paraformaldehyde. After 5 min permeabilization with 0.2% Triton X100, samples were incubated with primary antibodies (Anti-PI16 diluted 1 :1000, Anti-α-actinin diluted 1 :500 (Sigma-Aldrich, St. Louis, MO) and Hoechst for 30 min at 37⁰C. Incubation with secondary antibodies (Anti-Rabbit Alexa 555, Anti-Mouse Alexa 488, Invitrogen) was then for 30 min at 37⁰C. For determination of cell size cardiomyocytes were digitized by microscopy and cross-sectional areas were determined in a blinded way by computerized pixel counting >50 cells per condition.

Assessment of hypertrophy by isoleucine incorporation.

Neonatal rat cardiomyocytes were cultured in 24-well plates and transfected with a lacZ control or a PI16-expressing adenovirus. After 24h starvation without serum, cells were stimulated for 4Oh with isoproterenol/phenylephrine (5μM/50μM). 2Oh before harvesting [³H]isoleucine was added at a final concentration of 1 nCi/ml. After precipitation with 10% trichloroacetic acid and dissolving with 0.5 N NaOH, the incorporated radioactivity was counted in a scintillation counter.

Suppression of endogenous PM 6 by RNAi

Neonatal rat cardiomyocytes (NRCM) were transfected 24 h after isolation with RNAi using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were transfected using a nonhomologous scrambled RNAi (RNAi negative control with a medium GC content, Invitrogen) or a RNAi directed against Pl 16 (5'- AUG GAU GUU AGC UUC CUC CAC UCC C-3', final concentration 40 nmol, Invitrogen) or only exposed to the transfection reagent (control). After starving without serum for 24 h, [³H]isoleucine and isoproterenol/phenylephrine (2,5 μM/25 μM) or control buffer were added for isoleucine incorporation assays and cells were cultivated for another 40 h.

Generation of transgenic mice.

PI16-transgenic mice were generated by pronuclear injection of fertilized oocytes from FVB/N mice with a transgene construct containing the coding sequence of the mouse Pl 16 full-length under the control of the murine α-myosin heavy chain promotor. All mice were housed in a SPF facility. All animal experiments were approved by the responsible authorities.

Histological analyses.

Tissue sections (4 μm) of left ventricular myocardium were stained with hematoxylin/eosin for determination of cardiomyocyte cross-sectional areas. By digitizing the images and computerized pixel counting >200 individual cells per genotype and group were analyzed. Only nucleated cardiac myocytes from areas of transversely cut muscle fibres were included in the analyses by an investigator blinded to the genotype. Between 6 and 9 animals per group were analyzed.

In vivo hemodynamic analysis.

Left ventricular catheterization was performed as described previously ¹⁸. Briefly, a miniaturized pressure sensing catheter (1.4F Micro-tip catheter, Millar instruments, Houston, TX) was introduced via the right carotid artery under anesthesia with tribromoethanol. Increasing doses of dobutamine were infused via the left jugular vein. Chart software (Chart v5.0.2, ADInstruments, Spechbach, Germany) was employed for data recording (2000 Hz) and analysis of parameters of cardiac function.

Statistical analysis.

For comparison, unpaired t tests were performed using the Prism software package (GraphPad, San Diego, CA). Data are presented as mean ± s.e.m.. Differences were considered significant when p<0.05.

The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

Example 2: Supplementary methods

Bioinformatic search for homologues.

A BLAST search against the sequenced genomes of several species was performed with the protein sequence for mouse Pl 16. The identified proteins were only annotated as putative homologues if the mouse Pl 16 was found in a back search. Of the identified proteins, many were just seeming homologues that only contained the large SCP domain, as for example the Crisp (Cysteine rich protein)-family. To exclude such false-positive homologues, an evolutionary tree using CLUSTAL W³² was calculated with the protein sequences of more than 30 of the most similar proteins of several species by means of that real Pl 16 homologues were clearly separated. CLUSTAL W was then used for multiple alignment. The grade of homology was calculated taking the different sizes of the proteins into account, e.g. the short fish protein (Danio rerio) was aligned only with the corresponding part of mouse Pl 16.

RNA isolation and real-time quantitative PCR.

The quality of the isolated RNA was analyzed by denaturing agarose gel electrophoresis. First-strand cDNA was synthesized by using Superscript Il reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo-dT primers. PCR was performed with Sybr Green (Cambrex BioScience, East Rutherford, NJ) as fluorescent and 6- carboxy-X-rhodamine (Rox, Invitrogen) as reference dye using an ABI PRISM Sequence Detection System 7700 (Applied Biosystems, Foster City, Ca). Threshold cycle (Ct) values were determined using the Sequence Detector 1.7 software. Data were calculated with the 2-ΔΔCT method. After each experiment dissociation curves were analyzed to control for specificity of the amplification product. GAPDH was used as a reference. The primer sequences used for real-time PCR analysis are listed in table 1. PCR conditions using heat-activatable Taq polymerase (Hot Master Taq; Eppendorf, Hamburg, Germany) were as follows: 40 cycles of 94⁰C for 20 s (2 min initial cycle), 56⁰C for 20 s and 65⁰C for 35 s. Human Pl 16 was detected by TaqMan gene expression assay Hs00542137_m1 according to the manufacturer^'s instructions (Applied Biosystems).

Generation of a PI16-specific antibody and Western blot analysis.

The coding sequence of full-length Pl 16 lacking the first 23 amino acids (i.e. the signal sequence) was amplified by PCR from a mouse cardiac cDNA library and inserted into the pDEST15 expression vector (Invitrogen). The recombinant GST- tagged protein was insoluble and was purified from inclusion bodies in BL21 cells. Exon 5 of Pl 16 was amplified by PCR and inserted into the pDEST15 expression vector. The soluble exon 5 fragment was then purified by column chromatography from bacterial cell lysates. The purified full-length protein, the exon 5 peptide and a synthetized peptide corresponding to amino acid positions 84-94 of Pl 16 were then injected into rabbits (Immunoglobe, Himmelstadt, Germany) to generate polyclonal antibodies. The purified protein was then injected into rabbits (Immunoglobe, Himmelstadt, Germany) to generate polyclonal antibodies. The purified antigen coupled to HiTrap NHS-activated columns (GE Healthcare, Chalfont St Gilles, UK) was used for affinity purification of the antisera according to the manufacturer^'s instructions.

For Western blotting tissue or cell lysates containing 30 μg of protein were separated by electrophoresis on 10% SDS-PAGE gels and transferred to Immobilion-P membranes (Millipore Corporation, Billerica, MA). After blocking the membranes with 5% milk for 2 h, incubation with primary antibody overnight at 4⁰C (diluted 1 :5,000), followed by secondary anti-rabbit antibody for 1 h at room temperature (diluted 1 :10,000; Dianova, Hamburg, Germany) and chemiluminescent detection (ECL-Plus, GE Healthcare) were performed.

The present invention refers to the following tables:

Table 1: Primer sequences and product lengths of the mRNAs assessed by real-time PCR

Cxcl14, chemokine (C-X-C motif) ligand 14; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; Nppa, natriuretic peptide precursor type A; PI16, protease inhibitor 16; Psap, prosaposin;

Gene Primer sequences Product size

(bp)

CD14 forward 5--CATGGAGCGTGTGCTTGG-3^' U3 reverse 5^' -CGGATCTGAGAAGTTGC AGG A-3^"

CD 164 forward 5^" -GCAGCCCAACATCACCAC-3^" 119 reverse 5^' -GTTGAAGCTCGCACAGGTTT-S-

Cxcl14 forward 5^"-CACACTGCGAGGAGAAGATG-3^" 126 reverse 5--CCAGGCATTGTACCACTTGA-3¹

GAPDH human forward 5--GATCATCAGCAATGCCTCCT-3^' 140 reverse 5^"-GGGCCATCCACAGTCTTCT-3^"

GAPDH mouse forward 5^"-TGGCAAAGTGGAGATTGTTG-S^' 119 reverse 5--CATTATCGGCCTTGACTGTG-3^"

Nppa forward 5^"-TTTCAAGAACCTGCTAGACCAC-3^■ 98 reverse 5^' -CCCTGCTTCCTCAGTCTGCT-S^"

PI16 forward S^'-CCAGTGCCCTCTTGGCTAC-S^' 97 reverse 5^'-ACCTCGGTCACCCTTGGA-3^"

Psap forward 5- -GTGGACCAGTATTCCGAGGT-S- 120 reverse 5^' -GGGACCAG AGTCTTCATTGG-3^' Table 2: Genes identified by the secretion trap screen

54 genes containing a secretion signal were identified by a secretion trap screen using a cardiac cDNA library. Genes are depicted in descending order according to their probability of secretion. 30 of the identified proteins are putatively secreted and 9 membrane bound. 15 proteins have secretion signals but also one membrane domain and might be secreted only after shedding from the plasma membrane. Gene name, function and murine GenelD have been assigned according to Entrez Gene (NCBI). IEA means that the function is inferred only from electronic annotation. Prediction of signal sequence (SS) or signal anchor (SA) by the neural networks method and the hidden markov model of SignalP or if indicated only by the hidden markov model (HMM). The number of transmembrane helices was predicted by TMHMM Server 2.0. Prediction of non-classical secretion was carried out by SecretomP 1. "W" = warning e.g. prediction of signal sequence for classical secretion, a neural network (NN) score >0.6 suggests secretion including the odds that the sequence in fact is secreted.

function/process GenelD SignaleP TMHMM Secretome

(TMD) (score, odds) proteins with secretion signal, without transmembrane domain cystatin C protease inhibitor 13010 SS 0 W 0 95,7 1 apolipoprotein E lipid transport 11816 SS 0 W 0 92,6 5 nephroblastoma overexpressed gene growth factor activity 18133 SS 0 W 0 88,5 1 compl component 1, q subcomp , gamma complement activation 12262 SS 0 W 0 86,5 0 polypeptide follistatin-like 3 activin-inhibitor 83554 SS 0 W 0 86,4 7 lipoprotein lipase lipid metabolism 16956 SS 0 W 0 81,3 4 nephrin 1 role in glomerular permeability 170643 SS 0 W 0 77,2 7 protease inhibitor 16 peptidase activity (IAE) 74116 SS 0 W 0 76,2 6 prosaposin sphingolipid metabolism 19156 SS 0 W 0 73,2 3 glycine cleavage system protein H glycine catabolism (IEA) 68133 SS 0 + 0 72,

2 5 leucine-rich alpha-2-glycoproteιn granulocytic differentiation 76905 SS 0 W 071,2 3

CD14 antigen immune response 12475 SS 0 W 0 71,2 1 mannosidase, beta A, lysosomal glycoprotein catabolism 110173 SS 0 W 070,2 1 melanocyte proliferating gene 1 unknown 60315 SS 0 W 066,1 9

Ts translation elongation factor, mitochondrial translation elongation activity (IEA) 66399 SS 0 + 065,1 8 chemokine (C-X-C motif) ligand 14 chemokine, immune response 57266 SS 0 W 0 59,1 6 cytochrome c oxidase, subunit Vl a, polypeptide electron transport (IEA) 12862 SS (HMM) 0 W 0 81,3 8 2

RIKEN cDNA 2700059D21 gene unknown 433693 SS (HMM) 0 + 0 76,2 7 cytochrome c oxidase, subunit Vb electron transport (IEA) 12859 SS (HMM) 0 + 0 73,3 6

ATP synthase, F1 complex, alpha subunit, hydrogen ion transporter (IEA) 11946 SS (HMM) 0 - 0 56,1 3 isoform 1 ADP-πbosyltransferase 1 transferase activity 11870 SS 0 - 0 55,1 2 prohibitin DNA replication 18673 SS (HMM) 0 - 045,0 9 transforming growth factor beta 1 induced transcπption factor activity (IEA) 21807 SS (HMM) 0 - 0 29,0 6 transcnpt 4 protocadheπn alpha 6 unknown 12937 SS 0 0 19,

04 cysteine nch protein 2 cell proliferation, hemopoiesis 68337 0 + 0 81,3 5

TGF-beta1 -induced anti-apoptotic factor 1 apoptosis (IEA) 21842 0 + 0 78,3 2

RIKEN cDNA 0610038D11 gene unknown 67674 - 0 + 0 78,3 1 angiomotin-like 1 unknown (at tight-junction) 75723 - 0 + 0 68,2 0 properdin factor, complement alternative complement activation 18636 0 + 0 64,1 6

(IEA) ribosomal protein L36 protein biosynthesis (IEA) 54217 0 61,1 7 proteins with secretion signal and one transmembrane domain

UDP-N-acetyl-alpha-D-galactosamine transferase activity (IEA) 108760 SS (HMM) 1 + 0 86,4 5 natriuretic peptide precursor type A hormone 24602 SS 1 W 0 76,3 2 similar to Atrial natriuretic factor precursor hormone 230899 SS 1 W 0 76,3 3 UDP-GaI betaGlcNAc beta 1 ,4- carbohydrate metabolism (IEA) 56375 SS 1 W 0 66,2 0 galactosyltransferase, polypeptide 4 immediate early response 3 unknown 15937 SS + 0 65,

1 7

Neuropilin angiogenesis, heart development 18186 SS W 0 60,

1 4

RIKEN cDNA 9530068E07 gene unknown 213673 SS W 0 60,1 5 CD164 antigen cell adhesion 53599 SS - 0 47,1 2 TAP binding protein defense response 21356 SS - 040,

0 8 colony stimulating factor 2 receptor, alpha receptor activity 12982 SS - 0 36,

0 7

RIKEN cDNA 1500004A08 gene unknown (Kπngle domain) 216505 SS - 0 34,0 6 Phosphatidyhnositol Glycan, class T GPI anchor biosynthesis (IEA) 78928 SS - 0 32,0 7 MANSC domain containing 1 unknown 67729 SS - 0 27,0 5 RIKEN cDNA 9130213B05 gene unknown 231440 SS - 0 31,0 6 interieukin 6 signal transducer receptor of the notch signalling 16195 SS - 0 14, pathway 0 3 proteins with secretion signal and mutiple transmembrane domains

RIKEN cDNA 1110014C03 gene protein transport (IEA), golgi 68581 SS 2 W 0 88,

5 3

NADH dehydrogenase 3, mitochondπal oxidoreductase activity (IEA) 17718 SS 3 W 0 82,3 9 dolichyl-di-phosphooligosaccharide-protein glycotransferase activity 13200 SS 2 W 064,1 8 glycotransferase tumor differentially expressed 2 unknown 56442 SS 11 W 0 61,1 7

RIKEN CDNA 5630401J11 gene unknown 106489 SA 3 W 0 72,3 0 transmembrane protein 41 B hydrolase activity (IEA) 233724 SA 6 - 0 53,1 9

RIKEN cDNA 0610009E20 gene unknown 66048 SA 2 - 0 37,1 0

TM2 domain containing 1 GPCR (apoptosis induction) 94043 SS (HMM) 2 - 0 32,0 6 adiponectin receptor 1 lipid metabolism 72674 . 7 + 0 62,1 9

The present invention refers to the following nucleotide and amino acid sequences: SEQ ID No. 1:

Nucleotide sequence (mRNA) encoding mouse full-length Pl 16

ATGTTGCCGCCGCCACTGCTCCTCCTGCTGCTGCTGATTGCCACTGGCCCCACCACAGCCCTCA

CAGAGGACGAGAAGCAAACTATGGTGGATCTTCACAACCAGTACCGTGCCCAGGTGTCCCCGCC

AGCCTCAGATATGCTGCAGATGAGGTGGGATGACGAGCTGGCCGCCTTCGCCAAGGCCTACGCT

CAGAAGTGCGTGTGGGGCCACAACAAAGAACGCGGGCGGCGCGGAGAGAACTTGTTTGCCATC

ACGGACGAGGGCATGGACGTGCCGCTGGCCGTGGGGAACTGGCACGAGGAGCATGAGTATTAC

AATTTCAGCACGGCCACCTGCGATCCGAACCAGATGTGCGGCCACTACACTCAGGTAGTGTGGA

GCAAGACTGAGAGAATTGGCTGTGGCTCCCACTTCTGCGAGACGCTCCAGGGAGTGGAAGAAGC

TAACATCCATTTGCTGGTGTGCAACTATGAGCCTCCGGGGAACGTGAAGGGCCGTAAGCCCTAC

CAGGAGGGGACTCCTTGCTCCCAGTGCCCTCTTGGCTACAGCTGTGAGAACTCTCTCTGTGAGC

CCATGAGAAACCCGGAAAAGGCGCAGGATTCGCCTCCAAGGGTGACCGAGGTCCCTTCCACCC

GCGCAACTGAAGCCCCAAGCTCCAGGGAAACCGGTACTCCATCCCTAGCAACCTCTGAGACTCT

ACATTTCTCGGTAACAAAGGTCTCGGACTCCCTGGCAACCGAGTCCTCACCTGCAGTAGAAACAA

AGGCACCATCTTCCTTAGCAACCGAAGGCCCCTCCTCCATGGCAACAGAGGCTCAGGCTTTTGTA

ACTGAGGTCCCCTTGGTTTCTGCAAGGCACATGCAGCCCTCGGTGGATGAAGGGCCAGTTAACT

TCCTCACATCAACACATATCCCTGTCCCCAAATCTATGGACGAAGAAGCCAGCAAGTCGAGCGCA

ACCTCCGTGAGCCCAAAGAAATCGCTGTACCCCAAGATGTCCCTGACAGAGTCAGGAGAGTCCG

TACCCCAAATCCAGGAGGAGGCTGAACCCAAGGACGAGTTGTCTGAGCCCGAGGCCATATTGCC

CGAGGCAGAGGCCGCACCGACTGAGGCAGAGGTCGAGTTGCGGGAGCCCGAGGCTGAGTCTC

CCAAGGCCGAGTCGCCAGAGGCAGAGGCTGAGTCGCCTCTTTCCAGTGAGGCTTTGGTCCCAGT

TCTTCCAGCCCAGGAGCGTGGTGGGCAGAAGGCCTCACTGGAACACTCTGGCCACCCTGCCTC

CCCATCCCTGCCCACCTTCCCTAGTGCTTCGGGTAATGCCACAGGTGGGCGCACCCTGGCCCTA

CAATCATCCTGGACAGGTGCTGAGAACCCCGAAAAGGCCGACTGGGATTTGAAGAATTCTGCTC

ACGTGTGGGGACCTTTCCTGGGACTGCTGCTGCCTTCCCTGCTGCTGTTGGCTGGCATGGTCTG

A

SEQ ID No. 2:

Amino acid sequence of mouse full-length Pl 16

MLPPPLLLLLLLIATGPTTALTEDEKQTMVDLHNQYRAQVSPPASDMLQMRWDDELAAFAKAYAQKC

VWGHNKERGRRGENLFAITDEGMDVPLAVGNWHEEHEYYNFSTATCDPNQMCGHYTQWWSKTE

RIGCGSHFCETLQGVEEANIHLLVCNYEPPGNVKGRKPYQEGTPCSQCPLGYSCENSLCEPMRNPE

KAQDSPPRVTEVPSTRATEAPSSRETGTPSLATSETLHFSVTKVSDSLATESSPAVETKAPSSLATEG

PSSMATEAQAFVTEVPLVSARHMQPSVDEGPVNFLTSTHIPVPKSMDEEASKSSATSVSPKKSLYPK

MSLTESGESVPQIQEEAEPKDELSEPEAILPEAEAAPTEAEVELREPEAESPKAESPEAEAESPLSSEA

LVPVLPAQERGGQKASLEHSGHPASPSLPTFPSASGNATGGRTLALQSSWTGAENPEKADWDLKNS

AHVWGPFLGLLLPSLLLLAGMV SEQ ID No. 3:

Nucleotide sequence (mRNA) encoding human full-length Pl 16

ATGCACGGCTCCTGCAGTTTCCTGATGCTTCTGCTGCCGCTACTGCTACTGCTGGTGGCCACCA

CAGGCCCCGTTGGAGCCCTCACAGATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTA

CCGGGCCCAGGTATCCCCGACGGCCTCAGACATGCTGCACATGAGATGGGACGAGGAGCTGGC

CGCCTTCGCCAAGGCCTACGCACGGCAGTGCGTGTGGGGCCACAACAAGGAGCGCGGGCGCC

GCGGCGAGAATCTGTTCGCCATCACAGACGAGGGCATGGACGTGCCGCTGGCCATGGAGGAGT

GGCACCACGAGCGTGAGCACTACAACCTCAGCGCCGCCACCTGCAGCCCAGGCCAGATGTGCG

GCCACTACACGCAGGTGGTATGGGCCAAGACAGAGAGGATCGGCTGTGGTTCCCACTTCTGTGA

GAAGCTCCAGGGTGTTGAGGAGACCAACATCGAATTACTGGTGTGCAACTATGAGCCTCCGGGG

AACGTGAAGGGGAAACGGCCCTACCAGGAGGGGACTCCGTGCTCCCAATGTCCCTCTGGCTAC

CACTGCAAGAACTCCCTCTGTGAACCCATCGGAAGCCCGGAAGATGCTCAGGATTTGCCTTACCT

GGTAACTGAGGCCCCATCCTTCCGGGCGACTGAAGCATCAGACTCTAGGAAAATGGGTACTCCT

TCTTCCCTAGCAACGGGGATTCCGGCTTTCTTGGTAACAGAGGTCTCAGGCTCCCTGGCAACCAA

GGCTCTGCCTGCTGTGGAAACCCAGGCCCCAACTTCCTTAGCAACGAAAGACCCGCCCTCCATG

GCAACAGAGGCTCCACCTTGCGTAACAACTGAGGTCCCTTCCATTTTGGCAGCTCACAGCCTGC

CCTCCTTGGATGAGGAGCCAGTTACCTTCCCCAAATCGACCCATGTTCCTATCCCAAAATCAGCA

GACAAAGTGACAGACAAAACAAAAGTGCCCTCTAGGAGCCCAGAGAACTCTCTGGACCCCAAGA

TGTCCCTGACAGGGGCAAGGGAACTCCTACCCCATGCCCAGGAGGAGGCTGAGGCTGAGGCTG

AGTTGCCTCCTTCCAGTGAGGTCTTGGCCTCAGTTTTTCCAGCCCAGGACAAGCCAGGTGAGCT

GCAGGCCACACTGGACCACACGGGGCACACCTCCTCCAAGTCCCTGCCCAATTTCCCCAATACC

TCTGCCACCGCTAATGCCACGGGTGGGCGTGCCCTGGCTCTGCAGTCGTCCTTGCCAGGTGCA

GAGGGCCCTGACAAGCCTAGCGTCGTGTCAGGGCTGAACTCGGGCCCTGGTCATGTGTGGGGC

CCTCTCCTGGGACTACTGCTCCTGCCTCCTCTGGTGTTGGCTGGAATCTTCTGA

SEQ ID No. 4:

Amino acid sequence of human full-length Pl 16

MHGSCSFLMLLLPLLLLLVATTGPVGALTDEEKRLMVELHNLYRAQVSPTASDMLHMRWDEELAAFA

KAYARQCVWGHNKERGRRGENLFAITDEGMDVPLAMEEWHHEREHYNLSAATCSPGQMCGHYTQ

WWAKTERIGCGSHFCEKLQGVEETNIELLVCNYEPPGNVKGKRPYQEGTPCSQCPSGYHCKNSLC

EPIGSPEDAQDLPYLVTEAPSFRATEASDSRKMGTPSSLATGIPAFLVTEVSGSLATKALPAVETQAPT

SLATKDPPSMATEAPPCVTTEVPSILAAHSLPSLDEEPVTFPKSTHVPIPKSADKVTDKTKVPSRSPEN

SLDPKMSLTGARELLPHAQEEAEAEAELPPSSEVLASVFPAQDKPGELQATLDHTGHTSSKSLPNFPN

TSATANATGGRALALQSSLPGAEGPDKPSWSGLNSGPGHVWGPLLGLLLLPPLVLAGIF

SEQ ID No. 5:

Nucleotide sequence (mRNA) encoding mouse Pl 16 splice variant (w/o exon 5) ATGTTGCCGCCGCCACTGCTCCTCCTGCTGCTGCTGATTGCCACTGGCCCCACCACAGCCCTCA

CAGAGGACGAGAAGCAAACTATGGTGGATCTTCACAACCAGTACCGTGCCCAGGTGTCCCCGCC

AGCCTCAGATATGCTGCAGATGAGGTGGGATGACGAGCTGGCCGCCTTCGCCAAGGCCTACGCT

CAGAAGTGCGTGTGGGGCCACAACAAAGAACGCGGGCGGCGCGGAGAGAACTTGTTTGCCATC

ACGGACGAGGGCATGGACGTGCCGCTGGCCGTGGGGAACTGGCACGAGGAGCATGAGTATTAC

AATTTCAGCACGGCCACCTGCGATCCGAACCAGATGTGCGGCCACTACACTCAGGTAGTGTGGA

GCAAGACTGAGAGAATTGGCTGTGGCTCCCACTTCTGCGAGACGCTCCAGGGAGTGGAAGAAGC

TAACATCCATTTGCTGGTGTGCAACTATGAGCCTCCGGGGAACGTGAAGGGCCGTAAGCCCTAC

CAGGAGGGGACTCCTTGCTCCCAGTGCCCTCTTGGCTACAGCTGTGAGAACTCTCTCTGTGGTG

CTGAGAACCCCGAAAAGGCCGACTGGGATTTGAAGAATTCTGCTCACGTGTGGGGACCTTTCCT

GGGACTGCTGCTGCCTTCCCTGCTGCTGTTGGCTGGCATGGTCTGA

SEQ ID No. 6:

Amino acid sequenze of mouse Pl 16 splice variant (w/o exon 5)

MLPPPLLLLLLLIATGPTTALTEDEKQTMVDLHNQYRAQVSPPASDMLQMRWDDELAAFAKAYAQKC VWGHNKERGRRGENLFAITDEGMDVPLAVGNWHEEHEYYNFSTATCDPNQMCGHYTQWWSKTE RIGCGSHFCETLQGVEEANIHLLVCNYEPPGNVKGRKPYQEGTPCSQCPLGYSCENSLCGAENPEKA DWDLKNSAHVWGPFLGLLLPSLLLLAGMV

SEQ ID No. 7:

Nucleotide sequence (mRNA) encoding human Pl 16 splice variant (w/o exon 5)

ATGCACGGCTCCTGCAGTTTCCTGATGCTTCTGCTGCCGCTACTGCTACTGCTGGTGGCCACCA

CAGGCCCCGTTGGAGCCCTCACAGATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTA

CCGGGCCCAGGTATCCCCGACGGCCTCAGACATGCTGCACATGAGATGGGACGAGGAGCTGGC

CGCCTTCGCCAAGGCCTACGCACGGCAGTGCGTGTGGGGCCACAACAAGGAGCGCGGGCGCC

GCGGCGAGAATCTGTTCGCCATCACAGACGAGGGCATGGACGTGCCGCTGGCCATGGAGGAGT

GGCACCACGAGCGTGAGCACTACAACCTCAGCGCCGCCACCTGCAGCCCAGGCCAGATGTGCG

GCCACTACACGCAGGTGGTATGGGCCAAGACAGAGAGGATCGGCTGTGGTTCCCACTTCTGTGA

GAAGCTCCAGGGTGTTGAGGAGACCAACATCGAATTACTGGTGTGCAACTATGAGCCTCCGGGG

AACGTGAAGGGGAAACGGCCCTACCAGGAGGGGACTCCGTGCTCCCAATGTCCCTCTGGCTAC

CACTGCAAGAACTCCCTCTGTGGTGCAGAGGGCCCTGACAAGCCTAGCGTCGTGTCAGGGCTGA

ACTCGGGCCCTGGTCATGTGTGGGGCCCTCTCCTGGGACTACTGCTCCTGCCTCCTCTGGTGTT

GGCTGGAATCTTCTGA

SEQ ID No. 8:

Amino acid sequence of human Pl 16 splice variant (w/o exon 5)

MHGSCSFLMLLLPLLLLLVATTGPVGALTDEEKRLMVELHNLYRAQVSPTASDMLHMRWDEELAAFA KAYARQCVWGHNKERGRRGENLFAITDEGMDVPLAMEEWHHEREHYNLSAATCSPGQMCGHYTQ WWAKTERIGCGSHFCEKLQGVEETNIELLVCNYEPPGNVKGKRPYQEGTPCSQCPSGYHCKNSLC GAEGPDKPSWSGLNSGPGHVWGPLLGLLLLPPLVLAGIF

Additional references cited:

1. Towbin JA, Bowles NE. The failing heart. Nature. 2002;415:227-233.

2. Fredj S, Bescond J, Louault C, Potreau D. Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation. J Cell Physiol. 2005;202:891-899.

3. Gnecchi M, He H, Liang OD, MeIo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med. 2005;11 :367-368.

4. Uemura R, Xu M, Ahmad N, Ashraf M. Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling. Circ Res. 2006;98:1414-1421.

5. Bouzegrhane F, Thibault G. Is angiotensin Il a proliferative factor of cardiac fibroblasts? Cardiovasc Res. 2002;53:304-312.

6. Manabe I, Shindo T, Nagai R. Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy. Circ Res. 2002;91 :1103-1113.

7. Clark HF, Gurney AL, Abaya E, Baker K, Baldwin D, Brush J, Chen J, Chow B, Chui C, Crowley C, Currell B, Deuel B, Dowd P, Eaton D, Foster J, Grimaldi C, Gu Q, Hass PE, Heldens S, Huang A, Kim HS, Klimowski L, Jin Y, Johnson S, Lee J, Lewis L, Liao D, Mark M, Robbie E, Sanchez C, Schoenfeld J, Seshagiri S, Simmons L, Singh J, Smith V, Stinson J, Vagts A, Vandlen R, Watanabe C, Wieand D, Woods K, Xie MH, Yansura D, Yi S, Yu G, Yuan J, Zhang M, Zhang Z, Goddard A, Wood Wl, Godowski P, Gray A. The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment. Genome Res. 2003; 13:2265-2270.

8. Grimmond SM, Miranda KC, Yuan Z, Davis MJ, Hume DA, Yagi K, Tominaga N, Bono H, Hayashizaki Y, Okazaki Y, Teasdale RD. The mouse secretome: functional classification of the proteins secreted into the extracellular environment. Genome Res. 2003; 13: 1350-1359.

9. Brent MR. Genome annotation past, present, and future: how to define an ORF at each locus. Genome Res. 2005;15:1777-1786. 10. Klein RD, Gu Q, Goddard A₁ Rosenthal A. Selection for genes encoding secreted proteins and receptors. Proc Natl Acad Sci U S A. 1996;93:7108-7113.

11. Kramer A, Yang FC, Kraves S, Weitz CJ. A screen for secreted factors of the suprachiasmatic nucleus. Methods Enzymol. 2005;393:645-663.

12. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J MoI Biol. 2004;340:783-795.

13. Bendtsen JD, Jensen LJ, Blom N, Von Heijne G, Brunak S. Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des SeI. 2004;17:349-356.

14. Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J MoI Biol. 2001 ;305:567-580.

15. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 ;25:402- 408.

16. Buitrago M, Lorenz K, Maass AH, Oberdorf-Maass S, Keller U, Schmitteckert EM, Ivashchenko Y, Lohse MJ, Engelhardt S. The transcriptional repressor Nab1 is a specific regulator of pathological cardiac hypertrophy. Nat Med. 2005; 11 :837-844.

17. Rochais F, Vilardaga JP, Nikolaev VO, Bunemann M, Lohse MJ, Engelhardt S. Real-time optical recording of beta(1)-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol. J Clin Invest. 2007; 117:229-235.

18. Merkle S, Franz S, Schoen MP, Bauersachs J, Buitrago M, Frost RJA, Schmitteckert E, Lohse MJ, Engelhardt S. A Role for Caspase-1 in Heart Failure. Circ Res in press. 2007.

19. Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy and heart failure in betai -adrenergic receptor transgenic mice. Proc Natl Acad Sci U S A. 1999;96:7059-7064.

20. Hubbard T, Andrews D, Caccamo M, Cameron G, Chen Y, Clamp M, Clarke L, Coates G, Cox T, Cunningham F, Curwen V, Cutts T, Down T, Durbin R, Fernandez- Suarez XM, Gilbert J, Hammond M, Herrero J, Hotz H, Howe K, Iyer V, Jekosch K, Kahari A, Kasprzyk A, Keefe D, Keenan S, Kokocinsci F, London D, Longden I, McVicker G, Melsopp C, Meidl P, Potter S, Proctor G, Rae M, Rios D, Schuster M, Searle S, Severin J, Slater G, Smedley D, Smith J, Spooner W, Stabenau A, Stalker J, Storey R, Trevanion S, Ureta-Vidal A, Vogel J, White S, Woodwark C, Birney E. Ensembl 2005. Nucleic Acids Res. 2005;33:D447-453.

21. Schultz J, Milpetz F, Bork P₁ Ponting CP. SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A. 1998;95:5857-5864.

22. Tschumperlin DJ, Dai G, MaIy IV, Kikuchi T, Laiho LH, McVittie AK, Haley KJ, Lilly CM, So PT, Lauffenburger DA, Kamm RD, Drazen JM. Mechanotransduction through growth-factor shedding into the extracellular space. Nature. 2004;429:83-86.

23. Mulder NJ, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bradley P, Bork P, Bucher P, Cerutti L, Copley R, Courcelle E, Das U, Durbin R, Fleischmann W, Gough J, Haft D, Harte N, HuIo N, Kahn D, Kanapin A, Krestyaninova M, Lonsdale D, Lopez R, Letunic^' I, Madera M, Maslen J, McDowall J, Mitchell A, Nikolskaya AN, Orchard S, Pagni M, Ponting CP, Quevillon E, Selengut J, Sigrist CJ, Silventoinen V, Studholme DJ, Vaughan R, Wu CH. InterPro, progress and status in 2005. Nucleic Acids Res. 2005;33:D201-205.

24. Reeves JR, Xuan JW, Arfanis K, Morin C, Garde SV, Ruiz MT, Wisniewski J, Panchal C, Tanner JE. Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids. Biochem J. 2005;385:105-114.

25. Garde SV, Basrur VS, Li L, Finkelman MA, Krishan A, Wellham L, Ben-Josef E, Haddad M, Taylor JD, Porter AT, Tang DG. Prostate secretory protein (PSP94) suppresses the growth of androgen-independent prostate cancer cell line (PC3) and xenografts by inducing apoptosis. Prostate. 1999;38:118-125.

26. Eppig JT, BuIt CJ, Kadin JA, Richardson JE, Blake JA, Anagnostopoulos A, Baldarelli RM, Baya M, Beal JS, BeIIo SM, Boddy WJ, Bradt DW, Burkart DL, Butler NE, Campbell J, Cassell MA, Corbani LE, Cousins SL, Dahmen DJ, Dene H, Diehl AD, Drabkin HJ, Frazer KS, Frost P, Glass LH, Goldsmith CW, Grant PL, Lennon- Pierce M, Lewis J, Lu I, Maltais LJ, McAndrews-Hill M, McClellan L, Miers DB, Miller LA, Ni L, Ormsby JE, Qi D, Reddy TB, Reed DJ, Richards-Smith B, Shaw DR, Sinclair R, Smith CL, Szauter P, Walker MB, Walton DO, Washburn LL₁ Witham IT, Zhu Y. The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology. Nucleic Acids Res. 2005;33:D471-475. 27. Jensen LJ, Gupta R, Blom N₁ Devos D, Tamames J, Kesmir C, Nielsen H, Staerfeldt HH, Rapacki K, Workman C, Andersen CA, Knudsen S, Krogh A, Valencia A, Brunak S. Prediction of human protein function from post-translational modifications and localization features. J MoI Biol. 2002;319: 1257-1265.

28. Spinale FG. Matrix metalloproteinases: regulation and dysregulation in the failing heart. Circ Res. 2002;90:520-530.

29. Knowles JW, Esposito G, Mao L, Hagaman JR, Fox JE, Smithies O, Rockman HA, Maeda N. Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice. J Clin Invest. 2001 ; 107:975-984.

30. Holtwick R, van Eickels M, Skryabin BV, Baba HA, Bubikat A, Begrow F, Schneider MD, Garbers DL, Kuhn M. Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A. J Clin Invest. 2003; 111 :1399-1407.

31. Xu J, Kimball TR, Lorenz JN, Brown DA, Bauskin AR, Klevitsky R, Hewett TE, Breit SN, Molkentin JD. GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation. Circ Res. 2006;98:342-350.

32. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673- 4680.

Claims

1. Method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need a polypeptide selected from the group consisting of:

(c) a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440- 459 of SEQ ID NO: 4, or any one of corresponding fragments (by comparison of homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having Pl 16 function; and

2. Method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need one or more polypeptide(s), the expression of which is indicative for hypertrophy or a heart disease, or (a) variant(s) of said polypeptide(s).

3. Method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need a nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NO: 1 , 3, 5 and 7; (b) a nucleic acid encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 2, 4, 6 and 8;

(d) a nucleic acid encoding a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348- 368, 369-439 and 440^459 of SEQ ID NO: 4, or any one of corresponding fragments (by comparison of homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having PM 6 function;

(T) a nucleic acid encoding a polypeptide as defined in claim 1 , and, optionally, at least one further nucleic acid molecule encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or a variant of said further nucleic acid molecule.

4. Method for the treatment or prevention of hypertrophy or a heart disease comprising the step of administering to a subject in need one ore more nucleic acid molecule(s) encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or (a) variant(s) of said nucleic acid molecule(s).

5. Use of a polypeptide selected from the group consisting of:

(c) a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440- 459 of SEQ ID NO: 4, or any one of corresponding fragments (by comparison of homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having Pl 16 function; and (d) a polypeptide comprising an amino acid sequence at least 40%, 50%,

60%, 70%, 80%, 90% or 95% identical to the polypeptide of (a), (b) or

(c), optionally having Pl 16 function, and, optionally, at least one further polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or a variant of said further polypeptide, for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

6. Use of one or more polypeptide(s), the expression of which is indicative for hypertrophy or a heart disease, or (a) variant(s) of said polypeptide(s), for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

7. Use of a nucleic acid molecule selected from the group consisting of:

(d) a nucleic acid encoding a polypeptide comprising any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348- 368, 369-439 and 440-459 of SEQ ID NO: 4, or any one of corresponding fragments (by comparison of homology) of any one of SEQ ID NO: 2, 6 and 8, optionally said fragments having Pl 16 function;

(f) a nucleic acid encoding a polypeptide as defined in claim 5, and, optionally, at least one further nucleic acid molecule encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or a variant of said further nucleic acid molecule, for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

8. Use of one ore more nucleic acid molecule(s) encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, or (a) variant(s) of said nucleic acid molecule(s), for the preparation of a pharmaceutical composition for the treatment or prevention of hypertrophy or a heart disease.

9. The method or the use of any one of claims 3, 4, 7 and 8, wherein said nucleic acid is comprised by a vector.

10. The method or use of claim 9, wherein said vector is a(n) (adeno)viral vector.

11. Use of a polypeptide as defined in any one of claims 1 , 2, 5 and 6 or a nucleic acid molecule as defined in any one of claims 3 and 4 and 7 to 10, for the preparation of a diagnostic composition for detecting hypertrophy or a heart disease.

12. A method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the amount of expression or activity of a polypeptide as defined in claim 1 , and, optionally, of at least one further polypeptide, the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and

(b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein an increased (or decreased) amount of expression or activity of said polypeptide, and, optionally, an increased or decreased amount of expression or activity of said at least one further polypeptide, is indicative for said hypertrophy or heart disease.

13. A method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein an increased or decreased amount of expression or activity of said one ore more polypeptide(s) is indicative for said hypertrophy or heart disease.

14. A method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the presence or amount of expression of a nucleic acid molecule as defined in any one of claims 7 to 10, and, optionally, the presence or amount of expression of at least one further nucleic acid molecule encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and

15. A method for diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease in a subject, comprising:

(a) determining the presence or amount of expression of one or more nucleic acid molecule(s) encoding a polypeptide, the expression of which is indicative for hypertrophy or a heart disease, in a biological sample, like blood serum, of said subject; and

(b) diagnosing hypertrophy or a heart disease or a susceptibility to hypertrophy or a heart disease, wherein the presence/absence or an increased/decreased amount of expression of said one or more nucleic acid molecule(s) is indicative for said hypertrophy or heart disease.

16. The use of any one of claims 5, 6 and 11 or the method of any one of claims 1 , 2, 12 or 13, wherein an increased amount of expression or activity of said (further) polypeptide or said one or more polypeptide(s) is indicative for said hypertrophy or heart disease.

17. The use of any one of claims 5, 6 and 11 or the method of any one of claims 1 , 2, 12 or 13, wherein an decreased amount of expression or activity of said further polypeptide or said one or more polypeptide(s) is indicative for said hypertrophy or heart disease.

18. The use of any one of claims 7 to 11 or the method of any one of claims 3, 4, 14 or 15, wherein the presence or an increased amount of expression of said (further) nucleic acid molecule or said one or more nucleic acid molecule(s) is indicative for said hypertrophy or heart disease.

19. The use of any one of claims 7 to 11 or the method of any one of claims 3, 4, 14 or 15, wherein the absence or an decreased amount of expression of said further nucleic acid molecule or said one or more nucleic acid molecule(s) is indicative for said hypertrophy or heart disease.

20. The use of any one of claims 5, 6, 11 and 16 or the method of any one of claims 1 , 2, 12, 13 and 16, wherein said further polypeptide comprises CD14 (human GenelD according to Entrez Gene (NCBI): 929) or a variant thereof or Cxcl14 (GenelD: 9547) or a variant thereof.

21. The use of any one of claims 5, 6, 11 and 17 or the method of any one of claims 1 , 2, 12, 13 and 17, wherein said further polypeptide comprises CD164 (GenelD: 8763) or a variant thereof or Psap (GenelD: 5660) or a variant thereof.

22. The use of any one of claims 7 to 11 and 18 or the method of any one of claims 3, 4, 14, 15 and 18, wherein said further nucleic acid molecule comprises a nucleic acid encoding CD14 or a variant thereof or a nucleic acid encoding Cxcl14 or a variant thereof.

23. The use of any one of claims 7 to 11 and 19 or the method of any one of claims 3, 4, 14, 15 and 19, wherein said further nucleic acid molecule comprises a nucleic acid encoding CD164 or a variant thereof or a nucleic acid encoding Psap or a variant thereof.

24. The use of any one of claims 5 to 11 and 16 to 23 or the method of any one of claims 1 to 4 and 12 to 23, wherein said hypertrophy is selected from the group consisting of:

(a) cardiac hypertrophy or cardiomyocyte hypertrophy;

(b) hypertrophy of blood vessel cells;

(c) vascular disorders;

(d) a hypertrophy in context of a skin disease; and

(e) a hypertrophy of adipose tissue.

25. The use of any one of claims 5 to 11 and 16 to 23 or the method of any one of claims 1 to 4 and 12 to 23, wherein the heart disease is selected from the group consisting of:

(a) cardiac hypertrophy or cardiomyocyte hypertrophy;

(b) heart failure;

(c) (heart) fibrosis;

(d) atherosclerosis; and

(f) remodeling post-myocardial infarction.

26. Polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or 8;

(c) a polypeptide comprising a fragment of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment corresponds (by comparison of homology) to any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440-459 of SEQ ID NO: 4, and, optionally, wherein said fragment has PI16 function;

(d) a polypeptide comprising a fragment from amino acid position 31-173 (SCP domain), 174-213 or 214-237 of SEQ ID NO: 8, optionally having Pl 16 function; and

(e) a polypeptide comprising an amino acid sequence at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identical to the polypeptide of (a), (b), (c) or (d), optionally having Pl 16 function.

27. Nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 5 or 7;

(c) a nucleic acid encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6 or 8, wherein said fragment has PI16 function;

(d) a nucleic acid encoding a polypeptide comprising a fragment of any one of SEQ ID NO: 2, 4, 6 and 8, wherein said fragment corresponds (by comparison of homology) to any one of the fragments from amino acid position 31-173 (SCP domain), 174-347, 348-368, 369-439 and 440^59 of SEQ ID NO: 4, and, optionally, wherein said fragment has Pl 16 function;

(e) a nucleic acid encoding a polypeptide comprising a fragment from amino acid position 31-173 (SCP domain), 174-213 or 214-237 of SEQ ID NO: 8, wherein, optionally, said nucleic acid encodes a polypeptide having Pl 16 function;

(f) a nucleic acid of at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identity to the nucleic acid of (a), (b), (c), (d) or (e), wherein, optionally, said nucleic acid encodes a polypeptide having PI16 function; and

(g) a nucleic acid encoding a polypeptide as defined in claim 26.

28. Vector comprising the nucleic acid molecule of claim 27.

29. The vector of claim 28, wherein said vector is a(n) (adeno)viral vector.

30. Host cell comprising the polypeptide of claim 26, the nucleic acid molecule of claim 27 or the vector of claim 28 or 29.

31. Pharmaceutical or diagnostic composition comprising polypeptide of claim 26, the nucleic acid molecule of claim 27, the vector of claim 28 or 29 or the host cell of claim 30.

32. Non-human transgenic animal specifically expressing in the heart a nucleic acid molecule encoding a polypeptide as defined in claim 1 or 2.

33. The non-human transgenic animal of claim 32, wherein said nucleic acid molecule is expressed under the control of a heart-specific promoter.

34. The non-human transgenic animal of claim 32 or 33, wherein said nucleic acid molecule is expressed under the control of a cardiomyocyte specific promoter.

35. The non-human transgenic animal of any one of claims 32 to 34, wherein said nucleic acid molecule is expressed under the control of the α-myosin heavy chain (αMHC) promoter.

36. The non-human transgenic animal of any one of claims 32 to 35, comprising the nucleic acid construct as depicted in Fig. 12.

37. A nucleic acid construct comprising a nucleic acid molecule encoding a polypeptide as defined in claim 1 or 2 or a nucleic acid molecule according to claim 27 and a promoter as defined in any one of claims 33 to 35.

38. A nucleic acid construct as depicted in Fig. 12.

39. A vector comprising the nucleic acid construct of claim 37 or 38.

40. A host cell comprising the nucleic acid construct of claim 37 or 38 or the vector of claim 39.

41. The host cell of claim 40, which is an embryonic stem cell.

42. The host cell of claim 40 or 41 , which is a mouse embryonic stem cell.

43. Use of the transgenic animal of any one of claims 32 to 36 for the screening and/or validation of medicaments.

44. The use of claim 43, wherein said medicaments are for the treatment or prevention of hypertrophy or a heart disease as defined in any one of the preceding claims.