# Patent

## Title
Muscle lamin A/C interacting protein, gene encoding same, and uses therefor

## Abstract

Peptide sequences of human and murine muscle lamin A/C interacting protein and nucleotide sequences encoding same and are provided. Uses of the muscle lamin A/C interacting protein are also provided herein.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of priority from U.S. provisional patent application Ser. No. 60/956,533, filed Aug. 17, 2007, the entire contents of which are herein incorporated by reference.


FIELD OF THE INVENTION
The present invention relates generally to muscle/brain gene expression in development and disease. More particularly, the present invention relates to muscle lamins and, specifically, a striated muscle and cardiac lamin A/C interacting protein, referred to herein as MLIP, and nucleotide sequences thereof.
BACKGROUND OF THE INVENTION
Lamins are intermediate filament proteins found only in the nuclei of all multicellular eukaryotes. They form stable filaments at the nuclear inner membrane and are fundamentally important for nuclear architecture, chromatin organization and transcriptional regulation of gene expression. Mammalian cells encode both A-type (LMNA) and B-type (LMNB) lamins, which are highly related but can be distinguished on a biochemical and functional basis. LMNA has 12 exons, is localized to human chromosome 1q21.2-q21.3 and generates two protein isoforms, Lamin A and Lamin C through alternative splicing of LMNA2. Together with the outer nuclear membrane and nuclear pore complexes, the inner nuclear membrane forms the nuclear envelope that separates the chromosomes from cytoplasm in eukaryotic cells [2]. Furthermore, Lamin A/C interacts with numerous other proteins, including tissue-specific transcription factors [7].
Laminopathies belong to a heterogeneous group of disorders caused by mutations in the lamin A/C gene (LMNA) that affects a specific combination of tissues, such as heart, skeletal muscle, tendons, neurons, adipocytes and bone. Over 180 different mutations in the LMNA gene have been described. The wide clinical heterogeneity caused by mutations in the LMNA gene supports the hypothesis that Lamin A/C protein performs multiple functions in different tissues. The diseases caused by the wide spectrum of LMNA gene mutations are characterized by the extreme variability of the clinical phenotypes, ranging from cardiac and skeletal myopathies to partial lipodystrophy, peripheral neuropathy, and premature aging. No clear genotype-phenotype correlation has been clarified, since the same mutation can cause different diseases in unrelated families [8-10] and even amongst family members [11, 12]. A recent study, using hierarchical cluster analysis for assembling laminopathies into classes based on organ system involvement, uncovered a non-random relationship between the class of laminopathy and the mutation. These positions were strongly associated (p<0.0001) with the nuclear localization signal sequence of Lamin A/C [13].
One of the seven known laminopathies results in dilated cardiomyopathy (DCM) and is associated with at least eight different clustered missense in the rod 1 domain of Lamin A/C. Alteration of lamin A/C interaction(s) with heart specific factor(s) may be responsible for the pathogenesis of DCM laminopathies. However, the molecular pathogenesis from mutations in the LMNA gene to dilated cardiomyopathy with conduction disease is relatively unknown. Further, the molecular mechanisms for the relationship between tissue specificity of laminopathies and mutations in the LMNA gene are not understood. There remains a need to understand how these different pathologies arise from alterations in the same gene (LMNA) that is almost ubiquitously expressed in adult cells.
The diagnosis of the DCM type of laminopathy is particularly important because of the severity of the cardiac symptoms, which are characterized by conduction system defects, arrhythmias, left ventricular dysfunction, and dilation causing heart failure and subsequent death [14]. Conduction system disease may be observed in the absence of cardiomyopathy [9, 19, 20] or it may proceed cardiac dilation [21]. Severe progression of conduction system disease in laminopathies is typically characterized by sinus node dysfunction, progressive atrioventricular blockage, paroxysmal atrial fibrillation, and frequent premature ventricular beats [8, 9, 17, 20, 22-27]. About half of affected patients suffer sudden cardiac death due to lethal ventricular tachyarrhythmias, despite pacemaker implantation [8, 17, 25, 27-29]. Fibrofatty infiltration of the sinoatrial and the atrioventricular node, as well as the atrioventricular bundle have been described in humans with LMNA mutations as histopathological correlation to their cardiac conduction system disease [22, 24, 25, 30].
Several hypotheses have been proposed for the pathogenesis of laminopathies and most research has been focused on the ‘mechanical stress’ and ‘altered gene regulation’ hypotheses. The structural integrity of the nucleus may be affected by the expression of mutant A-type lamins. The fragility of the nuclear envelope is believed to contribute (in part) to pathologies in tissues subject to mechanical stresses, such as skeletal and cardiac muscle. The complete loss of A-type lamins supports this hypothesis.
Many of the proteins that are involved in chromatin organization, transcription and binding to DNA are either directly or indirectly associated with the nuclear envelope. Chromatin organization and transcriptional regulation of gene expression is, therefore, affected in specific ways due to the disruption of the nuclear envelope [34].
The mechanisms by which specific tissue are dramatically affected in laminopathies are not yet known. Knowledge of novel cardiac specific proteins that specifically interacts with lamin A/C would provide a means for diagnosing and treating the pathogenesis of cardiovascular disease and, more particularly, dilated cardiomyopathy with conduction disease.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel muscle specific protein involved in muscular, brain and/or cardiovascular disease or development, and a nucleotide sequence thereof.
In a first aspect of the present invention there is provided nucleotide sequences encoding human or murine muscle lamin A/C interacting protein (hMLIP, mMLIP).
In embodiments of the present invention, the nucleotide sequences comprise a) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47 to 74; b) a nucleotide sequence which is substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 47 to 74; c) a nucleotide sequence which is homologous to a sequence selected from the group consisting of SEQ ID NOs. 47 to 74; d) a nucleotide sequence which hybridizes to the complement of a sequence selected from the group consisting of SEQ ID NO: 47 to 74; e) a nucleotide sequence which encodes a peptide sequence selected from the group consisting of SEQ ID NOs: 7 to 16; f) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 80 to 106; g) a nucleotide sequence which is substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 80 to 106; h) a nucleotide sequence which is homologous to a sequence selected from the group consisting of SEQ ID NOs. 80 to 106; i) a nucleotide sequence which hybridizes to the complement of a sequence selected from the group consisting of SEQ ID NO: 80 to 106; or j) a nucleotide sequence which encodes a peptide sequence selected from the group consisting of SEQ ID NOs: 75 to 79.
According to the present invention, MLIP can interact with the rod 1 domain of lamin A/C. Preliminary experiments confirmed specific expression of MLIP in the hearts of E11.5 mouse embryos, neonatal and adult mouse hearts, striated muscles and brains. The full length human and mouse MLIP cDNAs have been cloned and at least four MLIP splice variants are evident in both human and mouse. MLIP is located at 6p12.1 of the human chromosome.
In another aspect of the present invention there is provided a transgenic animal comprising a gene sequence encoding cardiac lamin A/C interacting protein. The transgenic animal can comprise a DNA sequence selected from the group consisting of SEQ ID NOs: 1 to 6, 47 to 74 or 80 to 106.
In a further aspect of the present invention there is provided an antibody directed to a muscle lamin A/C interacting protein. The antibody may be directed to the muscle lamin A/C interacting protein encoded by: a) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47 to 74; b) a nucleotide sequence which is substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 47 to 74; c) a nucleotide sequence which is homologous to a sequence selected from the group consisting of SEQ ID NOs. 47 to 74; d) a nucleotide sequence which hybridizes to the complement of a sequence selected from the group consisting of SEQ ID NO: 47 to 74; e) a nucleotide sequence which encodes a peptide sequence selected from the group consisting of SEQ ID NOs: 7 to 16; f) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 80 to 106; g) a nucleotide sequence which is substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 80 to 106; h) a nucleotide sequence which is homologous to a sequence selected from the group consisting of SEQ ID NOs. 80 to 106; i) a nucleotide sequence which hybridizes to the complement of a sequence selected from the group consisting of SEQ ID NO: 80 to 106; or j) a nucleotide sequence which encodes a peptide sequence selected from the group consisting of SEQ ID NOs: 75 to 79.
The present invention also provides a method for detecting the presence or absence of a muscle lamin A/C interacting protein (MLIP) in a biological sample comprising the steps of: obtaining the biological sample from an animal, and providing a labelled antibody to MLIP to the sample, whereby presence or absence of the label indicates the presence or absence of the MLIP.
In yet another aspect of the present invention, there is provided a kit for detecting the presence or absence of a muscle lamin A/C interacting protein (MLIP) in a biological sample, the kit comprising an antibody to MLIP and instructions for use. In one embodiment, the kit is for detecting the presence or absence of a nucleotide sequence encoding muscle lamin A/C interacting protein (MLIP) in a sample and comprises one or more primers selected from the group consisting of SEQ ID NO: 17 to 46 and SEQ ID NOs: 93 to 94, together with instructions for use.
As shown herein, MLIP may be an important genetic modulator in cardiovascular (including brain and heart) and muscle development and disease.
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.



BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, wherein:
FIG. 1a shows relative MLIP RNA expression levels of four established αMHC-MLIP transgenic mouse lines with evidence of mild hypertrophy.
FIG. 1b shows preliminary heart to body weight ratios determined for each of the established αMHC-MLIP transgenic mouse lines.
FIGS. 2a-c show representative electrocardiographic profiles of a) αMHC-MLIP transgenic mouse Line 39 (n=6); b) αMHC-MLIP transgenic mouse Line 49 (n=4); and c) control mice (n=8).
FIG. 2d shows comparative heart rates in the MLIP transgenic mouse lines compared to control mice.
FIG. 2e shows comparative P-R intervals of MLIP transgenic mouse lines compared to control mice.
FIGS. 3a-c shows histological analysis of a) five week old wildtype mouse; b) a first five week old αMHC MLIP transgenic mouse line in accordance with the present invention; and c) a second five week old αMHC-MLIP transgenic mouse line in accordance with the present invention.
FIGS. 4a-b show endogenous MLIP localized to both the nucleus and cytosol of rat neonatal myocytes at a) 40× magnification and b) 100× magnification. FIG. 4c shows C2C12 cells co-stained with specific polyclonal antibodies for MLIP and PML. FIG. 4d shows in situ MLIP in mouse brain localized to the hippocampus and in FIG. 4e, endogenous MLIP (red) was localized to the nuclei and cytosol of rat hippocampal neurons and glial cells.
FIG. 5a illustrates two major splice variants of MLIP were cloned from mouse heart. FIG. 5b shows a western blot analysis of bacterial-expressed His6-MLIP and GST-Lamin recombinant proteins.
FIGS. 6a-b show specific expression of MLIP in a) mouse tissue and b) human tissue, by Northern analysis. FIG. 6c shows normalized tissue distribution of MLIP expression in adult mouse as determined by real time PCR. FIG. 6d shows relative mRNA levels of MLIP in different tissues.
FIGS. 7a-b show specific induction of MLIP expression during P19 cardiomyogenesis, illustrating P19 differentiation into a) cardiac cells in the presence of dimethyl sulfoxide (DMSO); and b) neuronal and glial cells in the presence of retinoic acid.
FIG. 8a shows an RT-PCR expression profile of MLIP during the critical phase of the perinatal heart's exit from the cell cycle. FIG. 8b shows a corresponding Western blot.
FIG. 9 shows an RT-PCR expression profile of MLIP for wild type hearts (closed circles) and MYBPC3 deficient hearts (open circles).
FIG. 10 shows relative adult tissue specific expression of MLIP as determined by qualitative RT-PCR.
FIG. 11 shows a Northern blot of MLIP expression in C2C12 cells during myotube formation.
FIG. 12 shows MLIP expression in C2C12 cells during myotube formation by indirect immunoflorescence staining with a MLIP specific antibody.
FIG. 13 shows MLIP co-localized with PML bodies in C2C12 cells by indirect immunoflorescence staining with a MLIP and PML specific antibodies.
FIG. 14 shows an amino acid sequence alignment of human MLIP proteins hMLIP-1, hMLIP-2, hMLIP-3, hMLIP-4 and hMLIP-5. The amino acid sequences were translated from the five different human MLIP nucleotide sequences cloned (SEQ ID NOs: 80 to 106) from a pooled human cDNA library.
FIG. 15 shows an amino acid sequence alignment of mouse MLIP proteins mMLIP-01, mMLIP-02, mMLIP-03, mMLIP-04, mMLIP-05, mMLIP-06, mMLIP-07, mMLIP-08, mMLIP-09 and mMLIP-10. The amino acid sequences were translated from the ten different mouse MLIP nucleotide sequences cloned (SEQ ID NOs: 47 to 74) from a pooled mouse heart cDNA library
FIG. 16 shows a nucleotide sequence alignment of human MLIP nucleotide sequences cloned (SEQ ID NOs: 80 to 106) from a pooled human cDNA library.
FIG. 17 shows a nucleotide sequence alignment of mouse MLIP nucleotide sequences cloned (SEQ ID NOs: 47 to 74) from a pooled mouse heart cDNA library.
FIG. 18 shows the results of direct cloning of MLIP. FIG. 18a shows RT-PCR results. FIG. 18b is an alternative splice map of MLIP.
FIG. 19 shows representative expression profiles a) for muscle specific genes, and b) following overexpression of MLIP.
FIGS. 20a-b show results of yeast-two hybrid of MLIP and LMO7.



DETAILED DESCRIPTION
Generally, the present invention provides novel human and mouse gene sequences encoding muscle lamin A/C interacting protein (hMLIP and mMLIP). The sequences comprise: a) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47 to 74; b) a nucleotide sequence which is substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 47 to 74; c) a nucleotide sequence which is homologous to a sequence selected from the group consisting of SEQ ID NOs. 47 to 74; d) a nucleotide sequence which hybridizes to the complement of a sequence selected from the group consisting of SEQ ID NO: 47 to 74; e) a nucleotide sequence which encodes a peptide sequence selected from the group consisting of SEQ ID NOs: 7 to 16; f) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 80 to 106; g) a nucleotide sequence which is substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 80 to 106; h) a nucleotide sequence which is homologous to a sequence selected from the group consisting of SEQ ID NOs. 80 to 106; i) a nucleotide sequence which hybridizes to the complement of a sequence selected from the group consisting of SEQ ID NO: 80 to 106; or j) a nucleotide sequence which encodes a peptide sequence selected from the group consisting of SEQ ID NOs: 75 to 79.
The genes in accordance with the present invention, and the proteins expressed therefrom, are useful for studies of regulation of myocyte development, proliferation, regeneration and/or disease and for identification of regulatory substances having impact on developmental or disease-associated outcomes, particularly in the cardiovascular sciences. Furthermore, transgenic animals comprising the gene can be used as a model to examine regulation of myocyte development, proliferation, regeneration or disease and identification of novel drug targets muscle and cardiovascular disease, and muscle regeneration. Loss or mis-regulation of MLIP may contribute to alterations in cardiac development. Further, loss or mis-regulation of MLIP may contribute to the onset and development of dilated cardiomyopathy with conduction disease. This is suggested by data obtained from investigating the effects of altered gene expression of MLIP in murine models as described herein. Preliminary investigation suggests that MLIP may be associated with the simultaneous development of both Dunnigans-type familial partial lipodystrophy (non-striated muscle laminopathy) and dilated cardiomyopathy. MLIP may also be involved in other muscular dystrophies, not necessarily cardiac related. Furthermore, MLIP expression in terminally differentiated cells of brain, muscle and heart may play a role in normal development, disease and regeneration.
Identification and initial characterization of a novel Lamin A/C interacting protein, MLIP: To identify heart specific proteins that interact with the rod 1 domain of lamin A/C, a yeast two-hybrid interaction screen was utilized. Proteins encoded by a human heart complementary DNA library (Clontech) were screened for their ability to interact with the rod 1 domain of Lamin A/C. Of the 3.5×106 independent clones screened, 232 clones were positive in the presence of the rod 1 domain of lamin A/C; thus, the DNA sequence of the 232 positive clones were determined. The MLIP clone, which was represented by 6 independent clones out of the 232, was found to be homologous to 49 expressed sequence tags (ESTs) present within GenBankTM online database. The clone was mapped to human chromosome 6p12.1 and subsequently identified as a putative open reading frame (C6orf142, GenBank accession number: NM—138569). The mouse homologue is mapped to mouse chromosome 9E.
The MLIP cDNA clone contains 13 exons that span over 247 kilobases and is predicted to translate a 458 amino-acid protein. Comparisons of the deduced 458-amino-acid protein, or the nucleic acid sequence with the GenBank database, revealed no substantial similarities with any other protein other than homologous forms in non-human genomes or EST databases. Initially, no structural or functional domain was identified within the primary amino acid sequence or the nucleic acid sequence of MLIP.
Mouse MLIP DNA shRNAi sequences are provided (SEQ ID NO: 1 to 6). Putative amino acid sequences corresponding to mouse MLIP DNA sequence fragments were determined (SEQ ID NOs: 7 to 16). PCR primers for mouse MLIP (SEQ ID NOs: 17 to 46) were constructed to generate PCR products forming contigs (SEQ ID NOs: 47 to 56 and 58 to 74) to construct the entire mouse MLIP gene (SEQ ID NO: 57). PCR primers for human MLIP (SEQ ID NOs: 93-94) were used to generate PCR products forming contigs of the various splice variants (SEQ ID NOs: 80 to 87 and 89 to 106) of the full length sequence of the human MLIP gene (SEQ ID NO: 88). Putative human peptide sequences were obtained (SEQ ID NOs: 75 to 79).
Alignments of the sequences are shown in FIGS. 14 to 17. FIG. 14 shows an amino acid sequence alignment of human MLIP proteins hMLIP-1, hMLIP-2, hMLIP-3, hMLIP-4 and hMLIP-5. The amino acid sequences were translated from the five different human MLIP nucleotide sequences cloned (SEQ ID NOs: 80 to 106) from a pooled human cDNA library. FIG. 15 shows an amino acid sequence alignment of mouse MLIP proteins mMLIP-01, mMLIP-02, mMLIP-03, mMLIP-04, mMLIP-05, mMLIP-06, mMLIP-07, mMLIP-08, mMLIP-09 and mMLIP-10. The amino acid sequences were translated from the ten different mouse MLIP nucleotide sequences cloned (SEQ ID NOs: 47 to 74) from a pooled mouse heart cDNA library. FIG. 16 shows a nucleotide sequence alignment of human MLIP nucleotide sequences cloned (SEQ ID NOs: 80 to 106) from a pooled human cDNA library. FIG. 17 shows a nucleotide sequence alignment of mouse MLIP nucleotide sequences cloned (SEQ ID NOs: 47 to 74) from a pooled mouse heart cDNA library.
The present invention also provides a method for detecting the presence or absence of a muscle lamin A/C interacting protein (MLIP) in a biological sample comprising the steps of: obtaining the biological sample from an animal, and providing a labelled antibody to MLIP to the sample, whereby presence or absence of the label indicates the presence or absence of the MLIP. The sequences can be detected using a kit according to another aspect of the present invention. For example, the present invention provides a kit for detecting the presence or absence of a muscle lamin A/C interacting protein (MLIP) in a biological sample, the kit comprising an antibody to MLIP and instructions for use. In another embodiment, the kit is for detecting the presence or absence of a nucleotide sequence encoding muscle lamin A/C interacting protein (MLIP) in a sample, the kit comprising one or more primers selected from the group consisting of SEQ ID NO: 17 to 46 and SEQ ID NOs: 93 to 94, together with instructions for use.
MLIP transgenic mice develop bradycardia with a prolonged P-R interval with abnormal myocardial morphology: Lamin A/C linked cardiomyopathies are known to develop cardiac conduction disease with dilation. Loss or mis-regulation of MLIP may contribute to alterations in cardiac development. Further, loss or mis-regulation of MLIP may contribute to the onset and development of dilated cardiomyopathy with conduction disease. To investigate this concept, four αMHC-MLIP transgenic mouse lines were generated. These mice possess the MLIP transgene driven by the αMHC promoter.
FIG. 1 shows relative MLIP RNA expression levels of the four αMHC-MLIP transgenic mouse lines with evidence of mild hypertrophy. FIG. 1a shows MLIP RNA concentrations from adult hearts of wildtype (WT, n=4) and each of the αMHC-MLIP transgenic mouse lines (n=2 per line) were determined by real-time PCR. All values were normalized to the mean concentration of MLIP RNA expression of wild-type control mice. FIG. 1b illustrates preliminary heart to body weight ratios which were determined for each of the established αMHC-MLIP transgenic mouse lines (*p<0.005 vs WT). These data established a 4 to 50-fold increase in MLIP RNA expression over wild-type control mice with a trend towards increased heart to body weight ratios at 5 weeks of age.
FIGS. 2a to 2c illustrate that MLIP transgenic mice develop bradycardia with a prolonged P-R interval. Representative electrocardiographic profiles of two αMHC-MLIP transgenic mouse lines and a control mouse are shown: FIG. 2a Line 39 (n=6), FIG. 2b Line 49 (n=4) and FIG. 2c control (n=8). Preliminary electrogardiographic studies of two of the βMHC-MLIP transgenic mouse lines reveal the progressive and significant appearance of both bradycardia with a prolonged P-R intervals in each of the lines.
FIG. 2d shows a significant reduction of heart rate (p<0.02) observed in both MLIP transgenic mouse lines compared to control mice. Further, FIG. 2e shows a significant increase (p<0.02) in the prolongation of the P-R interval of both MLIP transgenic mouse lines as compared to control mice.
FIGS. 3a to 3c show preliminary histological analysis of βMHC-MLIP transgenic mouse lines shows abnormal myocardial morphology. Five week old mouse hearts from a) wild-type and b-c) transgenic mice that over express the MLIP were fixed, sectioned and stained by H&E protocol.
To date, there has been no occurrence of sudden death or congestive heart failure phenotype with the oldest (16 weeks) transgenic mice.
Endogenous MLIP is localized to both the nucleus and cytosol of rat neonatal myocytes: Using an MLIP specific polyclonal antibody, the cellular localization of MLIP in rat neonatal cardiomyocytes was determined. As shown in FIG. 4a, endogenous MLIP appears to be localized in both the cytosol and nucleus. The cytosolic staining of MLIP is diffuse but may be associated with a cytosolic organelle. FIG. 4b shows that the nuclear staining MLIP is punctate in nature and suggests co-localization with PML bodies of the nucleus as well as the inner nuclear membrane where lamin A/C is found.
Endogenous MLIP is localized to mouse C2C12 cells and rat hippocampal glial and neuronal cells. As shown in FIG. 4c, C2C12 cells were co-stained with specific polyclonal antibodies for MLIP (green) and PML (red). FIG. 4d shows in situ MLIP in mouse brain (Allen Mouse Brain Institute) localized to the hippocampus and in FIG. 4e, endogenous MLIP (red) was localized to the nuclei (green) and cytosol of rat hippocampal neurons (map2, blue) and glial cells.
Within 12 hours of initiation of C2C12 differentiation there is an  ̃2.5-fold increase in MLIP mRNA expression that coincides with a transient Myf5 and MyoD up regulation, followed by an up regulation of myogenin and MLIP. FIG. 19ashows representative expression profiles for muscle specific genes, between 0 and 72 hours of differentiation. C2C12 cells transfected with pCDNA3.1-MLIP vector show that overexpression of MLIP results in subsequent up-regulation of MLIP, Pax7, MyoD and VGLL2 mRNA (FIG. 19b; ND=not detected). This appears to suggest that MLIP may regulate PAX7, a myogenic determinant during regenerative myogensis.
MLIP interacts with rod 1 domain of lamin A/C: In vitro pull-down assays were performed using recombinant hexa-histidine-MLIP and GST-lamin-rod I to determine if the observed interaction between MLIP and the rod I domain of lamin A/C, as observed in the yeast-hybrid assay, is a direct or indirect interaction. FIG. 5 illustrates that MLIP directly interacts with the rod I domain of Lamin A/C. As shown in FIG. 5a, two major splice variants of MLIP were cloned from mouse heart. Exon 3 of MLIP is absent in the short form of MLIP. Both the full length MLIP (His6-MLIP) and the short form of MLIP (His6-MLIP(ΔExon3)) were sub-cloned in frame with an N-terminal hexa-histadine tag of a pET100 vector and recombinant MLIP was subsequently expressed in E.coli. FIG. 5b shows the isolation of bacterial-expressed His6-MLIP and GST-Lamin recombinant proteins. Various combinations of His6-MLIP and GST-Lamin recombinant proteins were mixed together (as indicated) and incubated at room temperature for 60 min in 10 mM Phosphate buffer (pH7.4), 50 mM NaCl, 0.05% triton X-100. Ni2+-NTA sepharose beads were added to each reaction mixture (right panels) and complexes were isolated and washed by centrifugation in phosphate buffer (pH7.4), 50 mM NaCl, 0.05% triton X-100. Complexes were eluted by addition of SDS-PAGE loading buffer, boiled, and resolved by SDS-PAGE. Western analysis was performed using anti-GST (Cell Signaling) and anti-MLIP polyclonal antibodies. A 1:10 dilution of the total starting material was run on the same gel (left panels). Assay was repeated two additional times with similar results.
Heart-enriched expression of MLIP: FIGS. 6a to 6c show specific expression of MLIP in mouse and human. Distribution of MLIP expression by Northern analysis in a) mouse and b) human tissues revealed two transcripts. 10 ug of poly-A enriched RNA was loaded per lane. In FIG. 6c, normalized tissue distribution of MLIP expression in adult mouse was determined by real time PCR. MLIP expression profile by Northern analysis demonstrated that MLIP expression was primarily associated with the heart and brain in mouse and heart and skeletal muscle in human. Real-time PCR of MLIP RNA from various mouse tissues showed a similar distribution of MLIP expression with the heart having approximately 3 times more MLIP, with smooth muscle>skeletal muscle>heart (FIG. 6d).
Specific induction of MLIP expression during cardiomyogensis of P19 cells: Pluripotent P19 cells are a well established and extensively studied cell line that can be induced to differentiate neuronal/glial cells in the presence of retinoic acid, skeletal muscle cells (retinoic acid plus dimethyl sulfoxide (DMSO)), and cardiac muscle cells in the presence of DMSO.
FIGS. 7a to 7b show the specific induction of MLIP expression during P19 cardiomyogenesis. In FIG. 7a, P19 cell differentiate into cardiac cells in the presence of dimethyl sulfoxide (DMSO). In FIG. 7b, neuronal and glial cells in the presence of retinoic acid. MLIP expression during DMSO induced cardiomyogenesis of P19 cells increased dramatically at day 5 of DMSO induction and continued through day 9. The MLIP expression at day 5 was shown to be concurrent with a number of cardiac and striated muscle specific transcription factors such as GATA-4, MEF2C and Nkx2.5 [35]. This induction of MLIP expression by DMSO is specifically associated with cardiomyogensis of P19 cells since neuronal differentiated P19 cells did not express MLIP.
MLIP is expressed in a transient biphasic manner during the critical phase of the perinatal heart's exit from the cell cycle. FIG. 8a shows that shortly after birth, MLIP mRNA expression is up-regulated in the heart and peaks at 4 days post-birth followed by MLIP down-regulation by day 5. This transient expression of MLIP corresponds to the same period which the cardiomyocytes are exiting their last round of cell division with the absence of cytokinesis. The reactivation of MLIP after day 5 is associated with adult cardiomyocyte hypertrophic growth. Western blot analysis of endogenous MLIP through this period reveals a switch in LIP isoform distribution from a short MLIP form to a long MLIP form (FIG. 8b).
At least four alternative splice forms of MLIP have been identified through direct cloning or RACE analysis. FIG. 18 shows the results of direct cloning. In FIG. 18a, RT-PCR (left panel) was performed on mRNA isolated from mouse hearts using primers targeted to the 5′- and 3′-UTR of MLIP as defined by the EST database. The RT-PCR product was TA cloned into pCR-II plasmid and transformed into bacteria. Direct PCR was performed with primers targeting flanking regions of the MLIP insertion site amplified four different product sizes (right panel) with each PCR product sequenced. Based on these data, an alternative splice map of MLIP was constructed (FIG. 18b). It appears that the MLIP gene comprises 12 exons and is regulated by two putative promoters with exon 1a only observed in cDNA cloned from muscle and both exons 1a and 1b observed in cDNA cloned from brain.
Suppressed MLIP expression in the MYBPC3 mouse during the onset of DCM in the perinatal heart. Cardiac Myosin Binding Protein-C (MYBPC3) is a 1274 amino acid thick filament accessory protein component of the striated muscle sarcomere A band that constitutes 2% to 4% of the myofibril. Although there are four MYBPC3 genes in the human genome, only cardiac MYBPC3 is expressed in embryonic, neonatal, and adult hearts. MYBPC3 interacts with at least three sarcomere components: myosin heavy chain, actin, and titin. More than 30 cardiac MYBP3 gene mutations have been identified as causes of hypertrophic cardiomyopathy (HCM); an autosomal dominant disorder resulting from defective sarcomeres. The majority of cardiac MYBPC3 mutations are predicted to encode truncated proteins that lack portions of either the carboxyl myosin and/or titin binding domains. Mice that express mutant cardiac MYBPC3 to create murine HCM models have been produced. These mice, like humans bearing the same mutation, develop adult onset HCM. Homozygous mice that express two mutant alleles and no wild-type cardiac MYBPC3 develop LV dilation by 3 days of age and have all of the features of DCM, including LV chamber dilation with impaired fractional shortening.
FIG. 9 shows a 30-fold reduction in MLIP mRNA expression as determined by real-time PCR, observed by 1 day post-birth in the MYBPC3 deficient mouse as compared to wild-type mouse. During the period of abnormal myocyte proliferation in the MYBPC3 deficient mice, MLIP expression remains suppressed and then by 10 days MLIP expression has returned to normal levels.
MLIP interaction with LMO7. MLIP was screened against a human heart library in the yeast two-hybrid assay. LMO7, an emerin-binding protein that regulates transcription of emerin and many other muscle relevant genes (Holaska et. al. 2006), was identified as interacting with MLIP, and was confirmed in the mammalian two-hybrid assay (FIGS. 20a and 20b). Disrupting the LIP-LMNA interaction and/or the regulation of MLIP expression may result in a gain of function.
MLIP knock-out mice In another aspect of the present invention there is provided an MLIP knock-out mouse. Targeted disruption of MLIP in knock-out mice (such as by insertion of LacZ cDNA) may result in alterations of the following as compared to wild-type mice:
a) echocardiographic and electrocardiograpic (ECG) output;
b) histology, particularly in the hearts of genetically modified mice at a variety of key developmental stages. Heart sections likely demonstrate alterations/aberrations in myocardium morphology (H&E stain) and fibrosis (Masson Trichrome).
c) RNA and protein analysis will typically indicate the expression of classic markers for cardiomyopathies (ANF, BNP and βMHC). Expression profiling can be achieved by any method known in the art, such as Northern and Western blotting (for RNA and protein), or Southern blots or DNA chip analysis (for DNA analysis).
d) MLIP expression analysis—MLIP expression in the developing mouse embryo can be mapped by utilizing a MLIP specific polyclonal antibody which has been generated. These can be confirmed by the nlsLacZ expression in the MLIP-knockout mice. Embryos can be stained for LacZ expression and then sectioned to identify and confirm the tissue expression profile of MLIP.
The knock-out mice of the present may be generated using any known protocol. However, certain modifications may be envisioned by the person of ordinary skill in the art in the context of the present invention with respect to the expression of MLIP. One example of the knock-out mouse protocol is provided herein. The lacZ gene is fused to a nuclear localization signal sequence (nlslacZ) so as to increase the signal to noise ratio of lacZ detection. To generate the MLIP-nlslacZ targeting vector, a fragment containing at least exons 1 and 2 of the MLIP locus is isolated by screening a 129 mouse genomic library (Stratagene) using the total coding region of the mouse MLIP cDNA as a probe. The identified fragment is inserted into the multiple cloning site of pBluescript KS+. An ATG start codon in exon 1 is PCR-modified into an Ncol site, and a BspHI-Xhol nlslacZ cassette is ligated in-frame into exon 1 in between the Ncol and Sall sites. This subclone is flanked on the 3′ end with the PGK-hygromycin cassette, thereby deleting the coding sequence in exon 2. The Polyoma thymidine kinase gene obtained from a previously described vector [36] is added 3′ of the construct. Embryonic stem (ES) cells, such as those from the 129/Sv mouse line, for example, are transfected with the construct, and homologous recombinants screened by Southern analysis. ES cells with a homologous recombination are then injected into C57BI/6J blastocysts to generate heterozygous MLIP-nlslacZ mice. The mice are genotyped and separated into wild-type or heterozygous mice by Southern blot analysis and PCR based genotyping. Chimeric males are crossed to 129/SeEv females and germline transmission of the injected ES cell lines are monitored by detecting agouti mice among the F1 offspring and subsequent Southern blotting. Heterozygote mice are crossed to generate a homozygote null MLIP mouse. The colony can be expanded to provide sufficient mice for characterization.
Characterization MLIP mouse models: Mouse models generated can be initially characterized by the following protocols:
Immunocytochemistry: Cultured cells transiently expressing MLIP were plated on gelatin-coated coverslips. Cells are fixed with 4% paraformaldehyde in PBS on ice for 20 min, followed by three washes of PBS. The fixed cells are then blocked with 10% normal serum in PBS on ice for 60 min followed by a single wash in PBS. The cells will then be incubated in the appropriate primary anti-sera in 1.5% normal serum for 1-2 hours, washed three times in PBS, and then incubated for 60 min with the appropriate secondary anti sera conjugated to a flourophore (Molecular Probes) with 1.5% normal serum. Coverslips are then washed three times in PBS, mounted on glass slides, and analysed with a confocal microscope.
Northern analysis: Initially, total mouse RNA is isolated from heart, skeletal muscle, brain, liver and kidney at variety of developmental time points (E9.5 through E14.5, 1 day, 14 day and 8 weeks) using TRIzol reagent (Invitrogen). At least 10 μg of total RNA per lane is loaded on a 1% agarose gel (containing 0.42% MOPS, 5 mM sodium acetate, 0.7 mM EDTA, and 0.6% formaldehyde). Electrophoresis is performed in 1×MOPS buffer (0.42% MOPS, 5 mM sodium acetate, 0.7 mM EDTA) with a circulating pump at 40-60 constant volts for about 2-3 hours. The electrophoresis is stopped when the stop dye has migrated about 2⁄3-3⁄4 of the way through the gel. The RNA is then transferred overnight to a nylon membrane (pre-wet in 1×MOPS buffer) with a 10×SSPE stock solution (1.5 M NaCl, 0.1 M NaH2PO4, 10 mM EDTA, pH 7.0). The membrane is then be re-hybridized at 42° C. in 50% deionized formamide, 5×SSPE, 50 mM sodium phosphate buffer, pH 6.8, 1× Denhardt's solution, heat denatured salmon sperm DNA to 100-200 μg/ml, and 0.5% SDS for 3-4 hours. Hybridization occurs overnight with a radiolabelled probe at 42° C. in fresh prehybridization buffer with 7.5% dextran sulfate. 25 ng of a DNA probe is labelled with random primers, with MLIP cDNA fragment used as template. The next day, the membranes are washed twice in 2×SSPE, 0.1% SDS at room temperature, 10 minutes per wash, then for 30 minutes in 0.5× or 0.2×SSPE, 0.1% SDS at room temperature. The membrane is then be checked for radioactivity with a hand-held monitor and/or autoradiography before increasing the stringency. Initially, the membrane can be analysed by a phospho-imager and then exposed overnight against film.
RT-PCR and real time RT-PCR: cDNA is synthesized from total RNA samples (isolated for Northern analysis) by oligo (dT)-primed reverse transcription (Protoscript First Strand cDNA synthesis, New England Biolabs). To characterize MLIP expression in the heart through development, cDNA is subjected to either PCR amplification using HotStarTaq DNA polymerase (Qiagen) or real time quantitative PCR (qPCR). Primers are designed for the specific amplification of MLIP splice isoforms, such that the PCR product length will be 200 to 300 base pairs in length and overlap splice form specific exon boundaries.
To determine the relative abundance of MLIP isoforms in mouse tissue, qPCR is performed with SYBR green PCR master mix (Roche) using LightCycler 1.0 sequence detection system (Roche). Isoform-specific primers for MLIP [MLIP67 primers, 5′-TTCATCATCCTCAACAGCGT-3′ (forward) (SEQ ID No: 107), 5′-GGGTTGGGCTCATAAACTTC-3′ (reverse) (SEQ ID No: 108)] and [MLIP3 primers, 5′-TAGCTACTCGGCCCAAGTCT-3′ (forward) (SEQ ID No: 109), 5′-ATCCCATGAGGAATTTCAGG-3′ (reverse) (SEQ ID No: 110)] are used to analyze transcript abundance with mouse GAPDH transcript levels [GAPDH primers, 5′-GCAACAGGGTGGTGGACCT-3′ (forward) (SEQ ID No: 111), 5′-GCAACAGGGTGGTGGACCT-3′ (reverse) (SEQ ID No: 112)] serving as an internal control to compensate for differences in RNA recovery and used to normalize the values of transcript abundance of MLIP isoforms. All PCR reactions, cycled 40 times by a two-step cycle procedure (denaturation 95° C., 15s; annealing 65° C., 1 min) after the initial stages (50° C., 1s; 95° C. 10 min), can be performed in triplicate for each gene. To generate a standard curve, serially diluted heart cDNA is included in the 96-well plate along with cDNA from the various tissues.
In situ hybridization studies. Studies have been described previously [37]. A typical study which may be performed in the context of the present invention is briefly outlined herein. Eight week, 2 week, and newborn mice and E9.5 to E14.5 embryos are dissected free from the uterine muscle and studied. E0.5 is defined as noon on the day postcoitous when a vaginal plug is detected. Embryos are removed, washed in phosphate buffered saline (PBS), fixed overnight in 4% paraformaldhyde in PBS and either frozen (−20° C., 100% methanol) or kept in 70% ethanol at room temperature prior to paraffin embedding and histological analysis. Embedded sections are stained with hematoxylin and eosin or used for in situ hybridization as previously described[38]. Plasmids containing MLIP sequences are used as template for digoigenin (DIG) riboprobes, which are produced according to the manufacture's specifications (Roche). DIG-RNA probes are hybridized overnight at 70° C. and incubated with anti-DIG-AP fragment, and signal detected using Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl-□-D-galactopyranoside substrates (Roche)[39].
Electrocardiographic and Echocardiographic Analysis: Surface electrocardiograms can be obtained from anesthetized mice at 2 weeks, 2 months and 4 months of age. Subcutaneous electrodes are inserted in four configurations to obtain 4 electrocardiographic recordings: Lead 1 (right and left forelimb), Lead 2 (left forelimb and right hindlimb), Lead 3 (right forelimb and left hindlimb) and transthoracic (beneath skin of back and skin of chest at cardiac apex). After ECG monitoring, mice are then euthanized by harvesting the heart for biochemical analysis. To monitor the occurrence and frequency of supraventricular arrhythmias, telemetry electrocardiography is obtained using subcutaneous implantation of a radio-transducer and subcutaneous transthoracic electrodes). Devices are implanted under general anaesthesia in mice at 16 weeks of age, mice are allowed to recover for 1 week and are then monitored continuously for 24 hours. Mice are then euthanized and the heart is harvested for biochemical analysis. Mice are not kept with telemetry monitors longer than 3 weeks. To ascertain cardiac diastolic function it was necessary to measure intracardiac pressure/volume loops and diastolic pressures during the normal cardiac cycle in vivo. Intracardiac conductance catheters allow the accurate measurement of these parameters in the mouse heart. Pressure/volume loop measurements are obtained in anesthetized mice at 16 weeks of age by inserting a 1.4 French microcatheter down the carotid artery. This closed-chest preparation is minimally invasive and does not require mechanical ventilation. Echocardiography was performed with an Hewlett Packard SonosTM 4500 ultrasound machine and a 6-15 MHz linear array transducer on anesthetized mice with a heart rate greater than 450 bpm, as previously described [40].
Characterization of the regulation of MLIP function and expression in the heart during development and the onset of DCM-CCD.
This study tests the hypothesis that differential regulation of MLIP isoform expression is necessary for normal differentiation of cardiomyocytes. Expression in cultured cardiomyocytes of deleted and mutated reporter constructs of the MLIP promoter is analyzed to identify regulatory sequences and transcription factors that control MLIP mRNA levels. Temporal and spatial analysis of endogenous MLIP expression is characterized at different developmental time points of heart development using a variety of molecular methods: Northern, RT-PCR, real-time PCR, in situ hybridization and Western analysis. The investigation of just the levels of MLIP expression is not sufficient alone and the expression of each splice variant of MLIP needs to be taken into context as this can provide important information as to the function of MLIP.
Temporal and tissue specific expression of MLIP in the mouse: Specific tissue expression of total MLIP during mouse development can be investigated. The primary source of tissue is obtained from wild-type, in-bred 129/SeEv mice. The basis for this choice of mouse strain is that many of the genetically modified mouse models are generated in the 129/SeEv background. Total mouse RNA and protein are isolated from heart, skeletal muscle, brain, liver and kidney at a variety of developmental time points (E9.5 through E14.5, 1 day, 14 day and 8-12 weeks post birth) to be analyzed by northern analysis, real time PCR and Western blot. Once an MLIP expression profile is mapped throughout normal mouse development, a variety of published mouse models for DCM-CCD can be examined. These include mice carrying lamin missense mutations H222P[14] and N195K[41] and a lamin null allele [33]. Spatial expression can be elucidated by in situ hybridization and immunohistochemical studies of whole embryos (<E12.5) and tissue sections as described in general methods that would be known in the art.
FIG. 10 shows relative adult tissue specific expression of MLIP as determined by qualitative RT-PCR. FIG. 11 shows a Northern blot of MLIP expression in C2C12 cells during myotube formation. FIG. 12 shows MLIP expression in C2C12 cells during myotube formation by indirect immunoflorescence staining with a MLIP specific antibody.
FIG. 13 shows MLIP co-localized with PML bodies in C2C12 cells by indirect immunoflorescence staining with a MLIP and PML specific antibodies.
Chromatin Immunoprecipitation (ChIP) Analysis
A ChIP analysis was performed (SimpleChIPTM Enzymatic Chromatin IP Kit, Product Catalog Number 9002 and 9003, Cell Signaling Technology, Danvers, Mass., USA). C2C12 mouse myoblasts and both MLIP antibodies described herein were used. MLIP immunoprecipitated chromatin fragments were purified and PCR linkered/amplified. Following TA cloning and sequencing, 80 positive clones were identified and the genomic location of each of these fragments was determined.







TABLE 1







Genes that may be regulated by MLIP as determined by chromatin


immunoprecipitation








GENE
CELLULAR ROLE





Notch2
Development, Differentiation, Commitment Death,



Apoptosis, Proliferation, Survival, growth


CREM(ICER)
Death, Apoptosis, Proliferation, Growth


Sox5
Development, Commitment, Apoptosis


KIF5C
Biogenesis, Growth


PLCB1
Apoptosis, Proliferation, Growth


MET
Apoptosis, Proliferation, Growth


MMP3
Differentiation, Proliferation, Survival


RUNX1
Development, Differentiation, Death, Apoptosis,



Proliferation, Growth


Akt2
Differentiation, Cell Cycle Progression, Death,



Apoptosis, Proliferation, Survival, Growth


NEK7
—


FLI1
Differentiation, Apoptosis, Proliferation, Growth


PP2R3A
Cell Cycle Progression, Survival


GATA6
Differentiation, Apoptosis, Survival









Interestingly, in relation to the defined cellular role of A-type Lamins (LMNA) all the above genes share at least one of the following roles in the cell: differentiation, cell cycle progression, biogenesis and apoptosis.
MLIP alternative splice variant regulation and expression in both the normal mouse and mouse models for DCM-CCD. The expression of individual MLIP splice variants within the mouse heart at a variety of developmental time points (E9.5 through E14.5, 1 day, 14 day and 8-12 weeks post birth) for both normal and mouse models for DCM-CCD can be determined by real-time PCR. cDNA is synthesized from total RNA samples and subjected to real-time PCR. Primers have been designed and characterized for the specific amplification of each MLIP splice isoform with GAPDH as a normalization control between samples and dilutions. The quality of qPCR amplification is typically assessed by gel electrophoresis, as described in general methods known in the art.
MLIP promoter analysis. Examination of the 5′UTR of both mouse and human MLIP reveals a very high degree of homology. Deleted and mutated reporter constructs of the MLIP promoter are analyzed to identify regulatory sequences and transcription factors that control MLIP mRNA levels. A preliminary examination of the 5′ promoter region of the MLIP allele reveals several putative Tbx5, TEF-1, and Nkx 2.5 binding domains. To determine the minimal promoter region required for normal MLIP mRNA expression, a series of deletion constructs of the MLIP 5′UTR are generated and transiently transfected into cultured myocytes. Once the minimal MLIP 5′UTR is defined, putative muscle specific transcription factor binding domains can be mutated to determine their contribution to the regulation of MLIP expression. Once the important promoter elements have been mapped, site-directed mutagenesis is performed to test the effect of naturally occurring polymorphisms within the important regulatory sequences on the basal response of the MLIP promoter.
Plasmids and constructs: The putative promoter fragment for both human and mouse MLIP is obtained and sub-cloned into the pGL3-basic luciferase vector. A luciferase/MLIP 3′UTR chimeric construct in the pGL3-control vector that carries the SV40 enhancer and promoter and drives high levels of luciferase expression in cardiac myocytes is produced. Serial deletions and site-directed mutagenesis is performed following well-established protocols known in the art.
The MLIP specific antibody and genetically modified mice provide important reagents to characterize the biological role of MLIP. The regulation of MLIP function within the heart during normal development can be defined using these tools. In addition to defining the role of MLIP's function(s) in cardiomyocytes, the tools provide a powerful means for furthering the understanding the underlying mechanisms of cardiac specific laminopathies of DCM-CCD. As one possible embodiment of the present invention, expression of MLIP isoforms may be differentially regulated during specific developmental time points and the pathogenesis of DCM-CCD in mice. This may in turn effect sub-cellular localization of MLIP and consequently its function(s). Further, mis-expression of MLIP or loss of MLIP function can be found to result in altered cardiac structure and/or function in the mouse, based on the phenotypes generated by the genetically modified mice.
The spatial and temporal aspects of MLIP/Lamin interactions in real time in living cells can be determined using techniques known in the art, such as fluorescence resonance energy transfer (FRET) in living cells to measure real time flux of MLIP bound to Lamin A/C in response to stimuli. This may also provide a role of MLIP bound to Lamin A/C in cardiomyocyte physiology and the pathogenesis of Lamin associated DCM, as well as identifying the upstream signals involved in MLIP regulation, particularly in the roles of different heart specific transcription factors (Nkx 2.5 and Tbx5) on MLIP expression. Identification of specific MLIP interactors (other than Lamin A/C) can form the basis of defining the molecular mechanism of MLIP in the heart.
Loss or mis-regulation of MLIP may contribute to alterations in cardiac development. Further, loss or mis-regulation of MLIP may contribute to the onset and development of dilated cardiomyopathy with conduction disease. This is suggested by data obtained from investigating the effects of altered gene expression of MLIP in murine models as described herein. Preliminary investigation suggests that MLIP may be associated with the simultaneous development of both Dunnigans-type familial partial lipodystrophy (non-striated muscle laminopathy) and dilated cardiomyopathy, but the numbers reported are too low to be certain of their significance [31]. The finding of expression in brain tissue may indicate that MLIP could be involved in brain development, maintenance and/or pathology.
MLIP may also be involved in cardiomyocyte regeneration or in therapeutic development. For example, modulation of MLIP activity, either at the level of the gene or protein, may contribute to regulate cardiomyocyte or muscle differentiation. This can be important in regenerating heart or muscle tissue, which may be damaged from certain insults, such as infarct or the like. Certainly, within the context of the present invention, it is contemplated that MLIP may serve as a target for pharmaceutical agents.
The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto. All documents referred to herein are incorporated by reference.
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## Claims

1. An isolated human muscle lamin A/C interacting protein encoded by the nucleotide sequence as set forth in SEQ ID NO: 88.