Label‐free mass spectrometric analysis of the mdx‐4cv diaphragm identifies the matricellular protein periostin as a potential factor involved in dystrophinopathy‐related fibrosis

Proteomic profiling plays a decisive role in the identification of novel biomarkers of muscular dystrophy and the elucidation of new pathobiochemical mechanisms that underlie progressive muscle wasting. Building on the findings of recent comparative analyses of tissue samples and body fluids from dystrophic animals and patients afflicted with Duchenne muscular dystrophy, we have used here label‐free MS to study the severely dystrophic diaphragm from the not extensively characterized mdx‐4cv mouse. This animal model of progressive muscle wasting exhibits less dystrophin‐positive revertant fibers than the conventional mdx mouse, making it ideal for the future monitoring of experimental therapies. The pathoproteomic signature of the mdx‐4cv diaphragm included a significant increase in the fibrosis marker collagen and related extracellular matrix proteins (asporin, decorin, dermatopontin, prolargin) and cytoskeletal proteins (desmin, filamin, obscurin, plectin, spectrin, tubulin, vimentin, vinculin), as well as decreases in proteins of ion homeostasis (parvalbumin) and the contractile apparatus (myosin‐binding protein). Importantly, one of the most substantially increased proteins was identified as periostin, a matricellular component and apparent marker of fibrosis and tissue damage. Immunoblotting confirmed a considerable increase of periostin in the dystrophin‐deficient diaphragm from both mdx and mdx‐4cv mice, suggesting an involvement of this matricellular protein in dystrophinopathy‐related fibrosis.     

Subcellular Localization of Dystrophin Isoforms in Cardiomyocytes and Phenotypic Analysis of Dystrophin-deficient Mice Reveal Cardiac Myopathy is Predominantly Caused by a Deficiency in Full-length Dystrophin

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive muscle degenerative disorder that causes dilated cardiomyopathy in the second decade of life in affected males. Dystrophin, the gene responsible for DMD, encodes full-length dystrophin and various short dystrophin isoforms. In the mouse heart, full-length dystrophin Dp427 and a short dystrophin isoform, Dp71, are expressed. In this study, we intended to clarify the functions of these dystrophin isoforms in DMD-related cardiomyopathy. We used two strains of mice: mdx mice, in which Dp427 was absent but Dp71 was present, and DMD-null mice, in which both were absent. By immunohistochemical staining and density-gradient centrifugation, we found that Dp427 was located in the cardiac sarcolemma and also at the T-tubules, whereas Dp71 was specifically located at the T-tubules. In order to determine whether T tubule-associated Dp71 was involved in DMD-related cardiac disruption, we compared the cardiac phenotypes between DMD-null mice and mdx mice. Both DMD-null mice and mdx mice exhibited severe necrosis, which was followed by fibrosis in cardiac muscle. However, we could not detect a significant difference in myocardial fibrosis between mdx mice and DMD-null mice. Based on the present results, we have shown that cardiac myopathy is caused predominantly by a deficiency of full-length dystrophin Dp427.     

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomori''s tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.     

TNF‐α‐mediated signal transduction pathway is a major determinant of apoptosis in dilated cardiomyopathy

Although J2N-k strain of cardiomyopathic hamsters is an excellent model of dilated cardiomyopathy, the presence and mechanisms of apoptosis in the hearts of these genetically modified animals have not been investigated. This study examined the hypothesis that cardiac dysfunction and apoptosis in the cardiomyopathic hamsters were associated with tumour necrosis factor-alpha (TNF-α)-mediated signalling pathway involving the activation of some pro-apoptotic proteins and/or deactivation of some antiapoptotic proteins. Echocardiographic assessment of 31-week-old hamsters indicated an increase in the internal dimension of the left ventricle as well as decreases in the ejection fraction, fractional shortening and cardiac output without any evidence of cardiac hypertrophy. Increased level of TNF-α and apoptosis in cardiomyopathic hearts were accompanied by increased protein content for protein kinase C (PKC) -α and -ɛ isozymes as well as caspases 3 and 9. Phosphorylated protein content for p38 MAPK and NFκB was increased whereas that for Erk1/2, BAD and Bcl-2 was decreased in cardiomyopathic hearts. These results support the view that TNF-α and PKC isozymes may promote apoptosis due to the activation of p38 MAPK and deactivation of Erk1/2 pathways, and these changes may contribute toward the development of cardiac dysfunction in dilated cardiomyopathy.     

Cognitive flexibility deficits in a mouse model for the absence of full‐length dystrophin

Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full‐length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full‐length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full‐length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients.     

Proteomic profiling of liver tissue from the <Emphasis Type="Italic">mdx</Emphasis>-<Emphasis Type="Italic">4cv</Emphasis> mouse model of Duchenne muscular dystrophy

Background

Duchenne muscular dystrophy is a highly complex multi-system disease caused by primary abnormalities in the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle degeneration, this neuromuscular disorder is also associated with pathophysiological perturbations in many other organs including the liver. To determine potential proteome-wide alterations in liver tissue, we have used a comparative and mass spectrometry-based approach to study the dystrophic mdx-4cv mouse model of dystrophinopathy.

Methods

The comparative proteomic profiling of mdx-4cv versus wild type liver extracts was carried out with an Orbitrap Fusion Tribrid mass spectrometer. The distribution of identified liver proteins within protein families and potential protein interaction patterns were analysed by systems bioinformatics. Key findings on fatty acid binding proteins were confirmed by immunoblot analysis and immunofluorescence microscopy.

Results

The proteomic analysis revealed changes in a variety of protein families, affecting especially fatty acid, carbohydrate and amino acid metabolism, biotransformation, the cellular stress response and ion handling in the mdx-4cv liver. Drastically increased protein species were identified as fatty acid binding protein FABP5, ferritin and calumenin. Decreased liver proteins included phosphoglycerate kinase, apolipoprotein and perilipin. The drastic change in FABP5 was independently verified by immunoblotting and immunofluorescence microscopy.

Conclusions

The proteomic results presented here indicate that the intricate and multifaceted pathogenesis of the mdx-4cv model of dystrophinopathy is associated with secondary alterations in the liver affecting especially fatty acid transportation. Since FABP5 levels were also shown to be elevated in serum from dystrophic mice, this protein might be a useful indicator for monitoring liver changes in X-linked muscular dystrophy.

     

Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

Background

The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice.

Methods and Findings

Recombinant TAT-Utr and TAT-μUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-μUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290±920 U versus 5,950±1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%±5% versus 37%±4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%±5% versus 40%±8% drop; PBS versus TAT), and increased specific force production (9.7±1.1 N/cm² versus 12.8±0.9 N/cm²; PBS versus TAT).

Conclusions

These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.     

Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from <Emphasis Type="Italic">mdx</Emphasis> mouse muscle

The dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.     

The heart in Duchenne muscular dystrophy: early detection of contractile performance alteration

Progressive cardiomyopathy is a major cause of death in Duchenne muscular dystrophy (DMD) patients. Coupling between Ca²⁺ handling and contractile properties in dystrophic hearts is poorly understood. It is also not clear whether developing cardiac failure is dominated by alterations in Ca²⁺ pathways or more related to the contractile apparatus. We simultaneously recorded force and Ca²⁺ transients in field‐stimulated papillary muscles from young (10–14 weeks) wild‐type (wt) and dystrophic mdx mice. Force amplitudes were fivefold reduced in mdx muscles despite only 30 % reduction in fura‐2 ratio amplitudes. This indicated mechanisms other than systolic Ca²⁺ to additionally account for force decrements in mdx muscles. pCa‐force relations revealed decreased mdx myofibrillar Ca²⁺ sensitivity. ‘In vitro’ motility assays, studied in mdx hearts here for the first time, showed significantly slower sliding velocities. mdx MLC/MHC isoforms were not grossly altered. Dystrophic hearts showed echocardiography signs of early ventricular wall hypertrophy with a significantly enlarged end‐diastolic diameter ‘in vivo’. However, fractional shortening was still comparable to wt mice. Changes in the contractile apparatus satisfactorily explained force drop in mdx hearts. We give first evidence of early hypertrophy in mdx mice and possible mechanisms for already functional impairment of cardiac muscle in DMD.     

Ultrastructural and Functional Alterations of EC Coupling Elements in mdx Cardiomyocytes: An Analysis from Membrane Surface to Depth

A dilated cardiomyopathy (DCM) is associated with Duchenne muscular dystrophy (DMD). The loss of dystrophin leads to membrane instability and calcium dysregulation in skeletal muscle but effects of such a loss are not elucidated at cardiomyocytes level. We sought to examine whether membrane and transverse tubules damages occur in ventricular myocytes from mdx mouse model of DMD and how they impact the function of single excitation–contraction coupling elements. Scanning ion conductance microscopy (SICM) was used to characterize the integrity loss of living mdx cardiomyocytes surface. 2D Fourier transform analysis of labeled internal networks (transverse tubules, alpha-actinin, dihydropyridine receptors, ryanodine receptors) was performed to evaluate internal alterations. During calcium measurements, “smart microperfusions” of depolarizing solutions were applied through SICM nanopipette, stimulating single tubules elements. These approaches revealed structural membrane surface (39 % decrease for Z-groove ratio) and transverse tubules disorganization (21 % transverse tubules ratio decrease) in mdx as compared to control. These disruptions were associated with functional alterations (sixfold increase of calcium signal duration and twofold increase of sparks frequency). In DCM associated with DMD, myocytes display evident membrane alterations at the surface level but also in the cell depth with a disruption of transverse tubules network as observed in other cases of heart failure. These ultrastructural changes are associated with changes in the function of some coupling elements. Thus, these profound disruptions may play a role in calcium dysregulation through excitation–contraction coupling elements perturbation and suggest a transverse tubules stabilizing role for dystrophin.     

Proteomic profiling of the mitochondrial inner membrane of rat renal proximal convoluted tubules

The proximal convoluted tubule is the primary site of renal fluid, electrolyte, and nutrient reabsorption, processes that consume large amounts of adenosine‐5′‐triphosphate. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to the onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule. Separation by LC coupled with analysis by MS/MS (LC‐MS/MS) confidently identified 206 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N‐?‐acetyl‐lysine, seven of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron. The MS data have been deposited in the ProteomeXchange with the identifier PXD000121.     

Pilot study identifying myosin heavy chain 7, desmin,insulin‐like growth factor 7, and annexin A2 as circulating biomarkers of human heart failure

In‐depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac‐specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria, including myosin heavy chain 7, insulin‐like growth factor‐binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all four proteins. We verified antibody cross‐reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from four unaffected and four heart failure patients and demonstrated that all four proteins increased between twofold and 150‐fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human.     

epsilon-sarcoglycan replaces alpha-sarcoglycan in smooth muscle to form a unique dystrophin-glycoprotein complex.

The sarcoglycan complex has been well characterized in striated muscle, and defects in its components are associated with muscular dystrophy and cardiomyopathy. Here, we have characterized the smooth muscle sarcoglycan complex. By examination of embryonic muscle lineages and biochemical fractionation studies, we demonstrated that epsilon-sarcoglycan is an integral component of the smooth muscle sarcoglycan complex along with beta- and delta-sarcoglycan. Analysis of genetically defined animal models for muscular dystrophy supported this conclusion. The delta-sarcoglycan-deficient cardiomyopathic hamster and mice deficient in both dystrophin and utrophin showed loss of the smooth muscle sarcoglycan complex, whereas the complex was unaffected in alpha-sarcoglycan null mice in agreement with the finding that alpha-sarcoglycan is not expressed in smooth muscle cells. In the cardiomyopathic hamster, the smooth muscle sarcoglycan complex, containing epsilon-sarcoglycan, was fully restored following intramuscular injection of recombinant delta-sarcoglycan adenovirus. Together, these results demonstrate a tissue-dependent variation in the sarcoglycan complex and show that epsilon-sarcoglycan replaces alpha-sarcoglycan as an integral component of the smooth muscle dystrophin-glycoprotein complex. Our results also suggest a molecular basis for possible differential smooth muscle dysfunction in sarcoglycan-deficient patients.     

Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity

Selection on running capacity has created rat phenotypes of high-capacity runners (HCRs) that have enhanced cardiac function and low-capacity runners (LCRs) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCRs and LCRs from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCRs was six-fold greater than LCRs. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant (p<0.05; false discovery rate <10%, calculated using q-values) differences between HCRs and LCRs. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the β-oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCRs also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of α B-crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus, proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress.     

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X‐linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose‐derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X‐linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co‐cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)‐positive ASCs and DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle

Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabiolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and ncecrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance sperctroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (< 40–50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., decline in PCr/ATP) observed in adult mdx vs. control mice are quantilatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor.     

By‐passing the nonsense mutation in the 4CV mouse model of muscular dystrophy by induced exon skipping

Background

Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein‐truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg‐Dmd^mdx–4Cv/J (4^CV) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.

Methods

A series of 2′‐O‐methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.

Results

Compared to the original mdx mouse studies, induction of exon skipping from the 4^CV dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near‐full length dystrophin protein in cultured 4^CV myogenic cells and in vivo, after a single intramuscular injection.

Conclusions

By‐passing the protein‐truncating mutation in the 4^CV mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

Implications of protease activation in cardiac dysfunction and development of genetic cardiomyopathy in hamsters

It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.