A Small-Molecule Approach to Restore a Slow-Oxidative Phenotype and Defective CaMKIIß Signaling in Limb Girdle Muscular Dystrophy.

Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIß signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII (Myl2) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo, including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIß signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1.

Myostatin inhibition promotes fast fibre hypertrophy but causes loss of AMP-activated protein kinase signalling and poor exercise tolerance in a model of limb-girdle muscular dystrophy R1/2A.

KEY POINTS: Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. The effect of genetic and pharmacological inhibition of myostatin signalling on the disease phenotype in a mouse model of LGMD R1 (CAPN3 knockout mouse-C3KO) was studied. Inhibition of myostatin signalling in C3KO muscles resulted in significant muscle hypertrophy; however, there were no improvements in muscle strength and exacerbation of exercise intolerance concomitant with further reduction of muscle oxidative capacity was observed. Inhibition of myostatin signalling is unlikely to be a valid therapeutic strategy for LGMD R1. ABSTRACT: Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. There are currently no therapies available to patients. We sought to determine if induction of muscle growth, through myostatin inhibition, represents a viable therapeutic strategy for this disease. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. However, the effect of myostatin depends on the genetic and pathophysiological context and may not be efficacious in all contexts. We found that genetic inhibition of myostatin through overexpression of follistatin (an endogenous inhibitor of myostatin) in our LGMD R1 model (C3KO) resulted in 1.5- to 2-fold increase of muscle mass for the majority of limb muscles. However, muscle strength was not improved and exercise intolerance was exacerbated. Pharmacological inhibition of myostatin, using an anti-myostatin antibody, resulted in statistically significant increases in muscle mass; however, functional testing did not reveal changes in muscle strength nor endurance in treated C3KO mice. Histochemical and biochemical evaluation of follistatin overexpressing mice revealed a reduction in the percentage of oxidative fibres and decreased activation of AMP-activated protein kinase signalling in transgenics compared to C3KO muscles. Our data suggest that muscle hypertrophy, induced by myostatin inhibition, leads to loss of oxidative capacity, which further compromises metabolically impaired C3KO muscles and thus is unlikely to be a valid strategy for treatment of LGMD R1.

Spp1 (osteopontin) promotes TGFß processing in fibroblasts of dystrophin-deficient muscles through matrix metalloproteinases.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFß signaling. Spp1's effects on TGFß signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFß ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFß latent complex, decreased levels of active TGFß and reduced collagen expression. Correspondingly, we found reduced active TGFß in Spp1-/-mdxB10 and Mmp9-/-mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFß to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFß signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.

Calpain 3 and CaMKIIß signaling are required to induce HSP70 necessary for adaptive muscle growth after atrophy.

Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIß (CaMKIIß). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIß signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIß in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIß signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIß signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIß activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIß signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIß signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIß regulates remodeling in multiple contexts: endurance exercise and growth after atrophy.

Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.

In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors.

Failure to up-regulate transcription of genes necessary for muscle adaptation underlies limb girdle muscular dystrophy 2A (calpainopathy).

Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in the Calpain 3 (CAPN3) gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. In Capn3 knock-out mice (C3KO), Ca2+ release and Ca2+/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca2+-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild type (WT) and C3KO mice subjected to endurance exercise. In WT mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscles fail to release fatty acids from stored triacylglycerol. In conjunction with the defects in oxidative metabolism, C3KO mice demonstrate reduced exercise endurance. Failure to up-regulate genes in C3KO muscles is due, in part, to decreased levels of PGC1α, a transcriptional co-regulator that orchestrates the muscle adaptation response. Destabilization of PGC1α is attributable to decreased p38 MAPK activation via diminished CaMKII signaling. Thus, we elucidate a pathway downstream of Ca2+-mediated CaMKII activation that is dysfunctional in C3KO mice, leading to reduced transcription of genes involved in muscle adaptation. These studies identify a novel mechanism of muscular dystrophy: a blunted transcriptional response to muscle loading resulting in chronic failure to adapt and remodel.

Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

BACKGROUND: Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n',n'-tetraacetic acid (EGTA) to measure Ca(2+) fluxes. Muscles were subjected to both current and voltage clamp conditions. METHODS: Standard and confocal fluorescence microscopy was used to study Ca(2+) release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student's test with significance set at p < 0.05. RESULTS: We found that the peak value of Ca(2+) fluxes elicited by single action potentials was significantly reduced by 15-20 % in C3KO fibers, but the kinetics was unaltered. Ca(2+) release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca(2+) release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca(2+) release in C3KO mice but reduced peak Ca(2+) fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca(2+) release at all triads consistent with a homogenous reduction of functional voltage activated Ca(2+) release sites. CONCLUSIONS: Overall, these results suggest that decreased Ca(2+) release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2.

Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation.

Autolytic activation of calpain 3 proteinase is facilitated by calmodulin protein.

Calpains are broadly distributed, calcium-dependent enzymes that induce limited proteolysis in a wide range of substrates. Mutations in the gene encoding the muscle-specific family member calpain 3 (CAPN3) underlie limb-girdle muscular dystrophy 2A. We have shown previously that CAPN3 knockout muscles exhibit attenuated calcium release, reduced calmodulin kinase (CaMKII) signaling, and impaired muscle adaptation to exercise. However, neither the precise role of CAPN3 in these processes nor the mechanisms of CAPN3 activation in vivo have been fully elucidated. In this study, we identify calmodulin (CaM), a known transducer of the calcium signal, as the first positive regulator of CAPN3 autolytic activity. CaM was shown to bind CAPN3 at two sites located in the C2L domain. Biochemical studies using muscle extracts from transgenic mice overexpressing CAPN3 or its inactive mutant revealed that CaM binding enhanced CAPN3 autolytic activation. Furthermore, CaM facilitated CAPN3-mediated cleavage of its in vivo substrate titin in tissue extracts. Therefore, these studies reveal a novel interaction between CAPN3 and CaM and identify CaM as the first positive regulator of CAPN3 activity.

C3KO mouse expression analysis: downregulation of the muscular dystrophy Ky protein and alterations in muscle aging.

Mutations in CAPN3 gene cause limb-girdle muscular dystrophy type 2A (LGMD2A) characterized by muscle wasting and progressive degeneration of scapular and pelvic musculature. Since CAPN3 knockout mice (C3KO) display features of muscle pathology similar to those features observed in the earliest-stage or preclinical LGMD2A patients, gene expression profiling analysis in C3KO mice was performed to gain insight into mechanisms of disease. Two different comparisons were carried out in order to determine, first, the differential gene expression between wild-type (WT) and C3KO soleus and, second, to identify the transcripts differentially expressed in aging muscles of WT and C3KO mice. The up/downregulation of two genes, important for normal muscle function, was identified in C3KO mice: the Ky gene, encoding a protease implicated in muscle development, and Park2 gene encoding an E3 ubiquitin ligase (parkin). The Ky gene was downregulated in C3KO muscles suggesting that Ky protease may play a complementary role in regulating muscle cytoskeleton homeostasis in response to changes in muscle activity. Park2 was upregulated in the aged WT muscles but not in C3KO muscles. Taking into account the known functions of parkin E3 ligase, it is possible that it plays a role in ubiquitination and degradation of atrophy-specific and damaged proteins that are necessary to avoid cellular toxicity and a cellular stress response in aging muscles.

Satellite cell senescence underlies myopathy in a mouse model of limb-girdle muscular dystrophy 2H.

Mutations in the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) are responsible for the disease limb-girdle muscular dystrophy 2H (LGMD2H). Previously, we generated Trim32 knockout mice (Trim32-/- mice) and showed that they display a myopathic phenotype accompanied by neurogenic features. Here, we used these mice to investigate the muscle-specific defects arising from the absence of TRIM32, which underlie the myopathic phenotype. Using 2 models of induced atrophy, we showed that TRIM32 is dispensable for muscle atrophy. Conversely, TRIM32 was necessary for muscle regrowth after atrophy. Furthermore, TRIM32-deficient primary myoblasts underwent premature senescence and impaired myogenesis due to accumulation of PIAS4, an E3 SUMO ligase and TRIM32 substrate that was previously shown to be associated with senescence. Premature senescence of myoblasts was also observed in vivo in an atrophy/regrowth model. Trim32-/- muscles had substantially fewer activated satellite cells, increased PIAS4 levels, and growth failure compared with wild-type muscles. Moreover, Trim32-/- muscles exhibited features of premature sarcopenia, such as selective type II fast fiber atrophy. These results imply that premature senescence of muscle satellite cells is an underlying pathogenic feature of LGMD2H and reveal what we believe to be a new mechanism of muscular dystrophy associated with reductions in available satellite cells and premature sarcopenia.

Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3.

Calpain 3 (CAPN3) is a muscle-specific, calcium-dependent proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to titin in vitro. In this investigation, we tested the consequence of loss of titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-cysteine, cellular proteases acting on the autolytic sites of CAPN3, rather than autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to calpainopathy.

Calcium-dependent plasma membrane repair requires m- or mu-calpain, but not calpain-3, the proteasome, or caspases.

Mechanically damaged plasma membrane undergoes rapid calcium-dependent resealing that appears to depend, at least in part, on calpain-mediated cortical cytoskeletal remodeling. Cells null for Capns1, the non-catalytic small subunit present in both m- and mu-calpains, do not undergo calcium-mediated resealing. However, it is not known which of these calpains is needed for repair, or whether other major cytosolic proteinases may participate. Utilizing isozyme-selective siRNAs to decrease expression of Capn1 or Capn2, catalytic subunits of mu- and m-calpains, respectively, in a mouse embryonic fibroblast cell line, we now show that substantial loss of both activities is required to compromise calcium-mediated survival after cell scrape-damage. Using skeletal myotubes derived from Capn3-null mice, we were unable to demonstrate loss of sarcolemma resealing after needle scratch or laser damage. Isolated muscle fibers from Capn3 knockout mice also efficiently repaired laser damage. Employing either a cell line expressing a temperature sensitive E1 ubiquitin ligase, or lactacystin, a specific proteasome inhibitor, it was not possible to demonstrate an effect of the proteasome on calcium-mediated survival after injury. Moreover, several cell-permeant caspase inhibitors were incapable of significantly decreasing survival or inhibiting membrane repair. Taken together with previous studies, the results show that m- or mu-calpain can facilitate repair of damaged plasma membrane. While there was no evidence for the involvement of calpain-3, the proteasome or caspases in early events of plasma membrane repair, our studies do not rule out their participation in downstream events that may link plasma membrane repair to adaptive remodeling after injury.

Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle.

Mutations in the non-lysosomal cysteine protease calpain-3 cause autosomal recessive limb girdle muscular dystrophy. Pathological mechanisms occurring in this disease have not yet been elucidated. Here, we report both morphological and biochemical evidence of mitochondrial abnormalities in calpain-3 knockout (C3KO) muscles, including irregular ultrastructure and distribution of mitochondria. The morphological abnormalities in C3KO muscles are associated with reduced in vivo mitochondrial ATP production as measured by (31)P magnetic resonance spectroscopy. Mitochondrial abnormalities in C3KO muscles also correlate with the presence of oxidative stress; increased protein modification by oxygen free radicals and an elevated concentration of the anti-oxidative enzyme Mn-superoxide dismutase were observed in C3KO muscles. Previously we identified a number of mitochondrial proteins involved in beta-oxidation of fatty acids as potential substrates for calpain-3. In order to determine if the mitochondrial abnormalities resulted from the loss of direct regulation of mitochondrial proteins by calpain-3, we validated the potential substrates that were identified in previous proteomic studies. This analysis showed that the beta-oxidation enzyme, VLCAD, is cleaved by calpain-3 in vitro, but we were not able to confirm that VLCAD is an in vivo substrate for calpain-3. However, the activity of VLCAD was decreased in C3KO mitochondrial fractions compared with wild type, a finding that likely reflects a general mitochondrial dysfunction. Taken together, these data suggest that mitochondrial abnormalities leading to oxidative stress and energy deficit are important pathological features of calpainopathy and possibly represent secondary effects of the absence of calpain-3.

Osteopontin promotes fibrosis in dystrophic mouse muscle by modulating immune cell subsets and intramuscular TGF-beta.

Duchenne muscular dystrophy (DMD) is an X-linked, degenerative muscle disease that is exacerbated by secondary inflammation. Here, we characterized the immunological milieu of dystrophic muscle in mdx mice, a model of DMD, to identify potential therapeutic targets. We identified a specific subpopulation of cells expressing the Vbeta8.1/8.2 TCR that is predominant among TCR-beta+ T cells. These cells expressed high levels of osteopontin (OPN), a cytokine that promotes immune cell migration and survival. Elevated OPN levels correlated with the dystrophic process, since OPN was substantially elevated in the serum of mdx mice and muscle biopsies after disease onset. Muscle biopsies from individuals with DMD also had elevated OPN levels. To test the role of OPN in mdx muscle, mice lacking both OPN and dystrophin were generated and termed double-mutant mice (DMM mice). Reduced infiltration of NKT-like cells and neutrophils was observed in the muscle of DMM mice, supporting an immunomodulatory role for OPN in mdx muscle. Concomitantly, an increase in CD4+ and FoxP3+ Tregs was also observed in DMM muscle, which also showed reduced levels of TGF-beta, a known fibrosis mediator. These inflammatory changes correlated with increased strength and reduced diaphragm and cardiac fibrosis. These studies suggest that OPN may be a promising therapeutic target for reducing inflammation and fibrosis in individuals with DMD.

Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle.

Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation-contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle.

Molecular and cellular basis of calpainopathy (limb girdle muscular dystrophy type 2A).

Limb girdle muscular dystrophy type 2A results from mutations in the gene encoding the calpain 3 protease. Mutations in this disease are inherited in an autosomal recessive fashion and result in progressive proximal skeletal muscle wasting but no cardiac abnormalities. Calpain 3 has been shown to proteolytically cleave a wide variety of cytoskeletal and myofibrillar proteins and to act upstream of the ubiquitin-proteasome pathway. In this review, we summarize the known biochemical and physiological features of calpain 3 and hypothesize why mutations result in disease.

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice.

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.

Regulation of the M-cadherin-beta-catenin complex by calpain 3 during terminal stages of myogenic differentiation.

The cysteine protease calpain 3 (CAPN3) is essential for normal muscle function, since mutations in CAPN3 cause limb girdle muscular dystrophy type 2A. Previously, we showed that myoblasts isolated from CAPN3 knockout (C3KO) mice were able to fuse to myotubes; however, sarcomere formation was disrupted. In this study we further characterized morphological and biochemical features of C3KO myotubes in order to elucidate a role for CAPN3 during myogenesis. We showed that cell cycle withdrawal occurred normally in C3KO cultures, but C3KO myotubes have an increased number of myonuclei per myotube. We found that CAPN3 acts during myogenesis to specifically control levels of membrane-associated but not cytoplasmic beta-catenin and M-cadherin. CAPN3 was able to cleave both proteins, and in the absence of CAPN3, M-cadherin and beta-catenin abnormally accumulated at the membranes of myotubes. Given the role of M-cadherin in myoblast fusion, this finding suggests that the excessive myonuclear index of C3KO myotubes was due to enhanced fusion. Postfusion events, such as beta1D integrin expression and myofibrillogenesis, were suppressed in C3KO myotubes. These data suggest that the persistence of fusion observed in C3KO cells inhibits subsequent steps of differentiation, such as integrin complex rearrangements and sarcomere assembly.

Trim32 is a ubiquitin ligase mutated in limb girdle muscular dystrophy type 2H that binds to skeletal muscle myosin and ubiquitinates actin.

Trim32 belongs to the tripartite motif (TRIM) protein family, which is characterized by a common domain structure composed of a RING-finger, a B-box, and a coiled-coil motif. In addition to these motifs, Trim32 possesses six C-terminal NHL-domains. A point mutation in one NHL domain (D487N) has been linked to two forms of muscular dystrophy called limb girdle muscular dystrophy type 2H and sarcotubular myopathy. In the present study we demonstrate that Trim32 is an E3 ubiquitin ligase that acts in conjunction with ubiquitin-conjugating enzymes UbcH5a, UbcH5c, and UbcH6. Western blot analysis showed that Trim32 is expressed primarily in skeletal muscle, and revealed its differential expression from one muscle to another. The level of Trim32 expression was elevated significantly in muscle undergoing remodeling due to changes in weight bearing. Furthermore, expression of Trim32 was induced in myogenic differentiation. Thus, variability in Trim32 expression in different skeletal muscles could be due to induction of Trim32 expression upon changes in physiological conditions. We show that Trim32 associates with skeletal muscle thick filaments, interacting directly with the head and neck region of myosin. Our data indicate that myosin is not a substrate of Trim32; however, Trim32 was found to ubiquitinate actin in vitro and to cause a decrease in the level of endogenous actin when transfected into HEK293 cells. In conclusion, our results demonstrate that Trim32 is a ubiquitin ligase that is expressed in skeletal muscle, can be induced upon muscle unloading and reloading, associates with myofibrils and is able to ubiquitinate actin, suggesting its likely participation in myofibrillar protein turnover, especially during muscle adaptation.