Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin-proteasome pathway

Mutations in the non-lysosomal cysteine protease calpain 3 cause limb-girdle muscular dystrophy type 2A (LGMD2A). Our previous studies of the calpain 3 knockout mouse (C3KO) suggested a role for calpain 3 in sarcomere formation and remodeling. Calpain 3 may mediate remodeling by cleavage and release of myofibrillar proteins, targeting them for ubiquitination and proteasomal degradation. Loss of proper protein turnover may be the basis for this muscle disease. To test this hypothesis in vivo, we used an experimental model of hindlimb unloading and reloading that has been shown to induce sarcomere remodeling. We showed that the rate of atrophy and especially the rate of growth are decreased in C3KO muscles under conditions promoting sarcomere remodeling. In wild-type mice, an elevated level of ubiquitinated proteins was observed during muscle reloading, which is presumably necessary to remove atrophy-specific and damaged proteins. This increase in ubiquitination correlated with an increase in calpain 3 expression. C3KO muscles did not show any increase in ubiquitination at the reloading stage, suggesting that calpain 3 is necessary for ubiquitination and that it acts upstream of the ubiquitination machinery. We found upregulation of heat shock proteins in C3KO muscles following challenge with a physiological condition that requires highly increased protein degradation. Furthermore, old C3KO mice show evidence of insoluble protein aggregate formation in skeletal muscles. These studies suggest that accumulation of aged and damaged proteins can lead to cellular toxicity and a cell stress response in C3KO muscles, and that these characteristics are pathological features of LGMD2A.     

Mdm muscular dystrophy: interactions with calpain 3 and a novel functional role for titin's N2A domain

Human tibial muscular dystrophy and limb-girdle muscular dystrophy 2J are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2ADelta83), disrupting a putative binding site for CAPN3. To determine whether the muscular dystrophy in mutant mdm mice is caused by misregulation of CAPN3 activity, genetic crosses with CAPN3 overexpressing transgenic (C3Tg) and CAPN3 knockout (C3KO) mice were generated. Here, we report that overexpression of CAPN3 exacerbates the mdm disease, leading to a shorter life span and more severe muscular dystrophy. However, in a direct genetic test of CAPN3's role as a mediator of mdm pathology, C3KO;mdm double mutant mice showed no change in the progression or severity of disease indicating that aberrant CAPN3 activity is not a primary mechanism in this disease. To determine whether we could detect a functional deficit in titin in a non-disease state, we examined the treadmill locomotion of heterozygous +/mdm mice and detected a significant increase in stride time with a concomitant increase in stance time. Interestingly, these altered gait parameters were completely corrected by CAPN3 overexpression in transgenic C3Tg;+/mdm mice, supporting a CAPN3-dependent role for the N2A domain of TTN in the dynamics of muscle contraction.     

A Titan but not Necessarily a Ruler: Assessing the Role of Titin During Thick Filament Patterning and Assembly

The sarcomeres of striated muscle are among the most elaborate and dynamic eukaryotic cellular protein machinery, and the mechanisms by which these semicrystalline filament networks are initially patterned and assembled remain contentious. In addition to the acto‐myosin filaments that provide motor function, the sarcomere contains titin filaments, comprised of individual molecules of the giant Ig‐ and fibronectin domain‐rich protein titin. Titin is the largest known protein, containing many structurally distinct domains with a variety of proposed functions, including sarcomere stabilization, the prevention of over‐stretching, and returning to resting length after contraction. One molecule of titin, which binds to both the Z‐disk and the M‐line, spans a half‐sarcomere, and is proposed to serve as a “molecular ruler” that dictates the spacing of sarcomeres. The semirigid rod‐like A‐band region of titin has also been proposed to act as a scaffold for thick filament formation during muscle development, but despite decades of research, this hypothesis has not been rigorously tested. Recent studies in zebrafish have brought into question the necessity for the A‐band region of titin during the early stages of sarcomere patterning. In this review, we give an overview of the many different roles of titin in the development and function of striated muscle, and address the validity of the “molecular ruler” model of myofibrillogenesis in light of the current literature. Anat Rec, 297:1604–1614, 2014. © 2014 Wiley Periodicals, Inc.     

Titin/connectin-based modulation of the Frank-Starling mechanism of the heart

The basis of the Frank-Starling mechanism of the heart is the increase in active force when muscle is stretched. Various findings have shown that muscle length, i.e., sarcomere length (SL), modulates activation of cardiac myofilaments at a given concentration of Ca²⁺ ([Ca²⁺]). This augmented Ca²⁺ activation with SL, commonly known as “length-dependent activation”, is manifested as the leftward shift of the force-pCa (=−log [Ca²⁺]) relation as well as by the increase in maximal Ca²⁺-activated force. Despite the numerous studies that have been undertaken, the molecular mechanism(s) of length-dependent activation is (are) still not fully understood. The giant sarcomere protein titin/connectin is the largest protein known to date. Titin/connectin is responsible for most passive force in vertebrate striated muscle and also functions as a molecular scaffold during myofibrillogenesis. Recent studies suggest that titin/connectin plays an important role in length-dependent activation by sensing stretch and promoting actomyosin interaction. Here we review and extend this previous work and focus on the mechanism by which titin/connectin might modulate actomyosin interaction.     

A sandwich ELISA kit reveals marked elevation of titin N-terminal fragment levels in the urine of mdx mice

The mdx mouse is a model of Duchenne muscular dystrophy (DMD), a fatal progres-sive muscle wasting disease caused by dystrophin deficiency, and is used most widely in preclinical studies. Mice with dystrophin deficiency, however, show milder muscle strength phenotypes than humans. In human, the introduction of a sandwich enzyme- linked immunosorbent assay (ELISA) kit revealed a more than 700- fold increase in titin N- terminal fragment levels in the urine of pediatric patients with DMD. Notably, the urinary titin level declines with aging, reflecting progression of muscle wast-ing. In mouse, development of a highly sensitive ELISA kit has been awaited. Here, a sandwich ELISA kit to measure titin N- terminal fragment levels in mouse urine was developed. The developed kit showed good linearity, recovery, and repeatability in measuring recombinant or natural mouse titin N- terminal fragment levels. The titin N- terminal fragment concentration in the urine of mdx mice was more than 500- fold higher than that of normal mice. Urinary titin was further analyzed by extending the collection of urine samples to both young (3– 11 weeks old) and aged (56– 58 weeks old) mdx mice. The concentration in the young group was significantly higher than that in the aged group. It was concluded that muscle protein breakdown is active and persistent in mdx mice even though the muscle phenotype is mild. Our results pro-vide an opportunity to develop DMD treatments that aim to alleviate muscle protein breakdown by monitoring urinary titin levels.     

Muscle atrophy in Titin M-line deficient mice

We investigated the response to deletion of the titin M-line region in striated muscle, using a titin knockout model and a range of techniques that include histology, in situ hybridization, electron microscopy, and 2D gel analysis. We found that the loss of titin’s kinase domain and binding sites for myomesin and MURF-1 causes structural changes in the sarcomere that proceed from the M-line to the Z-disc and ultimately result in disassembly of the sarcomere. Disassembly goes along with central localization of nuclei (a hallmark for muscular dystrophy), up-regulation of heat-shock proteins, and induction of proteasome activity. While fiber type composition does not change in soleus and extensor digitorum longus muscle, fiber size is reduced. Animals die from complications of muscle atrophy at five weeks of age. In addition to the structural importance of the titin M-line region in any striated muscle, our data show how differences in M-line composition between heart and skeletal muscle affect sarcomere stability and function.These authors contributed equally to the study.     

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.     

Myotilin,the limb-girdle muscular dystrophy 1A (LGMD1A) protein,cross-links actin filaments and controls sarcomere assembly

The assembly and maintenance of the muscle sarcomere requires a complex interplay of actin- and myosin-associated proteins. Myotilin is a thin filament-associated Z-disc protein that consists of two Ig-domains flanked by a unique serine-rich amino-terminus and a short carboxy-terminal tail. It binds to alpha-actinin and filamin c and is mutated in limb girdle muscular dystrophy 1A (LGMD1A). Here we show that myotilin also directly binds F-actin, efficiently cross-links actin filaments alone or in concert with alpha-actinin and prevents filament disassembly induced by Latrunculin A. Myotilin forms dimers via its carboxy-terminal half, which may be necessary for the actin-bundling activity. Overexpression of full-length myotilin but not the carboxy-terminal half induces formation of thick actin cables in non-muscle cells devoid of endogenous myotilin. The expression of myotilin in muscle cells is tightly regulated to the later stages of in vitro myofibrillogenesis, when preassembled myofibrils begin to align. Expression of either amino- or carboxy-terminally truncated myotilin fragments but not wild-type myotilin in differentiating myocytes leads to myofibril disarray. The disease association and functional characteristics indicate an indispensable role for myotilin in stabilization and anchorage of thin filaments, which may be a prerequisite for correct Z-disc organization.     

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.     

Calpain 3 is a modulator of the dysferlin protein complex in skeletal muscle

Muscular dystrophies comprise a genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and weakness. Two forms of limb-girdle muscular dystrophy, 2A and 2B, are caused by mutations in calpain 3 (CAPN3) and dysferlin (DYSF), respectively. While CAPN3 may be involved in sarcomere remodeling, DYSF is proposed to play a role in membrane repair. The coexistence of CAPN3 and AHNAK, a protein involved in subsarcolemmal cytoarchitecture and membrane repair, in the dysferlin protein complex and the presence of proteolytic cleavage fragments of AHNAK in skeletal muscle led us to investigate whether AHNAK can act as substrate for CAPN3. We here demonstrate that AHNAK is cleaved by CAPN3 and show that AHNAK is lost in cells expressing active CAPN3. Conversely, AHNAK accumulates when calpain 3 is defective in skeletal muscle of calpainopathy patients. Moreover, we demonstrate that AHNAK fragments cleaved by CAPN3 have lost their affinity for dysferlin. Thus, our findings suggest interconnectivity between both diseases by revealing a novel physiological role for CAPN3 in regulating the dysferlin protein complex.     

Distinct roles for telethonin N‐versus C‐terminus in sarcomere assembly and maintenance

The N‐terminus of telethonin forms a unique structure linking two titin N‐termini at the Z‐disc. While a specific role for the C‐terminus has not been established, several studies indicate it may have a regulatory function. Using a morpholino approach in Xenopus, we show that telethonin knockdown leads to embryonic paralysis, myocyte defects, and sarcomeric disruption. These myopathic defects can be rescued by expressing full‐length telethonin mRNA in morpholino background, indicating that telethonin is required for myofibrillogenesis. However, a construct missing C‐terminal residues is incapable of rescuing motility or sarcomere assembly in cultured myocytes. We, therefore, tested two additional constructs: one where four C‐terminal phosphorylatable residues were mutated to alanines and another where terminal residues were randomly replaced. Data from these experiments support that the telethonin C‐terminus is required for assembly, but in a context‐dependent manner, indicating that factors and forces present in vivo can compensate for C‐terminal truncation or mutation. Developmental Dynamics 239:1124–1135, 2010. © 2010 Wiley‐Liss, Inc.     

Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.     

Hearing loss in the laminin-deficient dy mouse model of congenital muscular dystrophy

Sensorineural hearing loss is found in many inherited forms of muscular dystrophy. We investigated the dy mouse model, which has congenital muscular dystrophy due to a defect in laminin alpha 2, for evidence of cochlear dysfunction. Auditory brainstem response (ABR) audiometry to pure tones was used to evaluate 3-month-old homozygous dy/dy and age-matched C57 control mice. The average ABR thresholds to tone-burst stimuli for four frequencies (4, 8, 16, and 32 kHz) were determined and statistically compared by ANOVA. The dy/dy mice demonstrated elevated auditory thresholds ranging from 25 to 27 dB at each frequency tested (p<0.0001). Anatomic evaluations of the ears revealed pathology ranging from extensive connective tissue infiltration within the inner ear to possible minor defects in the cells of the organ of Corti. These anatomic and physiologic observations suggest that the extracellular matrix protein laminin plays a crucial role in normal cochlear function. Furthermore, the dy congenital muscular dystrophy mouse offers a novel model for evaluation of sensorineural hearing loss associated with muscular dystrophy.     

Physiological functions of the giant elastic protein titin in mammalian striated muscle

The striated muscle sarcomere contains the third filament comprising the giant elastic protein titin, in addition to thick and thin filaments. Titin is the primary source of nonactomyosin-based passive force in both skeletal and cardiac muscles, within the physiological sarcomere length range. Titin's force repositions the thick filaments in the center of the sarcomere after contraction or stretch and thus maintains sarcomere length and structural integrity. In the heart, titin determines myocardial wall stiffness, thereby regulating ventricular filling. Recent studies have revealed the mechanisms involved in the fine tuning of titin-based passive force via alternative splicing or posttranslational modification. It has also been discovered that titin performs roles that go beyond passive force generation, such as a regulation of the Frank-Starling mechanism of the heart. In this review, we discuss how titin regulates passive and active properties of striated muscle during normal muscle function and during disease.     

Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.

The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.     

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans,mice, and zebrafish

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.     

Mannose-binding lectin binds IgM to activate the lectin complement pathway in vitro and in vivo

Recent evidence has implicated a role for the MBL-dependent lectin pathway in gastrointestinal and myocardial ischemia/reperfusion (I/R)-induced injury. However, previous studies have implicated IgM and the classical pathway as initiators of complement activation following I/R. Thus, we investigated the potential interaction between MBL and IgM leading to complement activation. Using surface plasmon resonance, we demonstrate that MBL does bind human IgM. Subsequently, functional complement activation was demonstrated in vitro following sensitization of human RBCs with mouse anti-human CD59 IgM and more lysis was observed with MBL sufficient sera compared to MBL deficient (KO) sera. Similarly, treatment of human endothelial cells with mouse anti-human CD59 IgM, MBL and MASP-2 activated and deposited C4. These data suggest that the presence of both IgM and MBL can activate the lectin pathway in vitro. Serum ALT levels increased significantly in sIgM/MBL-A/C KO mice reconstituted with WT plasma compared to sIgM/MBL-A/C KO mice reconstituted with MBL-A/C KO plasma following gastrointestinal (G) I/R. Similarly, intestinal C3 deposition was greater in sIgM/MBL-A/C KO mice reconstituted with WT plasma compared to sIgM/MBL-A/C KO mice treated with MBL-A/C KO plasma. These data indicate for the first time that both IgM and MBL-A/C are required for GI/R-induced complement activation and subsequent injury.     

Transgenic overexpression of dystroglycan does not inhibit muscular dystrophy in mdx mice

Recently, there have been a number of studies demonstrating that overexpression of molecules in skeletal muscle can inhibit or ameliorate aspects of muscular dystrophy in the mdx mouse, a model for Duchenne muscular dystrophy. Several such studies involve molecules that increase the expression of dystroglycan, an important component of the dystrophin-glycoprotein complex. To test whether dystroglycan itself inhibits muscular dystrophy in mdx mice, we created dystroglycan transgenic mdx mice (DG/mdx). The alpha and beta chains of dystroglycan were highly overexpressed along the sarcolemmal membrane in most DG/mdx muscles. Increased dystroglycan expression, however, did not correlate with increased expression of utrophin or sarcoglycans, but rather caused their decreased expression. In addition, the percentage of centrally located myofiber nuclei and the level of serum creatine kinase activity were not decreased in DG/mdx mice relative to mdx animals. Therefore, dystroglycan overexpression does not cause the concomitant overexpression of a utrophin-glycoprotein complex in mdx muscles and has no effect on the development of muscle pathology associated with muscular dystrophy.