Girdle Muscular Dystrophy Type 2A Research Articles

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Quantitative Muscle MRI in Patients with Neuromuscular Diseases-Association of Muscle Proton Density Fat Fraction with Semi-Quantitative Grading of Fatty Infiltration and Muscle Strength at the Thigh Region.

(1) Background and Purpose: The skeletal muscles of patients suffering from neuromuscular diseases (NMD) are affected by atrophy, hypertrophy, fatty infiltration, and edematous changes. Magnetic resonance imaging (MRI) is an important tool for diagnosis and monitoring. Concerning fatty infiltration, T1-weighted or T2-weighted DIXON turbo spin echo (TSE) sequences enable a qualitative assessment of muscle involvement. To achieve higher comparability, semi-quantitative grading scales, such as the four-point Mercuri scale, are commonly applied. However, the evaluation remains investigator-dependent. Therefore, effort is being invested to develop quantitative MRI techniques for determination of imaging markers such as the proton density fat fraction (PDFF). The present work aims to assess the diagnostic value of PDFF in correlation to Mercuri grading and clinically determined muscle strength in patients with myotonic dystrophy type 2 (DM2), limb girdle muscular dystrophy type 2A (LGMD2A), and adult Pompe disease. (2) Methods: T2-weighted two-dimensional (2D) DIXON TSE and chemical shift encoding-based water-fat MRI were acquired in 13 patients (DM2: n = 5; LGMD2A: n = 5; Pompe disease: n = 3). Nine different thigh muscles were rated in all patients according to the Mercuri grading and segmented to extract PDFF values. Muscle strength was assessed according to the British Medical Research Council (BMRC) scale. For correlation analyses between Mercuri grading, muscle strength, and PDFF, the Spearman correlation coefficient (rs) was computed. (3) Results: Mean PDFF values ranged from 7% to 37% in adults with Pompe disease and DM2 and up to 79% in LGMD2A patients. In all three groups, a strong correlation of the Mercuri grading and PDFF values was observed for almost all muscles (rs > 0.70, p < 0.05). PDFF values correlated significantly to muscle strength for muscle groups responsible for knee flexion (rs = −0.80, p < 0.01). (4) Conclusion: In the small, investigated patient cohort, PDFF offers similar diagnostic precision as the clinically established Mercuri grading. Based on these preliminary data, PDFF could be further considered as an MRI-based biomarker in the assessment of fatty infiltration of muscle tissue in NMD. Further studies with larger patient cohorts are needed to advance PDFF as an MRI-based biomarker in NMD, with advantages such as its greater dynamic range, enabling the assessment of subtler changes, the amplified objectivity, and the potential of direct correlation to muscle function for selected muscles.

Over three decades of natural history of limb girdle muscular dystrophy type R1/2A and R2/2B: Mathematical modelling of a multifactorial study

We aimed to describe the natural history of Limb Girdle Muscular Dystrophy type 2A and 2B over more than three decades by considering muscular strength, motor, cardiac and respiratory function. 428 visits of nineteen 2A and twenty 2B patients were retrospectively analysed through a regression model to create the curves of evolution with disease duration of muscle strength (through Medical Research Council grading), motor function measure scale (D1, D2 and D3 domains) and cardio-pulmonary function tests. Clinically relevant muscular and motor function alterations occurred after the first decade of disease, while mild respiratory function alterations started after the second, with preserved cardiac function. Although type 2A showed relatively stronger distal lower limb muscles, while type 2B started with relatively stronger upper limb muscles, the corresponding motor functions were similar, becoming severely compromised after 25 years of disease. This was the longest retrospective study in types 2A and 2B. It defined curves of disease evolution not only from a neuromuscular, but also from functional, cardiac, and respiratory points of view, to be used to evaluate how the natural progression is changed by therapies. Due to slow disease progression, it was not possible to identify time sensitive endpoints.

REGISTRIES, CARE, QUALITY OF LIFE, MANAGEMENT OF NMD: P.340 Coalition to cure calpain 3: A patient organization committed to treating and ultimately curing limb girdle muscular dystrophy type 2A

Coalition to cure calpain 3 (C3) was founded in 2010 with a mission to fund research and clinical trials relevant to limb girdle muscular dystrophy type 2A (LGMD2A, also called LGMD R1 calpain 3-related), a form of calpainopathy, to support a network of families affected by this disease, and to raise awareness of LGMD2A in the global community. We are governed by a Board of Directors including two LGMD2A patients, a Scientific Advisory Board composed of academic scientists, and a Scientific Director. C3 provides support for promising research focused on understanding pathomechanisms of or finding treatments for LGMD2A. LGMD2A is characterized by proximal muscle weakness. Patients are usually diagnosed before their second decade and experience progressive skeletal muscle wasting in all muscles leading to loss of ambulation approximately 10 years after diagnosis. LGMD2A is caused by mutations in CAPN3, the gene which encodes the proteolytic enzyme calpain 3. Despite being the most common subtype of limb girdle muscular dystrophy, the pathophysiology of LGMD2A is still not well understood. Furthermore, the biological function of the calpain 3 enzyme is not entirely clear. C3 has funded research on the basic biology of calpain 3, has supported preclinical studies in LGMD2A models, and has sponsored the generation of tools for use in studying LGMD2A. C3 has also organized four workshops (in the US in 2011, 2018, and 2019, and in the Netherlands in 2013) that have been instrumental in bringing together experts in relevant fields to discuss basic science and clinical information pertaining to LGMD2A. A travel grant program, aimed at early career researchers, provides travel support for investigators to present on LGMD2A at major scientific conferences. Additionally, C3 has created the first global patient registry for LGMD2A. Our current priorities are to fund research efforts and support collaborations focused on finding a treatment for LGMD2A.

A muscle-specific calpain, CAPN3, forms a homotrimer

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta–EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.

P.394Coalition to cure calpain 3: a patient organization committed to treating and ultimately curing limb girdle muscular dystrophy type 2A

Coalition to cure calpain 3 (C3) was founded in 2010 with a mission to fund research and clinical trials relevant to limb girdle muscular dystrophy type 2A (LGMD2A, also called LGMDR1), a form of calpainopathy. The mission of C3 is to support a network of affected families, to raise awareness of LGMD2A in the global community and to support promising research focused on understanding pathomechanisms of or finding treatments for LGMD2A. We are governed by a Board of Directors including two LGMD2A patients, a Scientific Advisory Board composed of academic scientists, and a Scientific Director. Patients are usually diagnosed before their second decade. LGMD2A is initially characterized by proximal muscle weakness that progressively spreads to all muscles, leading to loss of ambulation approximately 10 years after diagnosis. LGMD2A is caused by mutations in CAPN3, the gene which encodes the proteolytic enzyme calpain 3. Despite being the most common subtype of limb girdle muscular dystrophy, the pathophysiology of LGMD2A is still not well understood. Furthermore, the biological function of the calpain 3 enzyme is not entirely clear. C3 has funded research on the basic biology of calpain 3, has supported preclinical studies in LGMD2A models, and has sponsored the generation of tools for use in studying LGMD2A. Additionally, C3 has created the first global patient registry for LGMD2A and is working in coordination with peer organizations to ensure that we reach a broad audience. C3 has also organized three workshops (in the US in 2011 and 2018, and in the Netherlands in 2013) that have been instrumental in bringing together experts in relevant fields to discuss basic science and clinical information pertaining to LGMD2A. A travel grant program, aimed at early career researchers, provides travel support for investigators to present on LGMD2A at major scientific conferences. Our current priorities are to fund research efforts and support collaborations focused on finding a treatment for LGMD2A.

Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy.

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives invitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

A Case of Limb Girdle Muscular Dystrophy Type 2A from India: Copy Number Variation Analysis using Targeted Amplicon Sequencing

A Case of Limb Girdle Muscular Dystrophy Type 2A from India: Copy Number Variation Analysis using Targeted Amplicon Sequencing

LGMD AUTOSOMAL RESSESSIVE AND DOMINANT: P.104Coalition to Cure Calpain 3: a patient organization committed to treating and ultimately curing limb girdle muscular dystrophy type 2A

Coalition to Cure Calpain 3 (C3) was founded in 2010 with a mission to fund research and clinical trials relevant to limb girdle muscular dystrophy type2A/calpainopathy (LGMD2A), to support a network of families affected by this disease, and to raise awareness of LGMD2A in the global community. We are governed by a Board of Directors including two LGMD2A patients, a Scientific Advisory Board composed of academic scientists, and a Scientific Director. C3 provides support for promising research focused on understanding pathomechanisms of or finding treatments for LGMD2A. LGMD2A is characterized by proximal muscle weakness. Patients are usually diagnosed before their second decade and experience progressive skeletal muscle wasting in all muscles leading to loss of ambulation approximately 10 years after diagnosis. LGMD2A is caused by mutations in CPN3, the gene which encodes the proteolytic enzyme calpain 3. Despite being the most common subtype of limb girdle muscular dystrophy, the pathophysiology of LGMD2A is still not well understood. Furthermore, the biological function of the calpain 3 enzyme is not entirely clear. C3 has funded research on the basic biology of calpain 3, has supported preclinical studies in LGMD2A models, and has sponsored the generation of tools for use in studying LGMD2A. C3 has also organized two workshops (in the US in 2011 and in the Netherlands in 2013) that have been instrumental in bringing together experts in relevant fields to discuss basic science and clinical information pertaining to LGMD2A, and has a third planned for October 2018. A travel grant program, aimed at early career researchers, provides travel support for investigators to present on LGMD2A at major scientific conferences. Additionally, C3 has created the first global patient registry for LGMD2A. Our current priorities are to fund research efforts and support collaborations focused on finding a treatment for LGMD2A.

Coincidence of hereditary motor and sensory neuropathy type 1A and limb lumbo-muscular dystrophy type 2A

A rare case of two neuromuscular disorders in a 29-year-old female is presented: autosomal dominant hereditary motor and sensory neuropathy type 1A (HMSN1A) due to PMP22 duplication and autosomal recessive limb girdle muscular dystrophy type 2A (LGMD2A) produced by CAPN3 common mutation c.550delA and novel c.575C>G (p.Thr192Ser).Walking difficulties appeared in 27 years, the patient had signs of both disorders, more of LGMD, but was not disabled. HMSN1A was inherited from her father whose disease was not recognized earlier.

P.2 - Coalition to cure calpain 3: A patient organization committed to treating and ultimately curing limb girdle muscular dystrophy type 2A

P.2 - Coalition to cure calpain 3: A patient organization committed to treating and ultimately curing limb girdle muscular dystrophy type 2A

A heterozygous 21-bp deletion in CAPN3 causes dominantly inherited limb girdle muscular dystrophy.

Limb girdle muscular dystrophy type 2A is the most common limb girdle muscular dystrophy form worldwide. Although strict recessive inheritance is assumed, patients carrying a single mutation in the calpain 3 gene (CAPN3) are reported. Such findings are commonly attributed to incomplete mutation screening. In this investigation, we report 37 individuals (age range: 21-85 years, 21 females and 16 males) from 10 families in whom only one mutation in CAPN3 could be identified; a 21-bp, in-frame deletion (c.643_663del21). This mutation co-segregated with evidence of muscle disease and autosomal dominant transmission in several generations. Evidence of muscle disease was indicated by muscle pain, muscle weakness and wasting, significant fat replacement of muscles on imaging, myopathic changes on muscle biopsy and loss of calpain 3 protein on western blotting. Thirty-one of 34 patients had elevated creatine kinase or myoglobin. Muscle weakness was generally milder than observed in limb girdle muscular dystrophy type 2A, but affected the same muscle groups (proximal leg, lumbar paraspinal and medial gastrocnemius muscles). In some cases, the weakness was severely disabling. The 21-bp deletion did not affect mRNA maturation. Calpain 3 expression in muscle, assessed by western blot, was below 15% of normal levels in the nine mutation carriers in whom this could be tested. Haplotype analysis in four families from three different countries suggests that the 21-bp deletion is a founder mutation. This study provides strong evidence that heterozygosity for the c.643_663del21 deletion in CAPN3 results in a dominantly inherited muscle disease. The normal expression of mutated mRNA and the severe loss of calpain 3 on western blotting, suggest a dominant negative effect with a loss-of-function mechanism affecting the calpain 3 homodimer. This renders patients deficient in calpain 3 as in limb girdle muscular dystrophy type 2A, albeit in a milder form in most cases. Based on findings in 10 families, our study indicates that a dominantly inherited pattern of calpainopathy exists, and should be considered in the diagnostic work-up and genetic counselling of patients with calpainopathy and single-allele aberrations in CAPN3.

312. Gene Correction of LGMD2A Patient-Specific iPS Cells for Targeted Autologous Cell Therapy

Limb girdle muscular dystrophy type 2A (LGMD2A), an incurable autosomal recessive disorder and the most common form of LGMD, is characterized by symmetric and progressive weakness of limb-girdle muscles leading to loss of ambulation. LGMD2A is caused by mutations in the Calpain 3 (CAPN3) gene which encodes a muscle-specific intracellular calcium-sensitive cysteine protease, hypothesized to act as a gate keeper for proper assembly and maintenance of the sarcomere. A cell therapy based on induced pluripotent stem (iPS) cells has the potential for treating muscular dystrophy. We have demonstrated that transplantation of skeletal muscle progenitors obtained from disease-free human iPS cells can restore muscle contractility in a mouse model for Duchenne Muscular Dystrophy. The goal of this study is to apply gene editing tools to correct the CAPN3 mutation in LGMD2A iPS cells, and to determine the feasibility of an iPS-cell based autologous cell therapy for LGMD2A. Here we investigated two approaches to genetically correct a LGMD2A patient-specific iPS cell line with homozygous deletion of exon 17 to 24 in the CAPN3 gene. The first approach consisted of knocking in the coding region of CAPN3 cDNA with SV40 polyadenylation signal sequence downstream in exon 1 of the endogenous CAPN3 gene by CRISPR-CAS9 induced double stranded break (DSB) mediated homology directed DNA repair from exogenous donor template, allowing for universal correction of all types of CAPN3 mutations. PCR, sequencing and Southern blot analysis on iPS colonies post- genome editing confirmed the integration of the cDNA in exon 1 of the CAPN3 gene. The second approach involved inserting the missing exons 17-24 with SV40 polyadenylation signal sequence downstream contiguous with the exon 16 of the endogenous CAPN3 gene. PCR and sequencing analysis on iPS colonies post-genome editing confirmed proper integration in this approach as well. LGMD2A iPS cell clones, genetically corrected using these two gene editing methods will be differentiated into skeletal muscle progenitors using conditional expression of Pax7, as previously described by our group, and evaluated in vitro and in vivo for restoration of full length CAPN3. For the latter, we have generated an immunodeficient LGMD2A mouse model by combining the CAPN3-KO mouse model with NOD/SCID and IL2Rg mutations. This mouse is ideal for studies on the transplantation of human cells. In summary, we have corrected the CAPN3 mutation in LGMD2A iPS cells for the first time, and ongoing transplantation studies with corrected iPS cells will provide proof-of-principal for a potential new cell therapy that can permanently restore CAPN3 function in all LGMD2A patients.

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

BackgroundMutations 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 Ca2+ 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 Ca2+ 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 Ca2+ fluxes. Muscles were subjected to both current and voltage clamp conditions.MethodsStandard and confocal fluorescence microscopy was used to study Ca2+ 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.ResultsWe found that the peak value of Ca2+ fluxes elicited by single action potentials was significantly reduced by 15–20 % in C3KO fibers, but the kinetics was unaltered. Ca2+ release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca2+ release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca2+ release in C3KO mice but reduced peak Ca2+ 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 Ca2+ release at all triads consistent with a homogenous reduction of functional voltage activated Ca2+ release sites.ConclusionsOverall, these results suggest that decreased Ca2+ release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

Protein and genetic diagnosis of limb girdle muscular dystrophy type 2A: The yield and the pitfalls

Limb girdle muscular dystrophy type 2A (LGMD2A) is the most frequent form of LGMD worldwide. Comprehensive clinical assessment and laboratory testing is essential for diagnosis of LGMD2A. Muscle immunoblot analysis of calpain-3 is the most useful tool to direct genetic testing, as detection of calpain-3 deficiency has high diagnostic value. However, calpain-3 immunoblot testing lacks sensitivity in about 30% of cases due to gene mutations that inactivate the enzyme. The best diagnostic strategy should be determined on a case-by-case basis, depending on which tissues are available, and which molecular and/or genetic methods are adopted. In this work we survey the current knowledge, advantages, limitations, and pitfalls of protein testing and mutation detection in LGMD2A and provide an update of genetic epidemiology.

G.P.229: The relationship of calpain 3 and titin in the M-band

Several published studies strongly implied the existence of functional relationship between calpain 3 and titin proteins. Calpain 3, the cysteine protease mutated in Limb Girdle Muscular Dystrophy type 2A (LGMD2A), has at least 2 different binding sites on titin, which is also one of calpain 3 substrates. Moreover, Tibial Muscular Dystrophy (TMD) and LGMD2J, both caused by the mutations in the last exons of the titin gene, are characterised by secondary reduction of calpain 3 expression. We also showed that the last domains of titin are not present in the TMD/LGMD2J muscle. Here, we tested several calpain 3 properties in vivo and in vitro conditions to better clarify the relationship between calpain 3 and titin in the M-band of the sarcomere, as well as its possible implication in the pathogenesis of LGMD2A and LGMD2J. An in vitro analysis validated the C-terminal part of the titin protein as a proteolytic substrate for calpain 3. Moreover, we demonstrated that TMD/LGMD2J mutations induce disruption of these cleavages in vitro while calpain 3 is still able to interact with titin. Interestingly and unexpectedly, we showed that the disappearance of the M-band titin seen in vivo as a consequence of TMD/LGMD2J mutations is still observed in the absence of calpain 3, indicating that this event is not caused by a calpain 3 proteolytic cleavage.

A Cardiomyopathy in a Patient With Limb Girdle Muscular Dystrophy Type 2A

Limb girdle muscular dystrophy (LGMD) is a muscular dystrophy with predominantly proximal distribution of weakness that spares the distal, facial, and extraocular muscles early in the course of the disease. Cardiac muscle may be affected, which may manifest as hypertrophic/dilated cardiomyopathy and cardiac dysrhythmias. Significant cardiac involvement has been documented frequently in LGMD2C-F, 2I, and LGMD1B forms of the disease, rarely in the LGMD1C and 2B subtypes, and thus far have not been reported in type 2A. We report a presentation of severe cardiomyopathy in an adult with muscular dystrophy type 2A. A 23-year-old black man was seen in hospital with complaints of decreased exercise tolerance, dyspnea on exertion, orthopnea, and some presycopal episodes for several weeks. The patient’s LGMD was diagnosed at the age of 21 years. No family history of similar problems was noted. Physical examination at the time revealed decreased …

Selective Pseudohypertrophy of Vastus Medialis Muscles Associated With Calpain 3 Deficiency

Calpain 3 deficiency causes limb girdle muscular dystrophy type 2A, which is one of the most common forms of limb girdle muscular dystrophy. Nevertheless, calpainopathy is not always associated with mutations in the specific gene and secondary reduction in protein expression has been described. We report a case of a 43-year-old man who complained of thigh muscle stiffness and had muscle hypertrophy of both vastus medialis with prolonged myotonic contraction by percussion. A muscle biopsy showed dystrophic features and calpain 3 deficiency was shown by immunoblot analysis although mutations in the specific gene were not found. Known cases of secondary calpain 3 protein deficiency were ruled out and mutations in MD1 and MD2 genes were excluded. This patient represents the first case of calpain 3 deficiency with selective pseudohypertrophy of vastus medialis muscles.

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.