Calpain 3 Is Important for Muscle Regeneration
Calpain 3 Is Important for Muscle Regeneration
Limb Girdle Muscular Dystrophy type 2A (LGMD2A) is an autosomal recessively inherited disorder characterized by symmetrical progressive proximal muscle weakness and atrophy that usually starts in the pelvic girdle musculature. However, the clinical course is characterized by great variability, ranging from severe forms with onset in the first decade and rapid progression to milder forms with later onset and slower course. Serum creatine kinase is usually highly elevated and the muscle morphology shows dystrophic features. The disorder is caused by a loss of functional calpain 3, a skeletal muscle specific isoform of the Ca-dependent calpain cysteine protease family. More than 460 pathogenic mutations, covering almost the entire length of the calpain 3 gene (CAPN3), have been discovered (Leiden Muscular Dystrophy Pages, http://www.dmd.nl). Recent research involving calpain 3 protease-inactive knock-in mice have demonstrated that calpain 3 appears to play a role in the Ca-efflux from the sarcoplasmatic reticulum in a way that does not involve the protease function of calpain 3, this may explain why dysfunctional calpain 3 leads to muscle weakness. However, the potential substrates of calpain 3 have been a major focus of LGMD2A research in the past few years, as it appears that calpain 3 may be involved in multiple aspects of muscle function and maintenance. Calpain 3 is anchored to the giant structural/scaffold protein titin in a stable and inactive manner, to keep it from degrading itself autolytically. For that very same reason, the substrates of calpain 3 is believed to be in close proximity, possibly bound to other parts of the sarcomeres. It is thought that calpain 3 is inactive most of the time, only to be activated and redistributed when sarcomeres are exercised beyond a threshold, leading to interaction with a number of proteins e.g. myosin light chain 1, suggesting a role for calpain 3 in sarcomere remodeling. Another important in vivo calpain 3 substrates that has been described is AHNAK, a very large protein involved in subsarcolemmal cytostructure and part of the dysferlin membrane repair complex, requires calpain 3 for it to be cleaved and the membrane repair to proceed. Hence, it is a key component in the repair of the wear and tear of skeletal muscle tissue. The search for calpain 3 substrates using cleavage site recognition have lead to a number of potential targets, one being Protein Inhibitor of Activated Stats 3 (PIAS3), an ubiquitously expressed E3 SUMO ligase implicated in many signaling pathways by modifying the localization and role in transcriptional regulation of transcription factors.
A recent study using a protease-inactive calpain 3 knock-in model, demonstrated that calpain 3 lacking protease functionality lead to muscular dystrophy, exacerbated by exercise. This group proposed that the protease activity of calpain 3 is required to protect muscle from degeneration under exercise-induced stress, and that loss of protease activity affected the dynamic distribution of calpain 3 during physical activity and its interaction with MARP2, a stress-response transcriptional regulator protein, in close proximity to calpain 3 on the N2A region of titin. MARP2 is upregulated during exercise in normal muscle, however, the protease-inactivity of calpain 3 resulted in decreased levels of MARP2 and abnormal levels of dysferlin in exercised calpain 3 knock-in mice, leading to the conclusion that the muscle membrane repair mechanism is greatly affected by the loss of calpain 3 protease activity even if the activation of satellite cells is not affected. Several studies have shown that calpain 3 deficiency leads to formation of abnormal sarcomeres, impairment of muscle contractile capacity and loss of the muscle fibers.
In order to balance the ongoing degeneration, muscle fibers must regenerate, but in patients with LGMD2A it is unknown how calpain 3 deficiency affects the regenerative response. We therefore investigated the level of regeneration in skeletal muscle of 22 patients with genetically confirmed LGMD2A by assessing commonly used markers of muscle regeneration. Internally nucleated fibers (INF) arise from muscle specific stem cells, satellite cells. These cells are activated during muscle degeneration and constitute the majority of regenerative response to muscle wasting. Activation of satellite cells ultimately leads to migration of nuclei to the damaged area of the muscle fiber. This process is evident in the regenerative phase after rhabdomyolysis. For more detailed analysis of muscle regeneration immunohistochemical staining for the developmental myogenic markers, neonatal myosin heavy chain (nMHC), vimentin, MyoD, and myogenin are employed as used in a recent study. MyoD and myogenin is also used as a diagnostic marker for rhadomyosarcomas. These markers are known to be upregulated during myogenesis and muscle fiber regeneration for a short period of time ranging from days to 1–3 weeks. Furthermore, we wanted to determine if apoptosis is present in muscle from patients with LGMD2A, as has been suggested previously. The morphology and level of regeneration in patients with two null alleles in CAPN3 were compared with those in patients with limb girdle muscular dystrophy type 2I (LGMD2I) and patients with Becker muscular dystrophy (BMD), who clinically and morphologically resemble patients with LGMD2A.
Background
Limb Girdle Muscular Dystrophy type 2A (LGMD2A) is an autosomal recessively inherited disorder characterized by symmetrical progressive proximal muscle weakness and atrophy that usually starts in the pelvic girdle musculature. However, the clinical course is characterized by great variability, ranging from severe forms with onset in the first decade and rapid progression to milder forms with later onset and slower course. Serum creatine kinase is usually highly elevated and the muscle morphology shows dystrophic features. The disorder is caused by a loss of functional calpain 3, a skeletal muscle specific isoform of the Ca-dependent calpain cysteine protease family. More than 460 pathogenic mutations, covering almost the entire length of the calpain 3 gene (CAPN3), have been discovered (Leiden Muscular Dystrophy Pages, http://www.dmd.nl). Recent research involving calpain 3 protease-inactive knock-in mice have demonstrated that calpain 3 appears to play a role in the Ca-efflux from the sarcoplasmatic reticulum in a way that does not involve the protease function of calpain 3, this may explain why dysfunctional calpain 3 leads to muscle weakness. However, the potential substrates of calpain 3 have been a major focus of LGMD2A research in the past few years, as it appears that calpain 3 may be involved in multiple aspects of muscle function and maintenance. Calpain 3 is anchored to the giant structural/scaffold protein titin in a stable and inactive manner, to keep it from degrading itself autolytically. For that very same reason, the substrates of calpain 3 is believed to be in close proximity, possibly bound to other parts of the sarcomeres. It is thought that calpain 3 is inactive most of the time, only to be activated and redistributed when sarcomeres are exercised beyond a threshold, leading to interaction with a number of proteins e.g. myosin light chain 1, suggesting a role for calpain 3 in sarcomere remodeling. Another important in vivo calpain 3 substrates that has been described is AHNAK, a very large protein involved in subsarcolemmal cytostructure and part of the dysferlin membrane repair complex, requires calpain 3 for it to be cleaved and the membrane repair to proceed. Hence, it is a key component in the repair of the wear and tear of skeletal muscle tissue. The search for calpain 3 substrates using cleavage site recognition have lead to a number of potential targets, one being Protein Inhibitor of Activated Stats 3 (PIAS3), an ubiquitously expressed E3 SUMO ligase implicated in many signaling pathways by modifying the localization and role in transcriptional regulation of transcription factors.
A recent study using a protease-inactive calpain 3 knock-in model, demonstrated that calpain 3 lacking protease functionality lead to muscular dystrophy, exacerbated by exercise. This group proposed that the protease activity of calpain 3 is required to protect muscle from degeneration under exercise-induced stress, and that loss of protease activity affected the dynamic distribution of calpain 3 during physical activity and its interaction with MARP2, a stress-response transcriptional regulator protein, in close proximity to calpain 3 on the N2A region of titin. MARP2 is upregulated during exercise in normal muscle, however, the protease-inactivity of calpain 3 resulted in decreased levels of MARP2 and abnormal levels of dysferlin in exercised calpain 3 knock-in mice, leading to the conclusion that the muscle membrane repair mechanism is greatly affected by the loss of calpain 3 protease activity even if the activation of satellite cells is not affected. Several studies have shown that calpain 3 deficiency leads to formation of abnormal sarcomeres, impairment of muscle contractile capacity and loss of the muscle fibers.
In order to balance the ongoing degeneration, muscle fibers must regenerate, but in patients with LGMD2A it is unknown how calpain 3 deficiency affects the regenerative response. We therefore investigated the level of regeneration in skeletal muscle of 22 patients with genetically confirmed LGMD2A by assessing commonly used markers of muscle regeneration. Internally nucleated fibers (INF) arise from muscle specific stem cells, satellite cells. These cells are activated during muscle degeneration and constitute the majority of regenerative response to muscle wasting. Activation of satellite cells ultimately leads to migration of nuclei to the damaged area of the muscle fiber. This process is evident in the regenerative phase after rhabdomyolysis. For more detailed analysis of muscle regeneration immunohistochemical staining for the developmental myogenic markers, neonatal myosin heavy chain (nMHC), vimentin, MyoD, and myogenin are employed as used in a recent study. MyoD and myogenin is also used as a diagnostic marker for rhadomyosarcomas. These markers are known to be upregulated during myogenesis and muscle fiber regeneration for a short period of time ranging from days to 1–3 weeks. Furthermore, we wanted to determine if apoptosis is present in muscle from patients with LGMD2A, as has been suggested previously. The morphology and level of regeneration in patients with two null alleles in CAPN3 were compared with those in patients with limb girdle muscular dystrophy type 2I (LGMD2I) and patients with Becker muscular dystrophy (BMD), who clinically and morphologically resemble patients with LGMD2A.
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