Abstract
BackgroundNonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). While calpain-3 is implicated in muscle cell differentiation, sarcomere formation, and muscle cytoskeletal remodeling, the physiological basis for LGMD2A has remained elusive.MethodsCell growth, gene expression profiling, and mitochondrial content and function were analyzed using muscle and muscle cell cultures established from healthy and calpain-3-deficient mice. Calpain-3-deficient mice were also treated with PPAR-delta agonist (GW501516) to assess mitochondrial function and membrane repair. The unpaired t test was used to assess the significance of the differences observed between the two groups or treatments. ANOVAs were used to assess significance over time.ResultsWe find that calpain-3 deficiency causes mitochondrial dysfunction in the muscles and myoblasts. Calpain-3-deficient myoblasts showed increased proliferation, and their gene expression profile showed aberrant mitochondrial biogenesis. Myotube gene expression analysis further revealed altered lipid metabolism in calpain-3-deficient muscle. Mitochondrial defects were validated in vitro and in vivo. We used GW501516 to improve mitochondrial biogenesis in vivo in 7-month-old calpain-3-deficient mice. This treatment improved satellite cell activity as indicated by increased MyoD and Pax7 mRNA expression. It also decreased muscle fatigability and reduced serum creatine kinase levels. The decreased mitochondrial function also impaired sarcolemmal repair in the calpain-3-deficient skeletal muscle. Improving mitochondrial activity by acute pyruvate treatment improved sarcolemmal repair.ConclusionOur results provide evidence that calpain-3 deficiency in the skeletal muscle is associated with poor mitochondrial biogenesis and function resulting in poor sarcolemmal repair. Addressing this deficit by drugs that improve mitochondrial activity offers new therapeutic avenues for LGMD2A.
Highlights
Nonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A)
A list of altered genes related to mitochondrial biogenesis, metabolism, lipid metabolism, and protein transport and their respective fold changes for myoblasts and myotubes is available in the supplemental excel file
In the present study, we demonstrated that the muscles lacking CAPN3 have a deficiency in mitochondrial biogenesis and mitochondrial activity, which results in the poor repair of injured myofiber sarcolemma
Summary
Nonsense or loss-of-function mutations in the non-lysosomal cysteine protease calpain-3 result in limb-girdle muscular dystrophy type 2A (LGMD2A). In addition to the two ubiquitous calpain isoforms CAPN1 and CAPN2, the skeletal muscles express CAPN3 protein known as p93 or calpain-3 [2] This 94-kDa Ca2+-dependent cysteine protease is known to localize to sarcomeres through binding the giant sarcomeric protein titin ( referred to as connectin) [3], its physiological functions are yet to be fully elucidated. Loss-of-function mutations in the CAPN3 gene lead to the autosomal recessive form of limb-girdle muscular dystrophy (LGMD) type 2A (LGMD2A). More than 440 different pathogenic mutations of the CAPN3 gene have been reported in LGMD2A patients [4] These mutations are distributed along the entire length of the gene product and include missense, nonsense, frame-shift, and deletion mutations [15,16,17,18]
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