Calpain-6 deficiency promotes skeletal muscle development and regeneration

Calpains are Ca2+-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.
Author Summary The calpains arose evolutionarily as a family of Ca2+-regulated intracellular proteases with divergent structures and functions. Unique among mammalian calpains, calpain-6 is “non-proteolytic”, and its physiological role has remained unknown. In this study, using Capn6 knock-out mice, we discovered that Calpain-6 is involved in skeletal muscle development and regeneration in vivo. Calpain-6 was predominantly expressed in embryonic muscles and bone cartilage. Capn6 knock-out promoted skeletal-muscle development as a result of increased progenitor cell differentiation. Furthermore, calpain-6 was detected in regenerating skeletal muscles, and suppressed this process. These results indicate that mammalian calpain-6 is a suppressive modulator for skeletal muscle differentiation and growth. Our findings indicate that calpain-6 might serve as a therapeutic target for muscular dystrophy/atrophy or as a useful tool in tissue engineering. For calpain studies, the physiological non-proteolytic role of calpain-6 may shed light on the elusive calpain functions that are independent of their well-studied proteolytic activities. In addition, as this structurally non-proteolytic feature is unique to the calpain-6 of placental mammals among vertebrates, our mutant mice might provide insight into the relationship between molecular and biological evolution.