Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration

Abstract

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.

Comparative transcriptome analysis of muscular dystrophy models Largemyd, Dmdmdx/Largemyd and Dmdmdx: what makes them different?

“…Calpain 6 has been considered as a differentiation and growth suppressor of skeletal muscle, since its deficiency can promote muscular differentiation in the development as well as in regeneration, modulating basal functions like cell division. 42 We found 55 DEGs for the age of 6 months. Among them are Col3a1, Col6a3 and Col8a1, all overexpressed, reflecting fibrosis formation.…”

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing

“…Generation of Capn6-and Capn9-deficient mice was performed as described previously (20,32). Ldlr −/− mice were maintained by homozygous breeding, as they have a common genetic background; the genotypes were determined by standard PCR-based genotyping with specific primers (Supplemental Table 3).…”

“…CAPN6 expression is reportedly detectable in fetal skeletal muscle and in the placenta (20); however, its expression in skeletal muscle vanishes during growth. A gene-targeting study showed that CAPN6 limits the development and regeneration of skeletal muscle (20), although the physiologic and pathophysiologic role of this molecule remains unclear. Hong et al noted that CAPN6 is induced during the differentiation of osteoclasts from BM-derived cells by receptor activator of NF-κB ligand, thereby accelerating bone-resorptive activity (30), suggesting that CAPN6 may function in the monocyte/macrophage lineage under certain conditions.…”

“…Relative necrotic core in the lesions was slightly decreased by Capn6 deficiency (Supplemental Figure 5H). We investigated CAPN6 systemic expression in HFD-fed Capn6 −/y Ldlr −/− mice using a LacZ reporter assay ( Figure 7D), as the mice express LacZ under the Capn6 native promoter (20). As a result, transcriptional activity of Capn6 is restricted in atherosclerotic plaques, but not in other organs.…”

“…CAPN6 was first cloned by Dear et al (29). CAPN6 expression is reportedly detectable in fetal skeletal muscle and in the placenta (20); however, its expression in skeletal muscle vanishes during growth. A gene-targeting study showed that CAPN6 limits the development and regeneration of skeletal muscle (20), although the physiologic and pathophysiologic role of this molecule remains unclear.…”