Impaired Muscle Regeneration in CAPN3-KO Mice Related to microRNA Dysregulation (S61.006)

OBJECTIVE: To advance pathogenic insight into the evolution of regeneration and fibrosis in the calpain-3 deficient (CAPN3-KO) mice to further understand limb girdle muscular dystrophy type 2A (LGMD2A). BACKGROUND: Studies in LGMD2A muscle biopsies showed microRNA dysregulation associated with inability of Pax7-positive satellite cells (SCs) to transit from proliferation to differentiation leading to impaired regeneration and fibrosis. DESIGN/METHODS: Muscle necrosis and regeneration were induced in CAPN3-KO and wild type (WT) mice by weekly cardiotoxin (CTX) injections into tibialis anterior and gastrocnemius muscles. Quantification studies 4 and 12 weeks post CTX included fiber type-specific diameters [slow twitch oxidative (STO) versus fast twitch glycolytic (FTG)], extent of connective tissue and muscle specific miRNAs levels. RESULTS: Muscle fiber diameters in regenerating CAPN3-KO were significantly smaller than WT mice at 4 and 12 weeks. STO fiber diameter at 12 weeks (23.1 ± 0.3 µm; n=1303) did not show a significant change compared to 4 weeks (24.1 ± 0.3 µm; n=1275) indicating that their radial growth was markedly impaired. FTG fiber radial growth was attenuated compared to WT but significantly better than STO fibers. Moreover, the number of STO fibers in CAPN3-KO was 2 fold greater than WT muscle. CAPN3-KO muscle fibrosis was also significantly increased, but the extent was not different between 4 and 12 weeks. miRNA expression levels (206,1,133a) were attenuated similar to LGMD2A biopsies. CONCLUSIONS: The absence of CAPN3 results in aberrant regeneration characterized by an increase in the number of small size STO (type I) fibers, consistent with defects in lateral fusion of myotubes and radial growth of regenerated fibers. These findings coupled with microRNA dysregulation point to inadequate differentiation of SCs predominantly in the type I fibers providing a rationale for microRNA overexpression to facilitate SC differentiation, skeletal muscle maturation, and promotion of normal muscle growth and regeneration. Study supported by: Nationwide Children`s Hospital Research Foundation Disclosure: Dr. Sahenk has received research support from Sarepta Therapeutics. Dr. Braganza has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Hussain has nothing to disclose. Dr. Meadows has nothing to disclose. Dr. Lewis has nothing to disclose. Dr. Flanigan has received personal compensation for activities with Pfizer Inc. Dr. Flanigan has received research support from PTC Therapeutics, Halo Therapeutics, and GlaxoSmithKline, Inc. Dr. Clark has nothing to disclose. Dr. Mendell has received research support from Sarepta Therapeutics Inc.