Caveolin 3 suppresses phosphorylation-dependent activation of sarcolemmal nNOS

Mutations of the caveolin 3 gene cause autosomal dominant limb-girdle muscular dystrophy (LGMD)1C. In mice, overexpression of mutant caveolin 3 leads to loss of caveolin 3 and results in myofiber hypotrophy in association with activation of neuronal nitric oxide synthase (nNOS) at the sarcolemma. Here, we show that caveolin 3 directly bound to nNOS and suppressed its phosphorylation-dependent activation at a specific residue, Ser1412 in the nicotinamide adenine dinucleotide phosphate (NADPH)-flavin adenine dinucleotide (FAD) module near the C-terminus of the reduction domain, in vitro. Constitutively active nNOS enhanced myoblast fusion, but not myogenesis, in vitro. Phosphorylation-dependent activation of nNOS occurred in muscles from caveolin 3-mutant mice and LGMD1C patients. Mating with nNOS-mutant mice exacerbated myofiber hypotrophy in the caveolin 3-mutant mice. In nNOS-mutant mice, regenerating myofibers after cardiotoxin injury became hypotrophic with reduced myoblast fusion. Administration of NO donor increased myofiber size and the number of myonuclei in the caveolin 3-mutant mice. Exercise also increased myofiber size accompanied by phosphorylation-dependent activation of nNOS in wild-type and caveolin 3-mutant mice. These data indicate that caveolin 3 inhibits phosphorylation-dependent activation of nNOS, which leads to myofiber hypertrophy via enhancing myoblast fusion. Hypertrophic signaling by nNOS phosphorylation could act in a compensatory manner in caveolin 3-deficient muscles.