Branched fibres in old dystrophic mdx muscle are associated with mechanical weakening of the sarcolemma, abnormal Ca2+ transients and a breakdown of Ca2+ homeostasis during fatigue

Abstract

In the dystrophinopathies, skeletal muscle fibres undergo cycles of degeneration and regeneration, with regenerated fibres displaying a branched morphology. This study tests the hypothesis that regenerated branched fibres are mechanically weakened by the presence of branches and are damaged by contractions which do not affect unbranched dystrophin-negative fibres. Experiments were carried out on single fast-twitch fibres and whole muscle from the dystrophin-negative mdx mouse. Fura-2 was ionophoresed into fibres to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Single branched mdx fibres have abnormal Ca(2+) kinetics, with the [Ca(2+)](i) transient at the peak of the twitch depressed, are damaged by fatiguing activation, resulting in a breakdown of Ca(2+) homeostasis, and break at branch points when submaximally activated in skinned fibre experiments. When old intact isolated mdx muscles, with >90% branched fibres, are eccentrically activated with a moderate eccentric protocol there is a 40 +/- 8% reduction in maximal force. Isolated single fibres from these muscles show areas of damage at fibre branch points. This same eccentric protocol causes no force loss in either littermate control muscles or mdx muscles with <10% branched fibres. I present a two-stage hypothesis for muscle damage in the dystrophinopathies, as follows: stage 1, the absence of dystrophin disrupts ion channel function, causing an activation of necrotizing Ca(2+)-activated proteases, which results in regenerated branched fibres; and stage 2, branched fibres are mechanically damaged during contraction. These results may have implications when considering therapies for boys with Duchenne muscular dystrophy. In particular, any therapy aimed at rescuing the defective gene will presumably have to do so before the number of branched fibres has increased to a level where the muscle is mechanically compromised.