Mechanisms of Plasma Membrane Repair in Striated Muscle

Abstract

Sarcolemma instability is a hallmark of multiple types of muscular dystrophy and is typically caused by two major pathological mechanisms: increased susceptibility of the sarcolemma to contraction-induced damage or decreased capacity to repair the sarcolemma after contraction-induced damage. The former mechanism results from mutations in critical transmembrane and cytoskeletal proteins of the dystrophin-glycoprotein complex (DGC), a key component of the membrane-stabilizing costameres. The latter mechanism is due to loss of function mutations in key membrane repair proteins, such as dysferlin, that lead to aberrant membrane resealing after membrane injury. In both cases, the respective vulnerabilities of the plasma membrane result in muscle fiber death and progressive muscle disease. Modulating membrane repair could represent a potential therapeutic target to improve sarcolemma integrity in either type of muscular dystrophy. However, while many putative membrane repair proteins have been identified, the mechanisms of membrane repair in striated muscle remain unclear. The primary model of membrane repair in muscle posits that membrane repair proteins form a complex at membrane wounds to facilitate the fusion of cytoplasmic vesicles to repair the membrane barrier. Therefore, the goal of this thesis was to investigate the factors that regulate trafficking of the membrane repair protein dysferlin after injury – both to the wound and into cytoplasmic vesicles. These studies investigate dynamin-dependent endocytosis and cytoskeletal rearrangement after injury and their roles in dysferlin trafficking and ultimately membrane resealing. Dynamin-dependent endocytosis was interrogated prior to and after injury in isolated adult mouse skeletal muscle fibers using live cell imaging after in vitro laser injury in the presence of an endocytic marker, FM1-43, and pharmacological inhibitors of dynamin. We show that dynamin-dependent endocytosis is highly active at rest. We also show that injury results in a massive endocytic response, measured by FM1-43, and dynamin inhibition severely blunts FM1-43 uptake after injury in wildtype and dysferlin-deficient cells. For that reason, we measured calcium flux as a dynamin-independent measure of membrane repair and show that dysferlin-deficient cells have increased calcium uptake after injury, consistent with defective membrane repair. Interestingly, dynamin inhibition had no effect on dysferlin recruitment to membrane wounds or dysferlin endocytosis after injury, suggesting the two pathways are independent and the mechanisms for dysferlin endocytosis are still unclear. The contribution of subsarcolemmal cytoskeleton remodeling to membrane repair was investigated using live cell imaging approaches to monitor dynamic actin after membrane injury in conjunction with a muscle-specific knockout model of one of the major subsarcolemmal isoforms of actin, gamma-actin. We show that calcium drives actin polymerization at the wound, forming a stable structure that remains localized at the wound for minutes after injury. We also show that muscle-specific knockout of gamma-actin results in a membrane repair defect. However, defective membrane repair observed in this model is independent of dysferlin, as dysferlin trafficking to membrane wounds and dysferlin endocytosis are unaffected by genetic loss of gamma-actin. It remains possible that alternate forms of subsarcolemmal actin partially compensate or contribute to dysferlin trafficking to the wound, or alternatively, actin may play additional roles in stabilizing the membrane repair complex during or after resealing. These studies demonstrate that injury-induced dysferlin trafficking is independent of both dynamin-dependent endocytosis and gamma-actin. However, our data emphasize that both dynamin-dependent endocytosis and the subsarcolemma cytoskeleton are activated after muscle membrane injury and are required for efficient membrane resealing.