Syntaxin 4-enhanced plasma membrane repair isindependent of dysferlin in skeletal muscle.

Plasma membrane repair (PMR) restores membrane integrity of cells, preventing cell death in vital organs, and has been studied extensively in skeletal muscle. Dysferlin, a sarcolemmal Ca-binding protein, plays a crucial role in PMR in skeletal muscle. Previous studies have suggested that PMR employs membrane trafficking and membrane fusion, similar to neurotransmission. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion in neurotransmission with the help of synaptotagmin, a crucial Ca-binding protein. Interestingly, dysferlin shares structural similarity with synaptotagmin and was shown to promote SNARE-mediated membrane fusion in a liposome-based assay. However, whether dysferlin facilitates SNARE-mediated membrane fusion in PMR in muscle cells remains unclear. In this study, we aimed to test if SNARE-mediated PMR requires dysferlin in muscle cells with pharmacological and genetic approaches. TAT-NSF700, which disrupts disassembly of SNARE complexes, was used to disrupt functions of SNAREs in muscle cells. We found that human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) treated with TAT-NSF700 showed a higher loss of membrane integrity after repetitive mechanical strains. Moreover, laser-wounded mouse flexor digitorum brevis (FDB) fibers treated with TAT-NSF700 showed an increased Ca influx, but a decreased FM1-43 uptake, which depends on dynamin-regulated endocytosis as we previously showed in FDB fibers. Importantly, overexpression of STX4-mCitrine or eGFP-SNAP23 decreased Ca influx in laser-wounded FDB fibers. Further, overexpression of STX4-mCitrine also decreased Ca influx in laser-wounded dysferlin-deficient FDB fibers. Overall, these results suggest that disassembly of SNARE complexes is required for efficient PMR, and STX4-enhanced PMR does not require dysferlin in skeletal muscle.