Syntaxin 4-enhanced plasma membrane repair is independent 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 Ca²⁺-binding protein, plays a crucial role in PMR in skeletal muscle. Previous studies have suggested that PMR uses membrane trafficking and membrane fusion, similar to neurotransmission. Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion in neurotransmission with the help of synaptotagmin, a crucial Ca²⁺-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 the 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 Ca²⁺ 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 Ca²⁺ influx in laser-wounded FDB fibers. Furthermore, overexpression of STX4-mCitrine also decreased Ca²⁺ 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.<b>NEW & NOTEWORTHY</b> Dysferlin, a crucial Ca²⁺-binding protein in plasma membrane repair (PMR), shares homology with synaptotagmin, which binds Ca²⁺ and regulates SNARE-mediated vesicle fusion in neurons. Dysferlin was thus hypothesized to function as synaptotagmin in PMR. We demonstrate here that the activity of SNAREs is important for PMR, and overexpression of STX4 enhances PMR in both intact and dysferlin-deficient skeletal muscle. These data suggest that SNARE-mediated PMR may be independent of dysferlin in skeletal muscle.