Dysferlin is an integral membrane protein of the triad junction (TJ) linked to Limb Girdle Muscular Dystrophy R2, an autosomal recessive disease. Our studies of dysferlin-null mouse myofibers in vitro have shown that mild injury by hypoosmotic shock decreases the amplitude of the voltage-induced Ca2+ transient and generates Ca2+ waves via Ca2+-induced Ca2+ release (CICR). These effects are suppressed by drugs that stabilize the DHPR-RyR1 couplon or inhibit RyR1 Ca2+ leak, as well as by low concentrations (10nM) of BAPTA-AM. These results suggest that influx of Ca2+ into the TJ is required for damage to dysferlin-null fibers. This is supported by the fact that the C2A domain of dysferlin (Dysf-C2A), which binds Ca2+, rescues normal Ca2+ signaling and that the Ca2+-binding moiety, GCaMP6fu, incorporated into dysferlin in place of Dysf-C2A, localizes to TJs normally and suppresses abnormal Ca2+ signaling. We also found a role for PKCα. Chimeric constructs of the C2 domain of PKCα with Dysf-C2A concentrates at TJs of most fibers and is even more effective than Dysf-C2A alone in rescuing normal Ca2+ signaling. Consistent with this, PKCα concentrates at the TJ of WT fibers and co-immunoprecipitates with dysferlin from transfected COS7 cells. Moreover, activation of PKCα with PMA suppresses abnormal Ca2+ signaling in injured dysferlin-null fibers, while inhibition of PKCα with Gö6976 induces abnormal Ca2+ signaling, including Ca2+ waves, in injured WT fibers. These results suggest that dysferlin stabilizes DHPR-RyR1 coupling and suppresses Ca2+ leak by facilitating targeted phosphorylation by PKCα, but that activation of PKCα can shut down RyR1-mediated Ca2+ leak and thus compensate for dysferlin’s absence.
Support: The Jain Foundation and NIH (RO1 AR064268; RO1 AR082469).