Elevated Ca(2+) at the triad junction underlies dysregulation of Ca(2+) signaling in dysferlin-null skeletal muscle

Dysferlin-null A/J myofibers generate abnormal Ca(2+) transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca(2+) waves (Lukyanenko et al., J. Physiol., 2017). Ca(2+) waves are typically associated with Ca(2+)-induced Ca(2+) release, or CICR, which can be myopathic. We tested the ability of a permeable Ca(2+) chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10–50 nM BAPTA-AM suppressed all Ca(2+) waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca(2+) transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca(2+) to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca(2+). EGTA, which has an affinity for Ca(2+) similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6f(u) in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca(2+) signaling. GCaMP6f(u) introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca(2+) signaling. Our results suggest that leak of Ca(2+) into the triad junctional cleft underlies dysregulation of Ca(2+) signaling in dysferlin-null myofibers, and that dysferlin’s C2A domain suppresses abnormal Ca(2+) signaling and protects muscle against injury by binding Ca(2+) in the cleft.