Elevated Ca at the triad junction underlies dysregulation of Ca signaling in dysferlin-null skeletal muscle.

Dysferlin-null A/J myofibers generate abnormal Ca transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca waves (Lukyanenko et al., J. Physiol., 2017). Ca waves are typically associated with Ca-induced Ca release, or CICR, which can be myopathic. We tested the ability of a permeable Ca chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca 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 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. EGTA, which has an affinity for Ca 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 in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca signaling. GCaMP6f introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca signaling. Our results suggest that leak of Ca into the triad junctional cleft underlies dysregulation of Ca signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca signaling and protects muscle against injury by binding Ca in the cleft.