Targeting Dysferlin’s C2A Domain to the Triad Junction Restores Membrane Repair and Regulation of Ca2+ Signaling in Dysferlin-Null Skeletal Muscle


Pre-Clinical Research

Poster Number: T364


Robert Bloch, PhD, University of Maryland Baltimore

myofibers. When it is mutated or absent in dysferlinopathies (MMD1, LGMD2B and others), muscle becomes dystrophic, likely for two reasons: failure of sarcolemmal membrane repair and dysregulation of Ca2+ signaling due to Ca2+ leak into the TJ cleft. This promotes Ca2+ waves in myofibers that are indicative of Ca2+-induced Ca2+ release, which can be pathogenic. Although they do not accumulate at TJs, smaller versions of dysferlin, termed “nanodysferlins”, can support membrane repair of dysferlin-null A/J myofibers but fail to suppress abnormal Ca2+ signaling after osmotic shock injury. Expressed alone, the C2A domain of dysferlin restores both activities, though only when expressed at high levels. Here we identify an efficient way to target the C2A domain to the TJ to restore dysferlin’s activity in Ca2+ signaling and membrane repair, at much lower levels of expression. Methods: We introduced Venus-fusions of DysfC2A and other C2 domains, either alone or linked in tandem, into A/J fibers by electroporation. We tested their activities in membrane repair after laser wounding and in Ca2+ signaling before and after OSI, assessed by loading myofibers with Rhod2-AM and stimulating at 1 Hz. Results: DysfC2A, but not the homologous C2 domains, MyofC2A and PKCαC2, rescued membrane repair and Ca2+ signaling. PKCαC2 accumulated at TJs efficiently, however, especially as the tandem PKCαC2-PKCαC2 construct. Using this to target DysfC2A to TJs as Venus-PKCαC2-PKCαC2-DysfC2A, we found >75% fibers with the construct concentrated at TJs, even when expressed at low levels (assayed by relative Venus fluorescence). It fully restored membrane repair and normal Ca2+ signaling. This was true whether the construct contained the Romeo epitope of dysferlin or not. Replacing DysfC2A with an additional PKCα-C2 or with MyofC2A failed to rescue the Ca2+ signaling phenotype. Pathogenic DysfC2A mutants (-W55R, -V67D) were inactive as the PKCαC2-PKCαC2 chimera, whereas chimeras of polymorphic mutants (-V68L, -A84V) were active. Conclusions: Dysferlin’s C2A domain is efficiently targeted to TJs by coupling it to two C2 domains of PKCα. Even at very low levels of expression, this construct fully restores regulation of Ca2+ signaling after OSI and membrane repair after laser wounding. We are now testing it by transducing dysferlin-deficient muscle with AAV. Supported by the Jain Foundation.