BPS2026 - Role of dysferlin’s transmembrane and extracellular domains in regulating calcium release in murine skeletal myofibers

Dysferlin is a transverse tubule protein that concentrates at triad junctions. Its absence causes LGMD R2. We are studying the role of dysferlin by probing the changes in dysferlin-null muscle fibers caused by osmotic shock injury (OSI). OSI decreases the amplitude of voltage-induced Ca 2+ transients (VICTs) in dysferlin-null but not control myofibers (Kerr et al., Proc. Natl. Acad. Sci. USA , 2013). Dysferlin also modulates the coupling of excitation to Ca 2+ release after OSI (Lukyanenko et al., J. Physiol. 2017) by buffering Ca 2+ leaking through RyRs (Lukyanenko et al., Front. Physiol. 2022). In studies of the cytoplasmic domains of dysferlin (as well as corresponding point mutations), we found that the C2A domain plays a critical role in suppressing abnormal Ca 2+ release (Muriel et al., J. Physiol. 2022). Here, we report studies of the role of dysferlin’s transmembrane (TM) and extracellular (EC) domains. We used dysferlin-null, A/J mouse FDB myofibers transfected with plasmids expressing Venus-tagged (V) point mutants of dysferlin missing TM (VDysf-ΔTM) or EC (VDysf-ΔEC) sequences, with shortened and/or modified TM domains ( (VDysf-TMΔFILLL; VDysf-TM and VDysf-TMΔFILLL with all Phe converted to Leu), and: -R2042C, -A2066V, -A2066T, -Y2070C, -Y2070X, -Y2070MΔEC, -A2071V, -A2071X, -M2073V, -M2073X. Recovery of VITCs and frequency of Ca 2+ waves after OSI for all constructs except A2071X were statistically identical to WT VDysf (∼80% and ≤0.2 Hz, respectively). VDysf-A2071X recovered to ∼72%, which was statistically different. In addition, high VDysf-Y2070X expression altered morphology changes. All variants targeted triad junctions like WT VDysf. We conclude that TM and EC domains of dysferlin do not regulate targeting of dysferlin to triad junctions or coupling of excitation to Ca 2+ release in murine skeletal muscle following injury. Supported by the Jain Foundation and NIH (RO1 AR064268).