Role of Dysferlin's C2A Domain in Voltage-Induced Calcium Release After Osmotic Shock in Murine Skeletal Myofibers

Abstract

Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2+-transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers (Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). We recently showed using OSI that dysferlin modulates the coupling of excitation to Ca2+ release (Lukyanenko et al., J. Physiol. 2017). Here we studied dysferlin-null A/J mouse FDB myofibers expressing mutated fluorescent dysferlin chimeras to examine the role of dysferlin's C2A domain in that coupling. We studied dysferlin missing C2A as well as C2A point mutations (pathogenic: W52R, V67D; polymorphisms: V68L; A84V), and Venus-C2A alone, expressed with and without dysferlin's transmembrane (TM) domain. Dysferlin-ΔC2A, -W52R and -V67D reduced recovery of VICTs after OSI (to 37%, 43% and 31% of controls, respectively). Dysf-V68L (72%) and -A84V (72%) were statistically identical to WT Venus-dysferlin (69%). Dysferlin localization to t-tubules, examined with C-terminal pHluorin constructs by reducing extracellular pH to 6.5 showed ∼60% reduction of fluorescence for WT Dysf-pHluorin fluorescence at pH6.5, 10-15% for the negative control (N-terminal pHluorin), and 10%, 35% and ∼60% for Dysf-W52R, -V67D, and -V68L, -A84V, and -ΔC2A, respectively. Thus, all mutants except Dysf-W52R were incorporated into t-tubules either partially or as efficiently as wild type. Venus-C2A alone, expressed with or without the TM domain, did not support the recovery of the VICT after OSI (both ∼50%). However, Venus-C2A lacking the TM domain significantly reduced Ca2+ waves after OSI (10% vs 83% of Venus- C2A-TM). We conclude that C2A modulates coupling of excitation to Ca2+ release in murine skeletal muscle, and that changes in coupling (e.g., with the Dysf-W52R and -V67D mutations) may be sufficient for pathogenicity. Supported by the Jain Foundation, and NIH (RO1 AR064268).