Impaired Sarcoplasmic Reticulum Calcium Release In Skeletal Muscle Fibers From Myotubularin-Deficient Mice

Abstract

X-linked myotubular myopathy (XLMTM) is a disease characterized by severe skeletal muscle weakness leading to death during childhood. XLMTM results from mutations in the MTM1 gene, coding for Myotubularin, a phosphoinositide phosphatase believed to play a role in plasma membrane homeostasis. The mechanisms responsible for muscle function impairment in XLMTM are unknown. Here we studied the properties of excitation-contraction coupling in skeletal muscle fibers isolated from a mouse model of the disease. Experiments were performed under silicone-voltage-clamp conditions using indo-1 as Ca2+ indicator. In muscle fibers from 5-week-old MTM1-deficient mice, the amplitude of the voltage-activated Ca2+ transient was strongly reduced. For instance, in response to a 10 ms-long pulse from -80 to +10 mV, the peak Δ[Ca2+] was 0.52 ± 0.1 μM (n=14) in MTM1-KO fibers as compared to 1.4 ± 0.14 μM in WT fibers (n=14). Conversely, the rate of [Ca2+] decay after the end of the pulses was similar in the two strains suggesting overall preserved myoplasmic Ca2+ removal capabilities. The SR calcium content was also found to be unaltered, as estimated from indo-1 signals measured in fibers equilibrated with high intracellular EGTA and in the presence of a SR Ca2+ pump blocker. The reduced amplitude of the Ca2+ transient in MTM1-deficient fibers was associated with a twice reduction in the peak density of the voltage-activated slow Ca2+ current with no apparent concurrent change in the density of intramembrane charge movement. Finally, confocal imaging with di-8-anneps revealed local disruptions in the typical fluorescence banded pattern, indicative of alteration of t-tubule membrane. Overall results unravel a critical role of MTM1 in the proper function of E-C coupling and strongly suggest that defective RyR1-mediated SR Ca2+ release is responsible for the failure of muscle function in myotubular myopathy.