Spatially Localized Disruptions of Voltage Activated Calcium Release in Mtm1-Deficient Muscle Fibers

Abstract

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin (Mtm1) are responsible for myotubular myopathy. We previously showed that muscle fibers from Mtm1-deficient mouse suffer from defective excitation-contraction coupling. Here we measured voltage-clamp activated Ca2+ signals in fibers from the flexor digitorum brevis muscles of 4 week-old wild type (WT) and Mtm1-ko mice under line-scan confocal microscopy using the dye rhod-2. Ca2+ release in the diseased fibers was deficient over the full range of activation: fitting the voltage dependence of the peak rate of rise of the line-averaged rhod-2 F/F0 signals with a Boltzmann function gave mean values for maximum rate, midpoint voltage and slope of 0.29 ± 0.02 and 0.14 ± 0.02 F/F0.ms−1, −8.8 ± 1.5 and 0.95 ± 2.3 mV, 7.0 ± 0.4 and 9.2 ± 0.7 mV in WT (n=22) and Mtm1-ko fibers (n=15), respectively. Furthermore, the mean time to peak rate was significantly delayed by 5-10 ms in Mtm1-ko as compared to WT fibers. These global alterations were associated with severe spatial inhomogeneity of Ca2+ release in the diseased fibers with rhod-2 transients yielding localized disruptions along the scanned line including reduced peak amplitude but also delayed or slower rate of onset, suggestive of specific alteration of the early peak component of the rate of Ca2+ release. In fibers treated with wortmannin and LY294002, the properties of the line-averaged rhod-2 F/F0 signals were unchanged in WT fibers but the mean maximum rate of rise of the rhod-2 signal was enhanced by 60% in Mtm1-deficient fibers. Results show that Mtm1-deficiency provokes EC coupling failure through accumulation of spatially localized disruptions of Ca2+ release. They also suggest that pharmacological inhibition of PtdIns-3-kinase activity has the potency to alleviate defective EC coupling in the diseased fibers.