Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems.

Bidirectional communication between the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor (RYR1) in the sarcoplasmic reticulum (SR) is responsible for both skeletal-type excitation-contraction coupling (voltage-gated Ca(2+) release from the SR) and increased amplitude of L-type Ca(2+) current via the DHPR. Because the DHPR and RYR1 are functionally coupled, mutations in RYR1 that are linked to malignant hyperthermia (MH) may affect DHPR activity. For this reason, we investigated whether cultured myotubes originating from mice carrying an MH-linked mutation in RYR1 (R163C) had altered voltage-gated Ca(2+) release from the SR, membrane-bound charge movement, and/or L-type Ca(2+) current. In myotubes homozygous (Hom) for the R163C mutation, voltage-gated Ca(2+) release from the SR was substantially reduced and shifted ( approximately 10 mV) to more hyperpolarizing potentials compared with wild-type (WT) myotubes. Intramembrane charge movements of both Hom and heterozygous (Het) myotubes displayed hyperpolarizing shifts similar to that observed in voltage-gated SR Ca(2+) release. The current-voltage relationships for L-type currents in both Hom and Het myotubes were also shifted to more hyperpolarizing potentials ( approximately 7 and 5 mV, respectively). Compared with WT myotubes, Het and Hom myotubes both displayed a greater sensitivity to the L-type channel agonist +/-Bay K 8644 (10 microM). In general, L-type currents in WT, Het, and Hom myotubes inactivated modestly after 30-s prepulses to -50, -10, 0, 10, 20, and 30 mV. However, L-type currents in Hom myotubes displayed a hyperpolarizing shift in inactivation relative to L-type currents in either WT or Het myotubes. Our present results indicate that mutations in RYR1 can alter DHPR activity and raise the possibility that this altered DHPR function may contribute to MH episodes.

Bidirectional signaling between the sarcolemmal L-type Ca(2+) channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca(2+) release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation-contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K(+) depolarization. The new findings are: (a) For all three genotypes, Ca(2+) transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca(2+) or the addition of Ca(2+) entry blockers (nifedipine, SKF96365, and Ni(2+)) enhanced the rate of decay in all genotypes. (d) When Ca(2+) entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K(+) depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor.

Duchenne muscular dystrophy (DMD) is an irreversible muscle disease characterized by a progressive loss of muscle function, decreased ambulation, and ultimately death as a result of cardiac or respiratory failure. DMD is caused by the lack of dystrophin, a protein that is important for membrane stability and signaling in excitable cells. Although vascular smooth muscle cells (VSMCs) dysfunction occurs in many pathological conditions, little is known about vascular smooth muscle function in DMD. We have previously shown that striated muscle cells, as well as neurons isolated from dystrophic (mdx) mice have higher intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) concentrations and decreased cell viability in comparison with wild type (Wt). Experiments were carried out in isolated VSMCs from mdx (a murine model of DMD) and congenic C57BL/10SnJ Wt mice. We found elevated [Ca2+]i and [Na+]i in VSMCs from mdx mice compared to Wt. Exposure to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC3 and TRPC6 channel activator, induced a greater elevation of [Ca2+]i and [Na+]i in mdx than Wt VSMCs. The OAG induced increases in [Ca2+]i could be abolished by either removal of extracellular Ca2+ or by SAR7334, a blocker of TRPC3 and TRPC 6 channels in both genotypes. Mdx and Wt VSMCs were susceptible to muscle cell stretch-induced elevations of [Ca2+]i and [Na+]i which was completely inhibited by GsMTx-4, a mechanosensitive ion channel inhibitor. Western blots showed a significant upregulation of TRPC1 -3, -6 proteins in mdx VSMCs compare to age-matched Wt. The lack of dystrophin in mdx VSMCs produced a profound alteration of [Ca2+]i and [Na+]i homeostasis that appears to be mediated by TRPC channels. Moreover, we have been able to demonstrate pharmacologically that the enhanced stretch-induced elevation of intracellular [Ca2+] and concomitant cell damage in mdx VSMCs also appears to be mediated through TRPC1, -3 and -6 channel activation.

Duchenne muscular dystrophy (DMD) is an inherited muscular disorder caused by mutations in the dystrophin gene. DMD patients have hypoxemic events due to sleep-disordered breathing. We reported an anomalous regulation of resting intracellular Ca2+ ([Ca2+]i) in vascular smooth muscle cells (VSMCs) from a mouse (mdx) model of DMD. We investigated the effect of hypoxia on [Ca2+]i in isolated and quiescent VSMCs from C57BL/10SnJ (WT) and C57BL/10ScSn-Dmd (mdx) male mice. [Ca2+]i was measured using Ca2+-selective microelectrodes under normoxic conditions (95% air, 5% CO2) and after hypoxia (glucose-free solution aerated with 95% N2-5% CO2 for 30 min). [Ca2+]i in mdx VSMCs was significantly elevated compared to WT under normoxia. Hypoxia-induced [Ca2+]i overload, which was significantly greater in mdx than in WT VSMCs. A low Ca2+ solution caused a reduction in [Ca2+]i and prevented [Ca2+]i overload secondary to hypoxia. Nifedipine (10 µM), a Ca2+ channel blocker, did not modify resting [Ca2+]i in VSMCs but partially prevented the hypoxia-induced elevation of [Ca2+]i in both genotypes. SAR7334 (1 µM), an antagonist of TRPC3 and TRPC6, reduced the basal and [Ca2+]i overload caused by hypoxia. Cell viability, assessed by tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, was significantly reduced in mdx compared to WT VSMCs. Pretreatment with SAR7341 increases cell viability in normoxic mdx (p < 0.001) and during hypoxia in WT and mdx VSMCs. These results provide evidence that the lack of dystrophin makes VSMCs more susceptible to hypoxia-induced [Ca2+]i overload, which appears to be mediated by increased Ca2+ entry through L-type Ca2+ and TRPC channels.