Voltage sensor current, SR Ca2+ release, and Ca2+ channel current during trains of action potential-like depolarizations of skeletal muscle fibers.

In skeletal muscle, CaV 1.1 serves as the voltage sensor for both excitation-contraction coupling (ECC) and L-type Ca2+ channel activation. We have recently adapted the technique of action potential (AP) voltage clamp (APVC) to monitor the current generated by the movement of intramembrane voltage sensors (IQ ) during single imposed transverse tubular AP-like depolarization waveforms (IQAP ). We now extend this procedure to monitoring IQAP , and Ca2+ currents during trains of tubular AP-like waveforms in adult murine skeletal muscle fibers, and compare them with the trajectories of APs and AP-induced Ca2+ release measured in other fibers using field stimulation and optical probes. The AP waveform remains relatively constant during brief trains (<1 sec) for propagating APs in non-V clamped fibers. Trains of 10 AP-like depolarizations at 10 Hz (900 ms), 50 Hz (180 ms), or 100 Hz (90 ms) did not alter IQAP amplitude or kinetics, consistent with previous findings in isolated muscle fibers where negligible charge immobilization occurred during 100 ms step depolarizations. Using field stimulation, Ca2+ release did exhibit a considerable decline from pulse to pulse during the train, also consistent with previous findings, indicating that the decline of Ca2+ release during a short train of APs is not correlated to modification of charge movement. Ca2+ currents during single or 10 Hz trains of AP-like depolarizations were hardly detectable, were minimal during 50 Hz trains, and became more evident during 100 Hz trains in some fibers. Our results verify predictions on the behavior of the ECC machinery in response to AP-like depolarizations and provide a direct demonstration that Ca2+ currents elicited by single AP-like waveforms are negligible, but can become more prominent in some fibers during short high-frequency train stimulation that elicits maximal isometric force.

Into the spotlight: RGK proteins in skeletal muscle.

The RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTPases are potent endogenous inhibitors of voltage-gated Ca2+ channels (VGCCs). While the impact of RGK proteins on cardiac physiology has been investigated extensively, much less is known regarding their influence on skeletal muscle biology. Thus, the purpose of this article is to establish a basis for future investigation into the role of RGK proteins in regulating the skeletal muscle excitation-contraction (EC) coupling complex via modulation of the L-type CaV1.1 VGCC. The pathological consequences of elevated muscle RGK protein expression in Type II Diabetes, Amyotrophic Lateral Sclerosis (ALS), Duchenne's Muscular Dystrophy and traumatic nerve injury are also discussed.

Mechanisms of altered skeletal muscle action potentials in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction for which only palliative treatment is currently available. Previously, we discovered reduced skeletal muscle Cl- channel (ClC-1) and inwardly rectifying K+ channel (Kir) currents in R6/2 HD transgenic mice. To further investigate the role of ClC-1 and Kir currents in HD skeletal muscle pathology, we measured the effect of reduced ClC-1 and Kir currents on action potential (AP) repetitive firing in R6/2 mice using a two-electrode current clamp. We found that R6/2 APs had a significantly lower peak amplitude, depolarized maximum repolarization, and prolonged decay time compared with wild type (WT). Of these differences, only the maximum repolarization was accounted for by the reduction in ClC-1 and Kir currents, indicating the presence of additional ion channel defects. We found that both KV1.5 and KV3.4 mRNA levels were significantly reduced in R6/2 skeletal muscle compared with WT, which explains the prolonged decay time of R6/2 APs. Overall, we found that APs in WT and R6/2 muscle significantly and progressively change during activity to maintain peak amplitude despite buildup of Na+ channel inactivation. Even with this resilience, the persistently reduced peak amplitude of R6/2 APs is expected to result in earlier fatigue and may help explain the motor impersistence experienced by HD patients. This work lays the foundation to link electrical changes to force generation defects in R6/2 HD mice and to examine the regulatory events controlling APs in WT muscle.

Progressive Cl- channel defects reveal disrupted skeletal muscle maturation in R6/2 Huntington's mice.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction. Previously, we discovered reduced skeletal muscle chloride channel (ClC-1) currents, inwardly rectifying potassium (Kir) channel currents, and membrane capacitance in R6/2 transgenic HD mice. The ClC-1 loss-of-function correlated with increased aberrant mRNA processing and decreased levels of full-length ClC-1 mRNA (Clcn1 gene). Physiologically, the resulting muscle hyperexcitability may help explain involuntary contractions of HD. In this study, the onset and progression of these defects are investigated in R6/2 mice, ranging from 3 wk old (presymptomatic) to 9-13 wk old (late-stage disease), and compared with age-matched wild-type (WT) siblings. The R6/2 ClC-1 current density and level of aberrantly spliced Clcn1 mRNA remain constant with age. In contrast, the ClC-1 current density increases, and the level of aberrantly spliced Clcn1 mRNA decreases with age in WT mice. The R6/2 ClC-1 properties diverge from WT before the onset of motor symptoms, which occurs at 5 wk of age. The relative decrease in R6/2 muscle capacitance also begins in 5-wk-old mice and is independent of fiber atrophy. Kir current density is consistently lower in R6/2 compared with WT muscle. The invariable R6/2 ClC-1 properties suggest a disruption in muscle maturation, which we confirm by measuring elevated levels of neonatal myosin heavy chain (MyHC) in late-stage R6/2 skeletal muscle. Similar changes in ClC-1 and MyHC isoforms in the more slowly developing Q175 HD mice suggest an altered maturational state is relevant to adult-onset HD. Finally, we find nuclear aggregates of muscleblind-like protein 1 without predominant CAG repeat colocalization in R6/2 muscle. This is unlike myotonic dystrophy, another trinucleotide repeat disorder with similar ClC-1 defects, and suggests a novel mechanism of aberrant mRNA splicing in HD. These early and progressive skeletal muscle defects reveal much needed peripheral biomarkers of disease progression and better elucidate the mechanism underlying HD myopathy.