Impact of Rad-mediated inhibition of Cav1.1 on muscle composition and contractile function

单体G蛋白Rad(与糖尿病相关的RAS样蛋白)在骨骼肌中的表达升高 肌萎缩侧索硬化症(ALS)患者和神经肌肉疾病小鼠模型的研究。高架者 Rad的表达可能影响肌肉功能，因为Rad是一种结构性活性的 骨骼肌L型钙通道(CaV1.1)作为兴奋收缩的电压敏感器 (EC)耦合。已证实EC偶联受损会导致多发性肌无力。 神经肌肉疾病，如肌萎缩侧索硬化症。这种兴奋性的抑制(称为“EC解偶联”)是一种直接的 这是由于质膜中存在的功能CaV1.1通道数量较少所致。此外, 在体内靶向敲除CaV1.1会导致显著的肌肉萎缩。总而言之，这些观察结果 提示Rad可以钝化力量的产生，并能启动骨骼肌的形态变化。的确， 初步数据表明，Rad在正常小鼠骨骼肌中过表达会损害EC偶联， 导致严重萎缩并改变肌肉代谢特征。因此，这笔R03小额赠款的目的是 建议调查骨骼肌中CaV1.1的慢性抑制的更广泛的后果。 具体目标1将确定Rad诱导的EC偶联的长期损害是否表现为 产生肌肉力量。要做到这一点，否则正常的腓肠肌将感染编码骨骼肌靶向金星荧光蛋白-Rad融合结构的腺相关病毒(AAV1) (tMCK-V-Rad)。感染后2、4和6个月，tMCK-V-Rad降低绝对和/或 对神经和直接肌肉刺激的特定肌肉力量的产生将使用 活体测力。特定目标2将调查Rad表达升高可以推动 肌肉质量和组成与进行性神经退行性疾病中观察到的相似。在这些 实验中，将感染tMCK-V-Rad的正常小鼠后肢肌肉收获、冷冻、切片 并用针对I型、IIA型、IIX型和IIB型肌球蛋白重链的抗体来检测 Rad高表达导致肌肉纤维类型从更多的糖酵解纤维转变的假说 更多的氧化纤维。

Expression of the monomeric G protein Rad (Ras-like Associated with Diabetes) is elevated in skeletal muscle of Amyotrophic Lateral Sclerosis (ALS) patients and mouse models of neuromuscular disease. The elevated expression of Rad is likely to impact muscle function because Rad is a constitutively-active inhibitor of the skeletal muscle L-type Ca2+ channel (CaV1.1), which serves as the voltage-sensor for excitation-contraction (EC) coupling. It has been established that impaired EC coupling contributes to muscle weakness in multiple neuromuscular disorders such as ALS. This depression of excitability (termed “EC un-coupling”) is a direct consequence of a fewer number of functional CaV1.1 channels present in the plasma membrane. In addition, targeted knock-down of CaV1.1 in vivo causes significant muscle atrophy. Taken together, these observations suggest that Rad blunts force generation and can initiate morphological changes in skeletal muscle. Indeed, preliminary data indicate that overexpression of Rad in normal mouse skeletal muscle impairs EC coupling, causes profound atrophy and changes muscle metabolic profile. Thus, the purpose of this R03 small grant proposal is to investigate the broader consequences of chronic inhibition of CaV1.1 in skeletal muscle. Specific Aim 1 will determine whether long-term Rad-induced impairment of EC coupling manifests in reduced muscle force generation. To do so, otherwise normal gastrocnemius muscle will be infected with an Adeno-Associated Virus (AAV1) encoding a skeletal muscle-targeted Venus fluorescent protein-Rad fusion construct (tMCK-V-Rad). Two, four, and six months post-infection, the ability of tMCK-V-Rad to reduce absolute and/or specific muscle force generation in response to nerve and direct muscle stimulation will be evaluated using an in vivo force assay. Specific Aim 2 will investigate the idea that elevated Rad expression can drive changes in muscle mass and composition similar to those observed in progressive neurodegenerative disease. In these experiments, normal mouse hindlimb muscles infected with tMCK-V-Rad will be harvested, frozen, sectioned and probed with antibodies directed to Type I, Type IIA, Type IIX and Type IIB myosin heavy chains to test the hypothesis that elevated Rad expression causes a shift in muscle fiber-type profile from more glycolytic fibers towards more oxidative fibers.