Alpha-actinins in normal and diseased muscle

描述(申请人提供)：α-肌动蛋白是肌动蛋白结合蛋白，既包括组织肌动蛋白细丝的细胞骨架亚型，也包括代表肌肉Z线主要结构成分的肌小节亚型。肌节α-肌动蛋白与许多与Z线和细丝相关的结构和信号蛋白的相互作用，可能在骨骼肌中执行静态功能，以维持有序的肌纤维阵列，并可能在协调肌纤维结构和功能方面发挥调节功能。在人类中，有两个基因编码骨骼肌α-肌动蛋白，ACTN2和ACTN3。α-肌动蛋白-2存在于所有骨骼肌纤维中，而α-肌动蛋白-3仅在快速(类型2)纤维中表达。我们之前已经证明，由于ACTN3(R577X)中过早终止密码子的纯合性，α-肌动蛋白-3缺乏症在普通人群中很常见。α-肌动蛋白-3的缺失与疾病表型无关，很可能α-肌动蛋白-2能够“补偿”人类α-肌动蛋白-3的缺失。然而，由于我们发现专业短跑运动员的577XX(α-肌动蛋白-3缺失)基因频率与对照组相比非常低(p&lt；0.0001)，而优秀耐力运动员577XX的频率很高，所以在异构体功能上肯定有细微的差异。我们现在已经证明，转录上敲除α-肌动蛋白-2，而不是α-肌动蛋白-3，会导致纤维型基因表达的变化，并且α-肌动蛋白-2，而不是-3，在小鼠中是胚胎致死的。我们建议在这里测试的中心工作假设是，各种α-肌动蛋白亚型在结构上进化出细微的差异，使它们能够执行明显不同的功能。这项申请建议利用细胞培养和斑马鱼发育模型以及转基因和基因敲除小鼠的转录抑制和挽救实验，来研究α-肌动蛋白-2和-3的不同功能及其对快肌和慢肌发挥的独特生理作用的贡献。这项建议中描述的实验将有助于我们理解α-肌动蛋白亚型在正常骨骼肌生物学中的不同作用，以及这些亚型可能影响人类正常体能和表现变化的机制(S)。我们的结果还可能使我们深入了解α-肌动蛋白基因型别在调节和/或引起人类神经肌肉疾病中可能扮演的角色(S)。

DESCRIPTION (provided by applicant): The alpha-actinins are actin-binding proteins that include both cytoskeletal isoforms that organize actin filaments, and sarcomeric isoforms that represent major structural components of muscle Z-lines. Based on their interactions with many structural and signaling proteins associated with the Z-line and thin filament, sarcomeric alpha-actinins likely perform a static function in skeletal muscle to maintain the ordered myofibrillar array, as well as possible regulatory functions in coordinating myofiber structure and function. In humans, two genes encode the skeletal muscle alpha-actinins, ACTN2 and ACTN3. alpha-Actinin-2 is present in all skeletal muscle fibers, whereas alpha-actinin-3 is expressed only in fast (type 2) fibers. We have previously shown that alpha- actinin-3 deficiency is common in the general population due to homozygosity for a premature stop codon in ACTN3 (R577X). Absence of alpha-actinin-3 is not associated with a disease phenotype and it is likely that alpha- actinin-2 is able to "compensate" for the absence of alpha-actinin-3 in humans. Nevertheless, there must be subtle differences in isoform function as we have found that specialist sprint athletes have an extremely low frequency of the 577XX (alpha-actinin-3 null) genotype compared to controls, (p<0.0001), whereas elite endurance athletes have a high frequency of 577XX. We have now shown that transcriptional knockdown of alpha-actinin -2, but not alpha-actinin -3, results in changes in fiber type gene expression, and that knockout of alpha-actinin-2, but not -3, is embryonic lethal in mice. The central working hypothesis that we propose to test here is that the various alpha-actinin isoforms have evolved subtle differences in structure that allow them to perform distinctly different functions. This application proposes to utilize transcriptional knockdown and rescue experiments in cell culture and zebrafish developmental models, as well as transgenic and knockout mice, to study the differential functions of alpha-actinin-2 and -3 and their contributions to the unique physiological roles that fast and slow muscles perform. The experiments described in this proposal will make a significant contribution to our understanding of the differential roles of alpha-actinin isoforms in normal skeletal muscle biology, and the mechanism(s) by which these may influence normal human variations in fitness and performance. Our results may also lead to insights into the role(s) that alpha-actinin genotypes may play in modulating and/or causing human neuromuscular disease.