Tropin T and Excitation-Contraction Coupling in Aging Skeletal Muscle

Tropin T 和衰老骨骼肌中的兴奋-收缩耦合

• 批准号: 8882201
• 负责人: Osvaldo Delbono
• 金额: $ 37.59万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8882201
• 关键词: 5' Flanking Region; Accounting; Adult; Age; Aging; Atrophic; Calcium; Calcium Channel; Calpain; Cell Nucleus; Cells; Charge; Cleaved cell; Complex; Coupling; DNA Binding; Deterioration; Elderly; Excision; Exercise; FVB Mouse; Fast-Twitch Muscle Fibers; Fiber; Gel; Generations; Genes; Genetic Transcription; Health; Healthcare; Human; Laboratories; Length; Mass Spectrum Analysis; Mediating; Membrane; Messenger RNA; Molecular; Morbidity - disease rate; Movement; Mus; Muscle; Muscle Contraction; Muscle Fibers; Muscle Weakness; Nuclear; Peptides; Performance; Population; Process; Protein Isoforms; Proteins; Proteolysis; Publications; RNA; RNA Splicing; Regulation; Rodent; Role; Ryanodine Receptors; Sarcoplasmic Reticulum; Skeletal Muscle; Striated Muscles; Testing; Therapeutic; Time; Transcript; Tropomyosin; Troponin T; Variant; Work; age related; calpain inhibitor; design; disability; exhaust; flexor digitorum brevis; in vivo; knock-down; mortality; muscle form; novel; patch clamp; prevent; small hairpin RNA; tool; voltage; young adult

DESCRIPTION (provided by applicant): In aging rodents and humans, decreased muscle mass does not fully account for the decrease in strength, indicating that atrophy only partially explains muscle weakness. Publications from our laboratory and others support the concept that aging impairs muscle activation-contraction efficiency. Altered transmittal of membrane depolarization to SR Ca2+ release decreases specific force in a process termed excitation- contraction uncoupling (ECU). Previous works from our laboratory identified the mouse specific Cav1.1 subunit gene 5'-flanking sequences necessary for basal transcription and control of Cav1.1 expression. However, the mechanism leading to impaired Cav1.1 transcription with aging and its treatment is unknown. Troponin T (TnT) is known to mediate the interaction between the Tn complex and tropomyosin (Tm) in the myoplasm, which is essential for calcium-activated striated muscle contraction. We have preliminary evidence of a nontraditional role for TnT3, the TnT isoform expressed in fast-twitch muscle fibers. We found full-length (FL)-TnT3 and its fragments in both the nuclear and cytosolic fractions of myofibers isolated from mouse skeletal muscle. More important, the myonuclei from old FVB mice had less of the full-length protein and more of the COOH-terminal (CT) fragment than those of young mice. When we knocked down endogenous TnT3 by shRNA in muscle in vivo, the calcium channel a1 subunit, essential molecule for muscle contraction, was down-regulated at both the RNA and protein levels. The following specific aims will test the hypotheses that: (1) TnT3 regulates voltage-gated Ca2+ channel α1 subunit (Cav1.1) expression in fast adult myofibers, and (2) decreased nuclear FL-TnT3 and increased CT-TnT3 fraction result in decreased Cacna1 expression and impaired excitation-contraction coupling with aging. These hypotheses will be tested by the following specific aims. (1) To establish that TnT3 regulates Cav1.1 expression and excitation-contraction coupling. (2) To determine that TnT3 is enzymatically cleaved in aging skeletal muscle and (3) To determine whether inhibiting skeletal muscle μ-calpain prevents age-dependent increase in TnT3 fragmentation and reduced Cacna1 expression and sarcoplasmic reticulum Ca2+ release. The proposed studies will define a novel role for TnT3 as a regulator of Cav1.1 expression and a tool to ameliorate or prevent muscle weakness with aging.

描述（由适用提供）：在衰老的啮齿动物和人类中，肌肉质量降低并不能完全解释力量的降低，这表明萎缩仅部分解释了肌肉无力。我们实验室和其他人的出版物支持衰老会损害肌肉激活效率的概念。在称为兴奋 - 收缩解偶联（ECU）的过程中，膜沉积向SR Ca2+释放的变化会降低特定力。我们实验室的先前作品确定了小鼠特异性CAV1.1亚基基因5'-频流序列，用于基本转录和控制Cav1.1表达所必需的序列。但是，导致CAV1.1转录及其治疗的机制尚不清楚。众所周知，肌钙蛋白T（TNT）介导了肌瘤中TN复合物与肌球蛋白（TM）之间的相互作用，这对于钙激活的肌肉肌肉收缩至关重要。我们有初步的证据表明TNT3的非传统作用，TNT 3在快速肌纤维中表达的TNT同工型。我们在从小鼠骨骼肌中分离出的肌纤维的核和胞质分数中发现了全长（FL）-TNT3及其片段。更重要的是，来自旧FVB小鼠的肌核的全长蛋白质较少，而COOH末端（CT）片段的肌肉少于年轻小鼠的碎片。当我们通过体内肌肉中的shRNA击倒内源性TNT3时，钙通道A1亚基（肌肉收缩的必需分子）在RNA和蛋白质水平下都下调。以下具体目的将检验：（1）TNT3调节快速成年肌纤维的电压门控CA2+通道α1亚基（CAV1.1）表达，（2）（2）改善核FL-TNT3和CT-TNT3馏分增加导致Cacna1的表达和降低的CACNA1表达和降低兴奋的兴奋性兴奋能力，而兴奋的兴奋。这些假设将通过以下特定目标进行检验。 （1）确定TNT3调节CAV1.1表达和兴奋 - 收缩耦合。 （2）确定TNT3在衰老的骨骼肌中被裂解，（3）确定抑制骨骼肌μ-粘蛋白是否可以防止TNT3片段化的年龄依赖性增加，而CACNA1表达降低和sarcoplasmasmasic网状CA2+ CA2+释放。拟议的研究将定义TNT3作为CAV1.1表达的调节剂的新作用，并将其改善或预防随着衰老的肌肉无力减轻或预防肌肉无力的工具。