Regulation of Fiber Switching in Lobster Muscle

龙虾肌肉中纤维转换的调节

• 批准号: 0077422
• 负责人: Donald Mykles
• 金额: $ 30.11万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0077422&HistoricalAwards=false
• 关键词: Regulation; Fiber; Switching; Lobster; Muscle

Skeletal muscle is capable of significant changes in contractile properties in response to normal and pathological conditions. These changes are brought about by fiber switching (transformation), a remodeling process in which one set of fiber-type-specific isoforms of myofibrillar proteins (e.g., myosin, tropomyosin, and troponin) is replaced by another set in the contractile apparatus. Both hormones and innervation play key roles in determining the fiber type composition in muscles from such diverse organisms as crustaceans and mammals. At the molecular level, transformation requires the coordinated expression of dozens of genes located on different chromosomes. In mammalian muscle, calcium-dependent signal transduction pathways involving calcineurin, a calcium/calmodulin-dependent phosphatase, and calcium-dependent kinases (e.g., protein kinase C, CaM kinase) activate transcription factors that drive the expression of slow-type genes during fast-to-slow transformation.This project uses the American lobster, Homarus americanus, in which transformation can be experimentally manipulated, to investigate the regulation of myofibrillar protein expression by molting hormone (ecdysteroid) and motor neuron activity. Fibers in the claw closer muscles undergo a developmentally-regulated transformation as the isomorphic claws of larvae and juveniles differentiate into the heteromorphic cutter and crusher claws of the adult. This fiber switching occurs at the boundary between the central fast-fiber and the peripheral slow-fiber regions, and thus the transformation of a specific fiber is determined by its position within the muscle. The PI will determine the temporal and spatial expression of myofibrillar protein isoforms during the intermolt cycle in order to establish whether thin and thick myofilament proteins are coordinately expressed. Another goal is to determine the effect of chronically-elevated ecdysteroid on transformation in juvenile lobsters in vivo using ATPase histochemistry and in situ hybridization. Since preliminary results suggest that switching occurs during postmolt (when hemolymph ecdysteroid level is low), the hypothesis is that elevated ecdysteroid would delay or block transformation. Chronic low-frequency stimulation of fast fibers in juvenile claws in vitro will induce fast-to-slow transformation. Transcripts of slow-type genes will be detected in single fibers by RT-PCR and in situ hybridization. The PI will also determine how ecdysteroid and serotonin modulate electrically-induced transformation and will use pharmacological reagents to identify components of signal transduction pathways that mediate transcriptional activation.This research has broad application to understanding the control of fiber transformation in both invertebrates and vertebrates. Exercise, motor neuron activity, disuse, disease, and hormones, induce fiber switching in mammalian and avian skeletal muscle, but the molecular mechanisms are poorly understood. One of the long-range goals is to identify "master regulators", transcription factors that initiate and coordinate slow-to-fast and fast-to-slow transformation. Signal transduction enzymes and transcriptional regulators are highly conserved in diverse animal species. Thus, the study of transformation in lobster will provide important insights about the potential interaction between hormones and electrical activity on myofibrillar protein expression.

骨骼肌能够响应于正常和病理条件而显著改变收缩特性。 这些变化是由纤维转换（转化）引起的，这是一种重塑过程，其中一组肌原纤维蛋白的纤维类型特异性同种型（例如，肌球蛋白、原肌球蛋白和肌钙蛋白）被收缩器中的另一组取代。 激素和神经支配在决定甲壳类动物和哺乳动物等多种生物的肌肉纤维类型组成中起着关键作用。 在分子水平上，转化需要位于不同染色体上的数十个基因的协调表达。 在哺乳动物肌肉中，钙依赖性信号转导途径涉及钙神经磷酸酶、钙/钙调蛋白依赖性磷酸酶和钙依赖性激酶（例如，蛋白激酶C，CaM激酶）激活转录因子，驱动慢型基因的表达在快速到缓慢的转换。本项目使用美国龙虾，Homarus americanus，其中可以实验操作的转换，研究蜕皮激素（蜕皮激素）和运动神经元活性对肌原纤维蛋白表达的调节。 纤维的爪更近的肌肉进行发育调节的改造，作为同构的爪子的幼虫和青少年分化成异形切割机和破碎机爪的成年人。 这种纤维转换发生在中央快纤维和外围慢纤维区域之间的边界处，因此特定纤维的转换由其在肌肉内的位置决定。 PI将确定蜕皮周期中肌原纤维蛋白亚型的时间和空间表达，以确定薄肌丝蛋白和厚肌丝蛋白是否协调表达。 另一个目标是确定的影响，长期升高蜕皮激素转化幼龙虾在体内使用ATP酶组织化学和原位杂交。 由于初步结果表明，转换发生在蜕皮后（血淋巴蜕皮激素水平低时），假设是蜕皮激素升高会延迟或阻断转化。 慢性低频刺激离体幼爪的快纤维会引起快到慢的转变。 慢型基因的转录本将通过RT-PCR和原位杂交在单个纤维中检测。 PI还将确定蜕皮激素和5-羟色胺如何调节电诱导的转化，并将使用药理学试剂来识别介导转录activation.This研究的信号转导通路的组件有广泛的应用，以了解在无脊椎动物和脊椎动物的纤维转化的控制。 运动、运动神经元活动、废用、疾病和激素可诱导哺乳动物和鸟类骨骼肌纤维转换，但其分子机制尚不清楚。 长期目标之一是确定“主调节因子”，即启动和协调慢到快和快到慢转化的转录因子。 信号转导酶和转录调节因子在不同的动物物种中高度保守。 因此，龙虾转化的研究将为激素和电活动对肌原纤维蛋白表达的潜在相互作用提供重要见解。