Regulation of Fiber Switching in Lobster Muscle

骨骼肌能够在正常和病理条件下发生显著的收缩特性变化。这些变化是由纤维转换(转换)带来的，这是一种重塑过程，在这种重塑过程中，一组特定纤维类型的肌原纤维蛋白(如肌球蛋白、原肌球蛋白和肌钙蛋白)被收缩装置中的另一组取代。激素和神经支配在决定甲壳类和哺乳动物等不同生物肌肉中的纤维类型组成方面起着关键作用。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，钙依赖的信号转导通路包括钙/钙调蛋白依赖的磷酸酶钙调神经磷酸酶和钙依赖的激酶(如蛋白激酶C，CaM激酶)激活转录因子，在快速到缓慢的转化过程中驱动慢型基因的表达。本项目以美国龙虾Homarus americanus为实验对象，研究蜕皮激素(蜕皮类固醇)和运动神经元活性对肌原纤维蛋白表达的调节。随着幼体和幼体的同构爪子分化为成体的异形切割爪和碾碎爪，紧爪肌肉中的纤维经历了一种发育调节的变化。这种纤维转换发生在中央快纤维和周围慢纤维区域之间的边界上，因此，特定纤维的变化由其在肌肉中的位置决定。等电点将决定肌原纤维蛋白亚型在蜕皮周期中的时间和空间表达，以确定薄肌丝和厚肌丝蛋白是否协调表达。另一个目标是利用ATPase组织化学和原位杂交来确定长期升高的蜕皮类固醇对体内幼体龙虾转化的影响。由于初步结果表明蜕皮后发生转换(当血淋巴蜕皮类固醇水平较低时)，因此假设蜕皮类固醇水平升高会延迟或阻止转换。在体外，慢性低频刺激幼爪中的快纤维会引起由快到慢的转化。通过RT-PCR和原位杂交，将在单个纤维中检测到慢型基因的转录本。PI还将确定蜕皮激素和5-羟色胺如何调节电诱导的转化，并将使用药理试剂来确定介导转录激活的信号转导途径的成分。这项研究在理解无脊椎动物和脊椎动物纤维转化的控制方面具有广泛的应用。运动、运动神经元活动、停用、疾病和激素可诱导哺乳动物和鸟类骨骼肌的纤维转换，但其分子机制尚不清楚。长期目标之一是确定“主控调控因子”，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，对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的见解。

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.