Regulation of Fiber Switching in Lobster Muscle

Colorado State University, Fort Collins CO

Investigators

Abstract

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.