Andrew Ziman, Ph.D. - US grants

DESCRIPTION (provided by applicant): Skeletal muscle functions by translating electrochemical signals into physical movement through a process known as excitation-contraction (EC) coupling. EC-coupling involves the coordinated functions of the transverse tubules, sarcoplasmic reticulum (SR) and sarcomeres, mediated by Ca2+, and operates with great speed and efficiency because of the high degree of organization of key EC-coupling components. The Bloch laboratory has provided evidence of structural links between the sarcomere and the network compartment of the SR, governed by the binding of obscurin, which surrounds the sarcomere, and a small splice form of ankyrin1 (sAnk1, ank1.5), which is an integral protein of the SR membrane. Preliminary experiments suggest that an acute reduction in sAnk1 alters the concentration and distribution of SERCA, the primary protein responsible for cytosolic [Ca2+] removal, and disrupts the organization of the network SR in mature muscle fibers without having a significant effect on the organization of the junctional SR. The overall goal of this proposal is to address the hypothesis that sAnk1 is required for the normal assembly and maintenance and function of the SR in skeletal muscle. To assess the physiological role of sAnk1 in skeletal muscle EC- coupling, I will inhibit the expression of sAnk1 utilizing a specifically targeted siRNA construct characterized in the Bloch laboratory and re-express wild type sAnk1 protein after knockdown. I have developed a method for analyzing the dynamics of SR lumenal [Ca2+] in mammalian skeletal muscle by filling the SR lumen with the fluorescent Ca2+ indicator, Fluo-5N. I propose to use these reagents and methods to address two specific aims: (1) to characterize the lumenal Ca2+ stores in myofibers isolated from the flexor digitorum brevis muscle of the rat, including assays of the continuity of the stores by fluorescence recovery after photobleaching (FRAP), and Ca2+ release by activators of the ryanodine receptor and by electrical stimulation;and (2) to characterize the effects of altering the expression of sAnk1 on these properties of the lumenal Ca2+ stores. In addition to measuring changes in Ca2+ dynamics induced by knocking down and recovering sAnk1 protein expression, I will learn and then use molecular biological and biochemical methods to analyze changes in expression of relevant proteins (i.e. SERCA) brought about by re-expression of wild type sAnk1 after knockdown predict that even low level reductions in sAnk1 will disrupt EC-coupling by disrupting the normally continuous SR lumen and also by preventing efficient SR Ca2+ uptake through the apparent disruption of SERCA. The disrupted EC-coupling should be recovered by the re-expression of wild type sAnk1. These experiments should provide a quantitative assessment of the SR lumenal Ca2+ stores in adult skeletal myofibers and on the role of sAnk1 and its interaction with obscurin in their assembly and maintenance. They should also provide a rigorous test of my hypothesis. PUBLIC HEALTH RELEVANCE: My research will explore the role of a small intracellular membrane protein in skeletal muscle in organizing the stores of calcium that are necessary for muscle contraction. My studies should provide insights into the mechanisms underlying the organization of the calcium stores, changes in which have been implicated in a range of diseases in heart and skeletal muscle.