In skeletal muscle, SR Ca2+ release is controlled by the ryanodine sensitive Ca2+ release channel (ryanodine receptor; RyR). Important insights into the molecular basis of RyR function have been gained by examining elementary Ca2+ release events (ECRE); discrete, highly localized fluorescent transients in myocytes loaded with the Ca2+ indicator Fluo-4 . It is accepted that ECRE arise from small groups of RyR's which act as functional Ca2+ release units (CRU's). Recent consideration of the molecular architecture of the CRU in the context of the biophysical properties of ECRE have resulted in theories to explain the local modulation of RyR dependent Ca2+ release; all concluding that there is a dynamic modulation within and between RyRs that can regulate Ca2+ release at the level of the CRU. Despite these recent theories, and the growing body of knowledge on the molecular basis of RyR dependent Ca2+ release, a paucity of data exists on the molecular function of RyR secondary to muscle disorders/diseases for which altered SR Ca2+ has been identified.
Specific Aim 1 : Determine the role of luminal SR [Ca2+ ] on RyR dependent Ca2+ release within skeletal myocytes. To this end we will use established SR depletion and loading techniques to experimentally vary the SR Ca2+ content. We will then use Ca2+ sparks as a functional assay of the """"""""local activation of RyR as well as examining """"""""global"""""""" SR Ca2+ flux by evaluating whole cell Ca2+ release transients.
Specific Aim 2 : Evaluate the consequences of muscle fatigue and subsequent modified SR content on the properties of Ca2+ sparks. Based on a luminal model for RyR modulation, alterations in the releasable pool of SR Ca2+ should be reflected in the rate of occurrence or spatiotemporal properties of Ca2+ sparks as established in the first Aim. These experiments will employ in-vitro fatigue protocols of differing severity and will subsequently evaluate any decline in the rapidly releasable pool of Ca2+ in relation to the rate of occurrence and spatiotemporal properties of Ca2+ spark ECRE. ? ? ?
