PHOSPHATASES IN EXCITATION/CONTRACTION COUPLING

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Cardiac contraction is triggered by voltage-gated Ca2+-influx across the sarcolemmal membrane and regulated by the subsequent cellular [Ca2+] I transient. This [Ca2+] I transient depends on the amplification of the triggering Ca2+ influx by SR Ca2+ release and is a central feature in excitation-contraction (EC) coupling. Using molecular and cellular tools, the applicant has started to examine this process and has identified quantitatively an important element: protein phosphatase modulation. Preliminary results show a nearly three-fold decrease in the [Ca2+] I transient that can be attributable solely to the phosphorylation-state of SR proteins. The applicant proposes to extend this preliminary work with the proposed work, identifying specific cellular and molecular features of the heart cell that are responsible. The project will focus on the following specific inter-related questions. (1) How do protein phosphatases affect EC coupling in adult and neonatal rat heart cells? The PI will use isolated patch-clamped single cells and indo-1 to examine how serine/threonine protein phosphatases (PPs) alter EC coupling. (2) What is the identity of active intracellular phosphatases in adult and neonatal heart cells? This series exploits a sensitive assay to identify specific protein phosphatase activity and determine subcellular location in cells being studies. (3) How does inhibition of specific intracellular phosphatases affect EC coupling? A gene transfection technique will be used to target the inhibition of specific PPs to address this question. (4) How are ~Ca2+ sparks~, the elementary SR Ca2+ release events, altered by changing levels of specific PPs? Patch clamped single heart cells loaded with Ca2+ indicator fluo-3 will be imaged using confocal microscopy. Manipulations of PP levels, based on the above studies, will be used to modulate EC coupling.