Positive changes in intrinsic cardiac contractility produced by endurance training appear to be associated with training-induced changes in sarcolemmal (SL) processes that are involved in the regulation of transmembrane Ca2+ dynamics. This implies that the magnitude and/or timecourse of the inward Ca2+ current (Ica) and subsequent cytosolic Ca2+ (Ca2+)c dynamics during EC coupling are modified by training. Changes in (CA2+)c regulation would not only affect cardiac contractility at the level of the thin filament, but would also be expected to affect other Ca2+-dependent processes. Major objectives of this proposal are to examine the effect of training on (1)ICa and (Ca2+)c dynamics during EC coupling and (2) the Ca2+-dependent phosphorylation of cardiac myosin P-light chain (PLC).
The specific aims of the proposed experiments are (a) to directly assess indices of contractility and to measure the timecourse and magnitude of ICa and (Ca2+)c changes occurring in electrically paced left ventricular (LV) cardiac myocytes isolated from sedentary and trained female rats. Whether or not training-induced changes in contractility are associated with changes in ICa and/or (Ca2+)c dynamics will become apparent. Myocyte contractility will be determined by measuring myocyte shortening dynamics using a novel on-line technique of parallel optical pressing which resolves high speed variation of parallel spacing (muscle striations). Whole cell patch clamp techniques will be used to assess the characteristics of ICa in LV myocytes and (Ca2+)c dynamics will be determined using fura-2 and a recently developed fluorescence microscopy system suitable for (Ca2+)c measurements in single cells; (b) to examine the relationship between myosin PLC phosphate content and contractility in intact myocardium and the effect of training on this relationship. The Ca2+-dependent phosphorylation of myosin PLC enhances submaximal force generation in chemically skinned cardiac fibers; experiments are designed to reveal if the contractility of intact myocardium is enhanced by myosin PLC phosphorylation and if training-induced changes in cardiac contractility are associated with changes in PLC phosphate content. Contractility and PLC phosphorylation dynamics will be examined using LV papillary muscles isolated from trained and sedentary rats; (c) to utilize the information resulting from Aims (a) and (b) in order to test and refine an existing kinetic model which describes the concerted dynamic behavior of the enzyme systems responsible for regulating cardiac PLC phosphate content.
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