The overall long-term objective of this project is to better understand the cellular mechanisms that control nter- and intracellular pH and to determine the effects of pH (extra- and intracellular) on cardiac myocyte calcium handling. The results will have considerable basic physiological importance and are highly relevant to cardiac pathophysiological conditions. Experiments will be performed on atrial and Purkinje myocytes using confocal imaging and epifluorescence techniques to measure intracellular pH, calcium and sodium (epifluorescence only) in individual cells. Flash-photolysis of caged protons will be used to generate regional ncreases in intracellular [H]. Fluorescence measurements will be coupled with current-clamp and voltage- clamp recordings of action potentials and whole cell membrane ionic currents, respectively.
The specific aims i nclude: 1. Characterize intracellular pH regulation in atrial and Purkinje cells. A detailed functional characterization of sarcolemmal pH control transporters will be performed. The results will be used to construct computational models of intracellular pH regulation. 2. Characterize electrical effects of electrogenic NBC in atrial and Purkinje cells. The relationship between NBC current and action potential repolarization and its modulation by internal pH will be determined. 3. Characterize intra- and intercellular proton diffusion in atrial and Purkinje cells. The relationship between intracellular pH and intracellular proton mobility will be determined, as well as the relationship between intracellular pH and junctional proton permeability/flux in myocyte pairs. The results will yield new insight concerning spatial control of pH in these cell types. 4. Characterize the effect of acidosis on calcium handling in atrial and Purkinje cells. The effects of whole- cell extra- and intracellular acidosis on the Ca transient, Ca current and SR Ca handling will be determined. The ability of the heart to generate electrical signals and contract is strongly influenced by the pH of the fluids inside and outside of individual cells. This research is designed to better understand the consequences of excessive accumulation of acid and base on heart cell function and to determine how the cells are able to regulate their internal pH.
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