Cellular edema is a critical aspect of myocardial injury. Defects in cell volume regulation are associated with cell damage and contractile and electrical dysfunction during cardioplegia (elective cardiac arrest), ischemia and reflow. Despite its importance, volume regulation by cardiac cells is not well understood. This presents an acute problem for cardioplegia because the solution perfusing the heart must be designed provide myocardial protection during arrest. The goal of these studies is to understand the mechanisms that regulate cardiac cell volume under cardioplegic and physiologic conditions. Processes underlying cell volume control will be identified from measurements of cell volume and determinants of the intracellular ion activities of K+, Na+, Cl- and H+ with ion-selective microelectrodes (ISE) in both isolated ventricular myocytes and papillary muscles from adult and neonatal rabbit hearts. Measurements of cell volume will be made with three independent methods: (1) video microscopy will 'optically section' myocytes; (2) volume will be determined from the resistance to current flow around a single myocyte held in a specially designed pipette; and (3) changes in cytoplasmic compartment volume will be determined by loading papillary muscles or myocytes with tetramethylammonium (TMA+) and measuring the trapped TMA+ with an ISE. We will: (1) Test the hypothesis that isosmotic cardioplegic solutions are anisotonic and cause cell volume to change. (2) Evaluate the role of Na+/K+/2Cl- cotransport, Na+/Cl- cotransport, Na+-K+ pump, Na+-H+ exchange, Cl--HCO3- exchange, and Na+-Ca2+ exchange in the maintenance of cell volume under physiologic and cardioplegic conditions and during osmotic stress. Transport processes will be identified from the effects of transport inhibitors and selective omission of transported ions from the bathing media on both cell volume and intracellular ion activities. (3) Assess the role of mobile anions and the [K+] [Cl-] product in setting cell volume. (3) Test the hypothesis that cell volume control is different in neonatal and adult cells. (4) Determine the effect of altering pHi and metabolic inhibition on cell volume. (5) In a working heart model, compare the efficacy of standard cardioplegic solutions to that of 'improved' solutions designed to better regulate cell volume. The combination of intracellular ion activity measurements with novel techniques for determining cell volume in isolated myocytes and papillary muscles can provide unique information about the response of cardiac cells to perturbations that alter cell volume and allow identification of solute transport pathways that contribute to regulation of cell volume and, ultimately, to the integrity of the cell membrane. The significance of these experiments is that they will provide insight into a basic, critical, physiological processes that are virtually unexplored in heart. Knowledge of the factors responsible for cell volume control is a necessary prerequisite for understanding pathophysiological disturbances of cell volume, such as those occurring during cardioplegia and infarction.
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