Changes in intracellular pH occur under pathophysiologic conditions such as myocardial ischemia, hypoxia or acidosis that are associated with depressed myocardial function. The studies proposed seek to gain further understanding of the mechanisms involved in myocardial acid-base homeostasis and pH-induced changes in myocardial contractility. Intact cultured and freshly dissociated cardiac myocytes will be used for these studies because they allow accurate sequential measurements of transsarcolemmal ion fluxes, pHi, cytosolic free (Ca) and contractile state. Metabolic or respiratory acidosis or alkalosis will be produced using HEPES-buffered media with pH values ranging from 5.0 to 9.0, media with varied HCO3 or CO2 content, or NH4Cl. Using 45Ca flux measurements, we will first test the hypothesis that changes in pHo or pHi affect transsarcolemmal Ca movement in cultured cardiac myocytes. We will examine in relation to contractile responses, changes in pHo or pHi on 1) unidirectional Ca fluxes via slow Ca channels, Na-Ca exchange and the sarcolemmal Ca pump; 2) net Ca fluxes; and 3) cellular Ca pools. We will also test the hypothesis that Ca-H interactions occur within cardiac myocytes and examine the properties of this interaction. The Ca-H interaction within the cells will be studied by monitoring (Ca)i as pHi is varied and vice versa, using recently developed pH- and Ca-sensitive fluorescent dyes (BCECF and Fura-2). (Ca)i will be altered by exposure to low Nao, Ko, or Cao media, cardiac glycosides, or Ca channel agonists or antagonists. pHi will be altered using NH4Cl, or by varying CO2 content with adjusted (HCO3). Whether Na-H exchange plays a significant role in pH-mediated changes in Ca fluxes and (Ca)i will also be examined using ethylisopropylamiloride, a potent inhibitor of Na- H exchange, and by direct measurement of 24Na flux via Na-H exchange. The potential roles of sarcoplasmic reticulum and mitochondria in Ca-H interaction will be examined using specific agents that alter Ca movements in these organelles. Additional studies using experimental conditions to alter pHo alone or pHi alone will test the hypothesis that both pHo and pHi determine the inotropic state of cardiac myocytes during disturbances of acid-base balance. These studies are expected to provide important additional knowledge on the effect of pH on Ca homeostasis and the underlying mechanisms involved in acidosis- or alkalosis-mediated changes in myocardial contractility.
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