Clive M. Baumgarten - US grants

The proposed research will examine the basis for membrane potential (Em) in the atrio-ventricular node (AVN) of rabbit heart. Em in the AVN is lower than in atrium, ventricle or Purkinje fiber and is relatively insensitive to changes in extracellular potassium activity. These findings have been explained by postulating that intracellular chloride activity is greater than required by passive distribution at Em and that PCl/PK, the ratio of chloride permeability to potassium permeability, is relatively high. However, neither intracellular chloride activity nor PCl/PK are known. Alternatively, as I have postulated here, the intracellular potassium activity may be lower than in other cardiac tissues, and it may be strongly dependent on extracellular potassium activity. The experiments proposed will critically test these two hypotheses. Potassium ion-selective microelectrodes and chloride ion-selective microelectrodes will be used to measure the intracellular activities of potassium and chloride directly. PCl/PK will be calculated. The relationships between Em and both extracellular potassium and chloride activities will be determined. The control of intracellular potassium activity will be studied by varying its extracellular activity. The control of intracellular chloride activity will be studied by varying the extracellular activities of potassium and chloride. These experiments will lead to an understanding of the basis of Em in AVN and will provide the baseline data necessary to understand more complex electrical, pharmacological, and pathological events.

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.

lipid metabolism; heart metabolism; cellular pathology; cell morphology; ion transport; stretch receptors; myocardial ischemia /hypoxia; cell osmotic pressure; reperfusion; membrane permeability; cardiac myocytes; video microscopy; heart cell; laboratory rabbit; microelectrodes; voltage /patch clamp; fluorescent dye /probe;

DESCRIPTION (the applicant's description verbatim): Congestive heart failure (CHF) induces significant changes in cardiac myocyte size. Increased myocyte volume (hypertrophy) ultimately requires intracellular accumulation of osmolytes and water. Intracellular osmolarity is regulated in myocytes by multiple mechanisms, including transmembrane flux of ions through channels that are sensitive to changes in cell volume. We discovered that one of these ionic currents, the cell swelling-activated Cl- current (IC,lswell) is chronically activated under isosmotic conditions in ventricular myocytes from dogs with tachycardia-induced and rabbits with aortic regurgitation-induced CHF. Furthermore, we showed that the activity of ICl,swell and cell volume in CHF and control myocytes were regulated by protein kinase C (PKC) and protein phosphatases thought to control phosphorylation of ion channels responsible for lCl,swell. The overall objective is to understand how Icl,swell and cell volume are regulated in volume and pressure overload models of CHF and how hormonal and autocrine-paracrine factors implicated in the genesis of CHF contribute to this regulation. The effects of catecholamines, autocrine-paracrine factors including angiotensin II and cardiotrophin-1, and selected growth factors on Icl,swell and cell volume will be examined. Intracellular signaling pathways, including protein kinase C, tyrosine kinases, mitogen-activated protein kinases, and phosphatases, will be examined to evaluate their influence on lCl,swell and myocyte volume. Perforated patch voltage clamp and digital video microscopy will be used concurrently to quantify ionic currents and their effect on cell volume. Single myocytes isolated acutely from either sham operated or CHF animals will be studied because these cells better reflect the in vivo state during CHF than do cell culture models. Because no single model of CHF fully represents clinical CHF, pressure, tachycardia, and volume overload models of CHF will be used. Where appropriate, the effect of interventions on cell signaling pathways will be confirmed with western blot with phospho-antibodies. The following questions will be addressed: 1. Are lCl,swell behavior and its effect on myocyte volume different in pressure than volume overload CHF? 2. Is Icl,swell activated prior to onset of clinically apparent CHF in pressure and volume overload models? 3. Are lCl,swell and myocyte volume regulated by autocrine-paracrine factors that are important in the genesis of CHF? 4. Do intracellular signaling pathways that are important in CHF influence lCl,swell and myocyte volume? Knowledge of swelling-activated ion currents and how they influence myocyte volume in CHF may provide important insights into the pathophysiology of tachyarrhythmias and contractile and diastolic dysfunction that occur in CHF. Further, this work may lead to new approaches to treat or prevent CHF and thereby, reduce the morbidity and mortality of this common disease.