The long-term objective of this research is to understand the factors which influence and regulate the permeability of cardiac gap junctions. We will test the hypothesis that the regulatory pathways which operate to maintain junctional permeability under normal conditions fail during ischemia, leading to arrhythmias and irreversible injury. Primary cultures of neonatal rat myocytes and isolated gap junctions will be used to examine the regulation by calcium and protons of intercellular coupling and channel conductance, respectively. The impact of ischemia on intercellular coupling will also be examined. Specifically, in the intact cells the effects of several interventions designed to increase intracellular (Ca2+), (H+), or both on junctional permeability, and cellular calcium and proton levels will be addressed using pharmacological interventions to inhibit the organelles' functions. Alteration of intracellular coupling will be measured using dual cell voltage clamping techniques, or by measurement of dye transfer rates. Using patch clamp techniques, the effects of these ions and of lipid composition on the conductance of isolated channels will be determined, and the results compared to what is observed in the intact cells. The effects of ischemia on intracellular ion levels and cellular contractile activity will be correlated with alterations of junctional conductance and ion fluxes. Finally the role of lipid peroxidation in the generation of the ischemic effects will be examined. These studies will test several hypotheses. 1) That cardiac gap junctions are sensitive to both Ca2+ and H+, and that these ions can act in a synergistic fashion to block permeability. 2) That intracellular organelles serve to protect the junctions from elevated cytoplasmic levels of Ca2+ and H+. 3) That failure of gap junctions contributes to the onset of post-ischemic arrhythmias. 4) That post-ischemic failure of gap junctions occurs as a result of high intracellular levels of Ca2+ and/or protons or to damage to cellular membranes (sarcolemma, mitochondria, sarcoplasmic reticulum). Realization of our objectives will lead to a better understanding of the factors which influence gap junction function and of the role of gap junctions in the occurrence of arrhythmias.
