The cardiac sodium channel plays an essential role in the normal propagation of electrical impulses and in abnormal conduction and arrhythmogenesis. Many ionic channels are regulated by cellular metabolic processes, and it has been shown that the sodium protein channel is an excellent substrate for phosphorylation by both and it has been shown that the sodium protein channel is an excellent substrate for phosphorylation by both adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase and protein kinase C. The purpose of this research is to investigate the hypothesis that the cardiac sodium channel is subject to metabolic control. Particular emphasis will be placed on the processes of slow and ultra-slow inactivation of the sodium current (INa), which are more prominent in depolarized or abnormal tissue and are likely targets for regulation. This is supported by preliminary data from our laboratory showing that the kinetics and extent of block of these slow processes by flecainide is modulated by the beta-adrenergic agonist isoproterenol. Examination of this hypothesis has heretofore not been possible because voltage clamp of INa has required lowering experimental temperature, which may obviously affect cellular metabolism. We can now voltage clamp 5-10 mu2 cell- attached patches with successful control of INa at 37degreesC and can measure macroscopic sodium currents in intact cells using physiologic solutions at body temperature for the first time. We will study the effects on macroscopic INa and single channel activity of important cellular signal transduction mechanisms, including cAMP, guanosine 3',5'- cyclic monophosphate (cGMP), phosphatidylinositol turnover, and guanine nucleotide-binding (G) proteins, in guinea pig and neonatal rat and mice ventricular myocytes. We will also determine whether observed pharmacologic effects are due to phosphorylation or to direct modulation of the channel. The approaches we will use in these studies include: 1) elevation of the intracellular concentration of a second messenger (e.g., with isoproterenol, nitrovasodilators, carbachol); 2) direct stimulation of protein kinases (using cAMP/cGMP analogs, phorbol esters); and 3) intracellular administration of specific compounds (e.g., the catalytic subunits of cAMP/cGMP-dependent protein kinases, inositol 1,4,5- trisphosphate). We will also investigate the influence of metabolic processes on the interaction between the cardiac sodium channel and flecainide, an antiarrhythmic agent whose block is a function of slow and ultra-slow inactivation. Ultimately, greater knowledge regarding control of INa and especially the slow inactivation processes could lead to the development of safer, more effective antiarrhythmic agents, since drugs that selectively enhance development of slow inactivation as a primary mechanisms for block would leave normal tissue relatively unaffected. During Phase I (2 years), the candidate will work in the Stahlman Cardiovascular Research Program, (SCRP), directed by Luc M. Hondeghem, M.D., Ph.D., refining her knowledge of the technique of whole-cell voltage clamp and patch clamp as well as intracellular perfusion. For Phase II (3 years), the candidate will continue work on the current proposal in her own laboratory in close collaboration with Dr. Hondeghem and other members of the SCRP.
