Heart disease is the leading cause of death in diabetic patients, and considerable evidence is now available to support the existence of a specific diabetic cardiomyopathy that is independent of coronary artery disease and hypertension. Functional and biochemical data acquired from multicellular cardiac preparations of diabetic animals support the view that cellular mechanisms controlling cytosolic Ca2+ on a beat-to-beat basis are abnormal and contribute to impaired relaxation. The goal of this project is to characterize diabetes-induced changes in the expression and function of Ca2+ regulating proteins in isolated cardiac myocytes, and to determine the role of hyperglycemia in the pathogenesis and pathophysiology of diabetic cardiomyopathy. To test the hypothesis that abnormal Ca2+ handling occurs at the single cell level, biophysical assessment of excitation-contraction coupling will be carried out in ventricular myocytes isolated from diabetic rats. Voltage clamp techniques will be used to determine whole-cell Ca2+ and Na/Ca exchange currents as a measure of sarcolemmal L-type Ca2+ channel and Na/Ca exchanger function. Video edge-detection and Ca2+ fluorescence measurements (fura-2) will be used as additional means to evaluate Na/Ca exchange and to analyze SR Ca2+ ATPase and ryanodine receptor function. Abundance of mRNA for these Ca2+ regulating proteins will be determined using northern blot analysis and amount of protein will be assessed by western blot analyses. To test the hypothesis that hyperglycemia influences the expression and function of these Ca2+ regulating proteins, isolated ventricular myocytes from both diabetic and nondiabetic animals will be maintained in a """"""""diabetic-like"""""""" medium (high glucose) in short-term primary culture. We will determine the specific role of hyperglycemia on expression, modification and function of calcium regulating proteins. Preliminary data show that within days, normal myocytes cultured in the """"""""diabetic-like"""""""" medium exhibit prolonged relaxation, prolonged action potential durations, and slowed cytosolic Ca2+ clearing, similar to that of diabetic myocytes. These experiments will provide important new insights into the pathogenesis and early events of diabetic cardiomyopathy. Taken together, these experiments will resolve the role of Ca2+ regulating proteins in diabetic cardiomyopathy by studying their function and expression at the single cell level. The adult myocyte culture system provides a well-defined method from elucidating fundamental mechanisms of high glucose that may contribute to the development of myocyte dysfunction in diabetes.
