We propose to study alterations in Ca2+ homeostasis and excitation- contraction coupling that occur in isolated ventricular myocytes as a consequences of hypertrophy and/or failure. Our studies will utilize transgenic mice with heart failure; two novel rabbit models of heart failure and hypertrophy (pacing-induced heart failure, and rabbit myocardial infarction); and myocytes isolated from biopsy specimens obtained from human patients with normal ventricular function, and with severe heart failure. The cytosolic Ca2+ concentration will be quantitated by the fluorescent Ca2+ indicator fluo-3, intracellular [Na+] by SBFI, and contraction and relaxation by video motion analysis. Voltage clamp studies in single myocytes will be employed to quantitative Na/Ca exchanger density, L-type Ca2+ channel function, and SR Ca2+ content. Function of the SR CA ATPase will be assessed by the rate of sequestration of Ca2+ in single myocytes when the Na/Ca exchanger is disabled by techniques involving the use of a rapid solution switcher device. Function of the Ca2+ in single myocytes when the Na/Ca exchanger is disabled by techniques involving the use of a rapid solution switcher device. Function of the Ca2+ release channel, the ryanodine receptor, will be assessed by measuring whole cell Ca2+ gain (the rate of change in Ca2+ concentration divided by the magnitude of the L-type Ca2+ current), and Ca2+ spark morphology and probability determined with line scan confocal microscopy. Tissue activity of the calcium- calmodulin dependent kinase, CaM kinase II, will be measured in intact tissue experiments. These techniques will be employed to examine: the mechanisms and time course by which cytoskeletal abnormalities cause hypertrophy and failure; the mechanisms of heart failure produced by packing-induced failure, and the alteration in [Ca2+] homeostasis in peri- infarct myocytes; the extent to which abnormal Ca2+ homeostasis is restored by enhancement of SR Ca ATPase function (phospholamban knockout, transfection with adenoviral vectors driving the expression of adenyl cyclase); the extent to which failing human myocytes have alterations in SR Ca ATPase and Na/Ca exchanger activity similar to those observed in animal models of failure; and the extent to which decreased activation of CaM kinase II, perhaps induced by reduction of the magnitude of the [Ca2+] transient, is an important factor in contributing to the progression of heart failure. These studies build on and extend our previous work with these different systems over the past four years, during the initial cycle of our heart Failure SCOR grant.
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