In Europe about 4 million people die every year from cardiovascular diseases. The mechanisms underlying heart failure are still poorly understood. Experiments proposed here test the hypothesis that periods of rest following mechanical stress result in a more physiological adaptation in cardiac myocytes, compared with continuous stress, which leads to pathological adaptation. Adaptation is proposed to be mediated, at least in part, by nucleo-cytoplasmic shuttling proteins which translocate to the nucleus in response to various stimuli to alter gene expression. Aim 1: Cultured cardiac neonatal myocytes will be exposed to continuous cyclic stretch or given a period of 8 hours rest every 16 hours. The 8 hours of rest is to mimic sleep. Then to measure genes normally associated with pathological adaptation. The expression of atrial natriuretic peptide, CaATPase (SERCA2a) and alpha skeletal muscle actin will be measured. Aim 2: Determine the expression and subcellular distribution of the nucleo-cytoplamic shuttling proteins TRIP6, LPP, Clock and PER2 proteins from neonatal cardiac myocytes in continuously stretched and rested cells. Aim 3: Determine the function of Clock and Period 2 proteins in response to continuous and intermittent hypertrophic stimuli by blocking their nuclear cycling. Nuclear cycling is blocked by synthetic peptides containing a membrane translocating motif and the nuclear localization signal for Clock or PER2. In a second series of experiments, the genes will also be knocked down with SiRNA. We expect to find that rest periods will prevent the increase in atrial natriuretic peptide /alpha skeletal muscle genes and the decline in CaATPase proteins seen in maladaptive hypertrophy after continuous mechanical stress. Studying the recuperative processes of myocytes during rest will provide a better understanding of the mechanisms underlying improved function of failing hearts following mechanical unloading by left ventricular assist devices.
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