Contractile function in ventricles surviving substantial myocardial infarction (MI) is improved by exercise training. In the current grant period, we have identified many Ca2+ regulatory pathways that are abnormal in MI myocytes and that a program of high intensity sprint training (HIST) instituted shortly after MI can reverse most of these abnormalities. However, it is difficult to unambiguously pinpoint which one of the many beneficial effects of HIST is predominant in bringing about improved contractility in MI myocytes. Based on our observations on contractile abnormalities in Ml myocytes, in this grant renewal we will manipulate gene expression to focus on two Ca2+ regulatory pathways: Na+/ Ca2+ exchanger (NCX1) and sarcoplasmic reticulum Ca2+ ATPase (SERCA2). We hypothesize that: (1) contractile abnormalities in MI myocytes are primarily mediated by subnormal NCX1 and/or SERCA2 function; (2) HIST restores cell shortening in MI myocytes to normal primarily by improving NCX1 and/or SERCA2 function; (3) improvement in NCX1 function in MI myocytes by HIST is mediated by down regulation of phospholemman (PLM); and (4) improvement in SERCA2 activity in Ml myocytes by HIST is mediated by down regulation of phospholamban (PLB). We have established a paced adult rat cardiac myocyte culture model to preserve normal contractility in cultured myocytes. We will demonstrate that myocytes isolated from Sham-Sedentary (Sed), MI-Sed, Sham-HIST, and MI-HIST rats retain their phenotypic differences in culture. We will then upregulate and downregulate NCX1, SERCA2, PLB, and PLM by adenovirus-mediated gene transfer. Edge-detection microscopy, whole cell patch clamp, microfluorimetry, and Western blots will be used to measure cell shortening, membrane currents and SR Ca2+ contents, Ca2+ transients, and gene expression, respectively. Finally, catheter-based, in vivo gene transfer will be used to selectively up- or downregulate Ca2+ homeostatic pathways in intact hearts. In vivo cardiac function will be followed by echocardiography. Successful exogenous gene expression will be verified by immunoblotting and contractile function examined in myocytes isolated from these hearts.
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