CA++ HOMEOSTASIS IN ISOLATED HYPERTROPHIED HEART CELLS

Temple University, Philadelphia PA

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Abstract

DESCRIPTION (adapted from the applicant's description): The overall hypothesis of this proposal is that alterations in myocyte calcium handling are centrally involved in the abnormal contractility of failing myocytes and contribute to the progressive decline of cardiac function in CHF. Two specific hypotheses will be tested. The first hypothesis is that the "signature" defects in calcium handling in CHF are due to an increase in the relative transport rate and capacity of the Na/Ca exchanger (NCX) with respect to the sarcoplasmic reticulum (SR) rather than an increase or decrease in the absolute rate or capacity of either transport mechanism. This will be tested by (1) measuring the calcium transport capacities of the SR and NCX in single myocytes from normal, hypertrophied and failing feline hearts and from non-failing, failing and left ventricular assisted device-supported (LVAD) human hearts, and (2) manipulating the calcium transport capacity of the SR and/or NCX in feline and human myocytes transfected with SERCA2a, native or mutant phospholamban (PLB), and/or NCX. The second hypothesis is that calcium influx via reverse-mode NCX makes a greater than normal contribution to excitation-contraction (EC) coupling and SR calcium loading because NCX density increases and the density of the L-type calcium channels and t-tubules decreases in CHF. This will be tested by (1) determining if calcium influx via reverse mode NCX modulates calcium-induced calcium release (CICR), (2) determining if calcium influx via reverse mode NCX replaces calcium entry via L-type calcium channels as the primary source of trigger calcium in CICR, and (3) measuring the amount of L-type calcium current, NCX current and SR calcium loading that is lost when T-tubules are disrupted by osmotic shock from the surface membranes of non-failing and failing feline and human myocytes.