Alterations in expression of sarcoplasmic reticulum calcium ATPase, and the sodium calcium exchanger have been reported to occur during ventricular hypertrophy, and could have functional effects which may contribute to the progression from hypertrophy to failure. Work described in this project will test the hypotheses that 1) sarcoplasmic reticulum function is decreased, and 2) that sodium calcium exchange activity is increased in intact single ventricular myocytes isolated from failing ventricular myocardium. Ventricular myocytes will be obtained from normal and failing mouse, rabbit, and human ventricular myocardium. Ventricular myocytes from human myocardium will be isolated using a novel technique which employs a vibratome to cut 400 mum thin sections, in the presence of BDM to inhibit cutting injury. This facilitates enzymatic dissociation of Ca2+-tolerant cells from small biopsy specimens. Sodium calcium exchanger activity will be quantitated by measuring the maximum sodium calcium exchange current per unit cell surface area produced by abrupt exposure of voltage clamped isolated myocytes to zero sodium solution containing 2.7 mM calcium. A novel fast solution switcher is used to produce a change in the medium bathing a single myocyte within 7 milliseconds in these experiments. Sarcoplasmic reticulum function in isolated single myocytes will be assessed by inducing contraction of myocytes by abrupt exposure zero sodium, zero calcium KCI solution, and then measuring the rate of relaxation, and the rate of fall in cytosolic free calcium. The latter will be measured with indo-1. This approach allows assessment of the rate at which the sarcoplasmic reticulum can sequester calcium within a single ventricular myocyte under conditions in which, after initial calcium release is triggered by transient influx of calcium from the extracellular space, the sodium calcium exchanger is disabled by the elimination of extracellular sodium. In addition, in myocytes in which SR function is studied, the contractility of isolated myocytes will be assessed by measurement of fractional shortening at different pacing rates, with a video motion detection. This approach should allow determination of the extent to which isolated myocyte contractile performance is impaired in myocytes obtained from failing myocardium, as well as the possible contributions of altered sodium calcium exchanger and SR function.

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National Heart, Lung, and Blood Institute (NHLBI)
Specialized Center (P50)
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University of California San Diego
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