Effect of extracellular ions and modulators of calcium transport on survival of tert-butyl hydroperoxide exposed cardiac myocytes.

Abstract

The aim was to investigate the effects of extracellular ions and agents that modify calcium translocating pathways on oxidative stress induced cell injury. Survival of cardiac myocytes exposed to tert-butyl hydroperoxide (t-BHP, 0.2 to 2 mM) was estimated by their ability to maintain rod shaped morphology and exclude trypan blue, and by the release of lactate dehydrogenase (LDH). Mean life time (mu) of a myocyte was analysed in accordance with the Weibull distribution. In pCa 3.0 Ringer's solution, t-BHP exposed myocytes underwent time and concentration dependent hypercontracture. The fraction of rod shaped cells excluding trypan blue correlated with LDH release. The lifetime of a cell was increased upon reduction of [Ca]o. Myocytes exposed to t-BHP in pCa 7.0 or 8.0 Ringer's solution attained a rigor state and became permeable to trypan blue. t-BHP-induced hypercontracture was delayed on increasing [K]o or [Na]o. Reduction of [Na]o to 67.5 mM, but not to 13.5 mM, accelerated t-BHP induced hypercontracture. Early reduction of [Ca]o to a pCa of 8.0 had no protective effect if 1 mM calcium was reintroduced after half the duration of exposure. Verapamil (10 microM), nifedipine (1 microM), butanedione monoxamine (20 mM), and caffeine (10 mM) accelerated t-BHP-induced loss of rod shaped morphology. Ryanodine (1 microM), Trolox-C (1 microM), and butylated hydroxytoluene (BHT, 500 microM) had no effect on survival of t-BHP exposed myocytes. Survival was prolonged upon preincubation of the myocytes with BAPTA-AM or desferrioxamine or when the cells were exposed to t-BHP in the presence of La (1 mM) or N-propyl gallate (10 microM). Ferrous catalysed free radical reactions initiated by t-BHP lead to hypercontracture or rigor shortening of myocytes, depending upon [Ca]o. It is unlikely that t-BHP-induced hypercontracture is mediated by Ca influx through L- or T-type Ca channels or due to release of Ca from intracellular stores. Activation of Ca influx via Na-Ca exchange and/or increase in the passive membrane permeability to Ca is consistent with the observations reported. Myocyte rigor observed upon attenuation of Ca overload suggests contribution of Ca-independent processes.