EFFECT OF EXTRACELLULAR IONS AND MODULATORS OF CALCIUM-TRANSPORT ON SURVIVAL OF TERT-BUTYL HYDROPEROXIDE EXPOSED CARDIAC MYOCYTES

Objective: The aim was to investigate the effects of extracellular ions and agents that modify calcium translocating pathways on oxidative stress induced cell injury. Methods: 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. Results: 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 muM), nifedipine (1 muM), butanedione monoxamine (20 mM), and caffeine (10 mM) accelerated t-BHP-induced loss of rod shaped morphology. Ryanodine (1 muM), Trolox-C (1 muM), and butylated hydroxytoluene (BHT, 500 muM) 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 muM). Conclusions: 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.