SIMULATED ISCHEMIA AND REPERFUSION IN ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES

Objective: The objectives were (1) to develop a cellular model of simulated ischaemia and reperfusion in isolated ventricular myocytes; (2) to determine effects of simulated ischaemia and reperfusion on calcium current (I-Ca), transient inward current (I-Tl) and contraction; and (3) to determine whether pharmacological agents which alter intracellular sodium and calcium loading affect signs of calcium overload in reperfusion in this model. Methods: Electrical activity was recorded with conventional and voltage clamp techniques. Cell shortening was measured with a video edge detector. Myocytes were equilibrated in Tyrode solution, exposed to simulated ischaemia (hypoxia, acidosis, lactate, hyperkalaemia, glucose-free) for 20 min, and reperfused with Tyrode solution. Results: Ischaemia depolarised myocytes [-89(SEM I) to -67(4) mV, p<0.05], abbreviated action potential duration [APD(90), 257(14) to 188(12) ms, p<0.05], and abolished contractions. Contractions elicited by voltage clamp steps also were abolished in ischaemia; however, I-Ca decreased by only 51% [-0.98(0.08) to -0.50(0.06) nA, p<0.05]. Signs of calcium overload, including aftercontractions, oscillatory afterpotentials, and I-Tl, occurred in 69% of myocytes in reperfusion. Upon reperfusion, both APD(90) and I-Ca recovered slowly; however, contractions returned quickly and temporarily exceeded control. Amiloride during ischaemia and reperfusion lowered incidence of I-Tl in reperfusion, whereas nifedipine and lignocaine had no effect on I-Tl. Conclusions: This model of ischaemia and reperfusion in ventricular myocytes shows many features of multicellular preparations, such as membrane depolarisation and action potential duration shortening during ischaemia, and appearance of oscillatory afterpotentials upon reperfusion. Inhibition of contraction during ischaemia and recovery of contraction in reperfusion are independent of changes in APD(90) or I-Ca. Induction of aftercontractions, oscillatory afterpotentials, and I-Tl in reperfusion is associated with reduced peak I-Ca. Amiloride most probably decreased signs of calcium overload in early reperfusion by inhibiting sodium loading via Na+/H+ exchange. Additionally, amiloride may inhibit I-Tl directly by blocking Na+/Ca2+ exchange.