SIMULATED ISCHEMIA AND REPERFUSION IN ISOLATED GUINEA-PIG VENTRICULARMYOCYTES

Objective: The objectives were (1) to develop a cellular model of simu lated 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 intracellu lar sodium and calcium loading affect signs of calcium overload in rep erfusion in this model. Methods: Electrical activity was recorded with conventional and voltage clamp techniques. Cell shortening was measur ed with a video edge detector. Myocytes were equilibrated in Tyrode so lution, exposed to simulated ischaemia (hypoxia, acidosis, lactate, hy perkalaemia, glucose-free) for 20 min, and reperfused with Tyrode solu tion. 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-C a decreased by only 51% [-0.98(0.08) to -0.50(0.06) nA, p<0.05]. Signs of calcium overload, including aftercontractions, oscillatory afterpo tentials, and I-Tl, occurred in 69% of myocytes in reperfusion. Upon r eperfusion, both APD(90) and I-Ca recovered slowly; however, contracti ons returned quickly and temporarily exceeded control. Amiloride durin g 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 depolari sation and action potential duration shortening during ischaemia, and appearance of oscillatory afterpotentials upon reperfusion. Inhibition of contraction during ischaemia and recovery of contraction in reperf usion are independent of changes in APD(90) or I-Ca. Induction of afte rcontractions, 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 lo ading via Na+/H+ exchange. Additionally, amiloride may inhibit I-Tl di rectly by blocking Na+/Ca2+ exchange.