The purpose of this study was to develop an isolated tissue model in which arrhythmic activity could be generated in response to conditions encountered in ischemia followed by reperfusion, and in which intracellular recordings could be used to identify and study arrhythmogenic mechanisms. Isolated canine Purkinje fiber-papillary muscle preparations were superfused with modified Tyrode's solutions. Tissues were exposed to conditions observed in ischemia (hypoxia, acidosis, elevated lactate, zero substrate for 40 minutes). Superfusion with Tyrode's solution of "normal" composition was then reinstituted. Transmembrane recordings from Purkinje and muscle tissues were made, using standard microelectrode techniques. Ischemic conditions caused loss of membrane potential, shortened action potentials, depressed excitability, and progressive bidirectional conduction block between muscle and Purkinje tissues. Spontaneous activity, probably reentrant in origin, was observed. Return to nonischemic conditions resulted in a multiphasic sequence of responses in Purkinje fibers: prompt hyperpolarization, progressive depolarization to unresponsiveness, and final repolarization to control. The depolarization phase was accompanied by oscillatory afterpotentials which initiated extrasystoles. Final repolarization included a phase of automaticity at low membrane potentials, during which Purkinje tissue functioned as a parasystolic focus. Elevation of potassium concentration to 10 mM during the ischemic period did not alter the sequence of electrophysiological events during ischemic conditions or upon reperfusion. This study demonstrates that ischemia followed by reperfusion elicits an orderly sequence of electrophysiological events which may constitute important mechanisms of arrhythmia in vivo.

Na+/H+ exchange, an electroneutral cotransport system, is activated by reperfusion of the ischaemic heart. While activation can restore intracellular pH following an acid load, the concomitant increase in intracellular Na+ can also aggravate existing derangements of ionic homeostasis, particularly with respect to calcium overload, and result in exacerbation and acceleration of tissue injury, a phenomenon which has been termed the pH paradox. In addition, Na+/H+ exchange has been shown to participate in the activation of both platelets and neutrophils, factors widely acknowledged to participate in ischaemic and reperfusion injury. All studies thus far reported (summarised in the table) have shown desirable and beneficial effects of Na+/H+ exchange inhibitors on various cellular processes which contribute to myocardial reperfusion injury. These multiple effects of Na+/H+ exchange inhibitors are unique and unmatched by any other group of pharmacological agents. They offer the hope of superior tissue protection and salvage, with limited potential for toxicity, following reperfusion protocols. We propose, therefore, that activation of the Na+/H+ exchanger mediates reperfusion injury and that suppression of the exchanger will be of superior benefit in reduction of such injury during restoration of flow. The rapid development of new and highly specific Na+/H+ exchange inhibitors offers substantial promise for the use of these agents as adjunct therapy in numerous reperfusion protocols.

Previous studies have shown that the inotropic response of the heart to beta-adrenergic stimulation declines with aging. This alteration has been attributed partly to an age-related impairment in the activation of the beta-adrenoceptor-G protein-adenylate cyclase complex. To further understand the mechanisms underlying the age-related deficit, the present study compared beta-adrenergic-mediated contractile response, cAMP accumulation, and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins in isolated perfused hearts from adult (6-8 months) and aged (28-30 months) Fischer 344 rats. In isometrically contracting, electrically paced (240 beats per minute) hearts perfused at constant flow rate (9 ml/min per gram ventricle), the baseline contractile performance differed significantly between adult and aged hearts. Thus, contraction duration was prolonged (approximately 15%, p < 0.001) in the aged relative to the adult heart, and this was due to increases in time to peak tension and relaxation time. Further, developed peak tension, normalized per gram ventricular wet weight, was significantly lower (approximately 20%, p < 0.05) in the aged compared with the adult heart. In these isolated perfused heart preparations, beta-adrenergic stimulation with isoproterenol (ISO, 0.001-1 microM) evoked concentration-dependent positive inotropic and lusitropic responses, both of which were significantly lower (15-20%, p < 0.05-0.001) in the aged compared with the adult heart. These age-related differences were manifested as relatively smaller ISO-induced increases in 1) developed peak tension, 2) maximum rate of tension development (+dT/dt), and 3) maximum rate of relaxation (-dT/dt) in the aged compared with the adult heart. The ISO-induced abbreviation of time to half relaxation was also less marked in the aged heart. Under similar experimental conditions, ISO (0.1 microM)-induced increase in tissue cAMP content was also lower (approximately 18%, p < 0.05) in the aged heart. ISO (0.1 microM)-induced phosphorylation of the sarcoplasmic reticulum protein phospholamban and myofibrillar protein troponin I was significantly diminished (approximately 38% and 25% decline, respectively, for phospholamban and troponin I; p < 0.05-0.001) in the aged compared with the adult heart. No significant age-related difference was, however, evident in ISO-induced phosphorylation of C protein of myofibrils. These data suggest that age-related decrements in beta-adrenergic-mediated cAMP accumulation and phosphorylation of phospholamban and troponin I contribute to the diminished contractile responses of the aged heart to beta-adrenergic stimulation.

Effects of cytochrome P-450 metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETS; 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET), were examined in isolated guinea pig hearts and ventricular myocytes. Addition of 1-16 ng/ml EETs to normal isolated hearts produced no effects on contractility or coronary pressure. In hearts subjected to 60 min of low-flow ischemia, impairment of contractility and declines in heart rate and coronary perfusion pressure were similar in the presence or absence of 1 ng/ml EETs. However, in the presence of either 5,6- or 11,12-EET, recovery was delayed for the first 10 min only. No significant differences were found in any group regarding heart rate, coronary perfusion pressure, or energy metabolite content after 30 min of reperfusion. In myocytes, both 5,6- and 11,12-EET (100 pg/ml, 1.0 ng/ml, and 20 ng/ml) significantly increased cell shortening as well as intracellular calcium concentrations, whereas 8,9- or 14,15-EET was without effect on these parameters. These results describe for the first time the direct effects of various EETs on cardiac cell function as well as their ability to modulate some of the myocardial responses to postischemic reperfusion. The results suggest a potential role for these substances in the response of the heart to pathological insult.

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The effects of the phorbol ester, phorbol myristate acetate (PMA) were examined on function and energy metabolism in the isolated working heart of the rat. At a concentration of 10−9 m. PMA produced a rapid loss in cardiac function in terms of aortic flow rate (AFR) and coronary flow rates (CFR) whereas a similar concentration of 4α‐phorbol 12,13‐didecanoate was ineffective. At a concentration of 10−10 m., the PMA‐induced depression was more gradual but nevertheless very pronounced with an almost total loss in AFR after 30 min perfusion. The reduction in CFR was more moderate than that observed with respect to AFR. The protein kinase C (PKC) inhibitor (±)‐l‐O‐hexadecyl‐2‐O‐acylglycerol significantly attenuated the loss in AFR and CFR following addition of PMA. Two inhibitors of Na+/H+ exchange, amiloride and quinacrine, totally prevented the reduction in AFR. Although the PMA‐induced depression in CFR was also attenuated by both amiloride and quinacrine, these effects were not significant, probably reflecting the less pronounced effect of PMA on this parameter. Nifedipine, a dihydropyridine calcium channel blocker reduced PMA toxicity to a similar degree as Na+/H+ exchange inhibition whereas the calcium channel agonist Bay K 8644 was without effect. Tissue content of energy metabolites including high energy phosphates, total adenine nucleotides or lactate were not significantly affected by PMA perfusion. We conclude that PKC activation is necessary for phorbol ester‐induced cardiac dysfunction. The consequence of PKC stimulation includes (1) Na+/H+ exchange activation and a subsequent elevation in intracellular calcium [Ca2+]i via Na+/Ca2+ exchange and (2) PKC‐dependent phosphorylation of the calcium channel, both of which would produce toxicity by elevation of [Ca2+]i. Pharmacological manipulation of any of these steps prevents PMA toxicity by virtue of a reduction in the accumulation of [Ca2+]i. PMA effects or their prevention are unrelated to any changes in energy metabolism.

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