Protective effects against hydrogen peroxide-induced toxicity by activators of the ATP-sensitive potassium channel in isolated rat hearts.

Activation of ATP-sensitive (KATP) channels has been shown to exert protective effects on the ischemic and reperfused myocardium. Reactive oxygen species are thought to mediate, at least in part, this form of cardiac injury. Using isolated perfused rat hearts, we therefore studied whether KATP activation exerts any effect on the direct deleterious effects of either 200 microM hydrogen peroxide or a free radical generating system consisting of purine plus xanthine oxidase in terms of function and energy metabolite status. On their own, hydrogen peroxide or the combination of purine plus xanthine oxidase treatment resulted in a time-dependent depression of myocardial contractility, which reached over 90% after 30 min perfusion, an effect which was associated with approximately 1000% elevation in left ventricular end-diastolic pressure (LVEDP). The KATP channel opener cromakalim (0.5 microM) significantly attenuated the hydrogen peroxide-induced loss in systolic function throughout the treatment period, and reduced the elevation in LVEDP with significant attenuation 10, 15 and 20 min after hydrogen peroxide addition. Contractile dysfunction produced by hydrogen peroxide was associated with significantly reduced tissue ATP, creatine phosphate and glycogen content to approximately 70, 60 and 70% of control, respectively. The depletion of these metabolites was significantly attenuated to 35, 23 and 23% of control, respectively, in the presence of cromakalim. The protective effects of cromakalim against contractile dysfunction, as well as depletion in intermediary energy metabolites, was abolished in the presence of the KATP channel antagonist glibenclamide (1 microM). However, glibenclamide on its own failed to alter the cardiac response to hydrogen peroxide with respect to any parameter. The responses to the free radical generating system consisting of purine plus xanthine oxidase was unaffected by cromakalim. Our study shows that KATP channel activation selectively protects against the cardiotoxic influence of hydrogen peroxide, and may explain, in part, the salutary effects of KATP activators in myocardial ischemia.

Modulation of the electrophysiological effects of ischemia reperfusion by methylisobutyl amiloride.

We studied the effect of the Na+/H+ exchanger inhibitor methylisobutyl amiloride (MIA, 1 microM) on action potential characteristics and arrhythmias induced by: (a) reperfusion following regional ischemia in rat hearts and (b) realkalization after lactate acidosis in rabbit hearts. We also determined the effect of MIA on the incidence of transient inward currents (ITIs) induced by acidosis-realkalization in rabbit cardiocytes. Ligation of the LAD coronary artery for 10 min depolarized the resting potential from -78 +/- 1.9 mV to -66.9 +/- 1.0 mV and depressed the action potential but did not induce overt arrhythmias. Delayed afterdepolarizations were observed during ischemia in 50% of untreated hearts whereas reperfusion produced severe ventricular tachyarrhythmias in all of them. MIA reduced the incidence of arrhythmias to 27% and their duration to less than 1 min. MIA increased action potential duration by 38 +/- 4.1%. BaCl2 produced a similar APD lengthening and had an antifibrillatory effect. Acidic reperfusion induced bradycardia and reduced severity of arrhythmias. In rabbit hearts, MIA increased the action potential duration by 61 +/- 4.3% and abolished arrhythmias on realkalization. Eleven out of 18 cells developed transient inward currents during acidosis-realkalization and seven of them underwent irreversible injury. MIA prevented the appearance of ITIs, had no effect on ICa,L but decreased the outward component of IK1 by 50%. Our results suggest that the protective effect of MIA is in part due to changes in cellular electrical activity that modulate Na+ and Ca2+ entry via different pathways.

Transient ischemia in the presence of an adenosine deaminase plus a nucleoside transport inhibitor confers protection against contractile depression produced by hydrogen peroxide. Possible role of glycogen.

We previously reported that adenosine A1 receptor activation protects against the cardiodepressant effects of hydrogen peroxide in isolated rat hearts. The present study examined whether a transient ischemic period of 5 min duration, which preconditions the heart against ischemic and reperfusion-induced dysfunction, can bestow protection against 30-min exposure to hydrogen peroxide in isolated rat hearts. Transient ischemia on its own failed to alter the cardiac response to hydrogen peroxide. However, when transient ischemia was carried out in the presence of the nucleoside transport inhibitor S-(4-Nitrobenzyl)-6-thioguanosine and the adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)adenine, a significant attenuation of the hydrogen peroxide-induced loss in contractility was evident and this was associated with significant preservation of tissue glycogen content. The protective effect of the transient ischemia/drug combination on both functional changes and glycogen levels was abolished by the adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine as well as by glibenclamide, a blocker of the ATP-sensitive potassium channel (KATP). To further assess the role of glycogen in the protection against hydrogen peroxide, we compared the effects of the adenosine A1 agonist N6-cyclopentyl adenosine (CPA) and insulin. While both treatments protected against hydrogen peroxide the effect of insulin was superior to any other treatment. Moreover, while all protective modalities preserved glycogen stores after hydrogen peroxide treatment, the protection afforded by insulin was also associated with significantly elevated glycogen levels prior to hydrogen peroxide administration. No protection by either CPA or insulin was evident in the absence of exogenous glucose. Taken together, our results demonstrate that a brief period of ischemia with concomitant administration of agents which increase interstitial adenosine levels protects against hydrogen peroxide toxicity. The effect is mediated by activation of adenosine A1 receptors and is linked to KATP stimulation. Moreover, our results are strongly suggestive of an important role of glycogen preservation in bestowing protective effects against hydrogen peroxide cardiotoxicity.

Signal transduction mechanisms in the ischemic and reperfused myocardium.

The cellular mechanisms regulating myocardial dysfunction during ischemia and subsequent reperfusion are complex. As can be determined from this review, it is clear that signal transduction pathways are altered during these conditions, which may explain, in part, the pathophysiology of ischemia and reperfusion. With respect to beta-adrenoceptor signal transduction, adaptive changes during ischemia and reperfusion ensure that this critical pathway for the regulation of cardiac function remains intact. Additionally, although the relative contribution of alpha 1-adrenoceptors to the regulation of cardiac function is minimal in normal myocardium, these receptors clearly exacerbate conditions associated with the generation of arrhythmias during reperfusion. It is likely that this enhancement of arrhythmogenesis is related to the activation of NHE by a PKC-dependent mechanisms. The importance of non-receptor-mediated signal transduction as a mediator of ischemia and reperfusion injury has long been established with respect to products of membrane lipid breakdown. As discussed, recent evidence now suggests that other compounds formed during ischemia and reperfusion, such as reactive oxygen species and NO, are also linked to cellular second messenger systems. In conclusion, as signal transduction is critical for normal myocardial function, signal transduction pathways are of even more importance during ischemia and reperfusion. There is an increasing interest in the role of non-receptor-mediated signal transduction as a mediator of ischemia and reperfusion injury and it is hoped that these pathways may represent new levels for therapeutic intervention.

Age-related changes in electrophysiological responses to muscarinic receptor stimulation in rat myocardium.

Recent studies have demonstrated that the negative chronotropic and inotropic responses of the heart to cholinergic muscarinic receptor stimulation are strikingly enhanced with aging in the rat model. The present study investigated the electrophysiological basis of this phenomenon by determining the effects of a muscarinic receptor agonist, carbachol, on transmembrane action potential parameters in right atrial tissue and right ventricular free wall preparations from adult (6-8 months old) and aged (26-28 months old) Fischer 344 rats. In addition, the effect of carbachol on atrioventricular conduction time (AVT) was determined in isolated perfused beating hearts. The results showed the following. The baseline maximum diastolic potential (MDP: adult, -76.4 +/- 1.8 mV; aged, -66.8 +/- 1.5 mV; p < 0.05; n = 5) but not the action potential duration measured at 95% repolarization (APD95: adult, 40.0 +/- 5.0 ms; aged, 47.4 +/- 6.7 ms; n = 5) differed significantly in aged compared with adult atrium. No significant age-related difference was evident in baseline MDP measured in ventricular epicardium (adult, -69.8 +/- 0.5 mV; aged, -69.0 +/- 1.1 mV; n = 6) or endocardium (adult, -72.5 +/- 1.4 mV; aged, -73.0 +/- 1.2 mV; n =6). The baseline action potential duration measured at 50% repolarization (APD50) differed significantly with age in ventricular endocardium (adult, 11.6 +/- 2.2 ms; aged, 23.0 +/- 4.6 ms; p < 0.05; n =6) but not in epicardium (APD50: adult, 8.1 +/- 0.4 ms; aged, 13.0 +/- 2.3 ms; n = 6). Superfusion with carbachol (0.1 nM - 10 mu M) resulted in concentration-dependent hyperpolarization of MDP in atrium; the magnitude of hyperpolarization differed significantly with age (2.5-fold higher in the aged; p < 0.05; n = 5). Carbachol caused concentration-dependent shortening of APD50; this effect differed significantly with age in the ventricle (2-fold greater in the aged; p < 0.05; n = 6) but not in the atrium. Carbachol prolonged the AVT in atrial-paced (240 beats/min) hearts; the magnitude of carbachol-induced increase in AVT did not differ significantly with age. These results are consistent with the possibility that in the aging heart, greater hyperpolarization at the level of the right atrium (likely involving pacemaker cells) and greater shortening of APD50 at the level of ventricular myocytes may contribute to the enhanced cholinergic-triggered bradycardia and negative inotropic response, respectively.

Role of protein kinase C in mediating effects of hydrogen peroxide in guinea-pig ventricular myocytes.

The present study examined the effects of hydrogen peroxide (H2O2) on intracellular calcium transients and unloaded cell shortening in the presence of the protein kinase C (PKC) inhibitors 1-(5-isoquinolinesulfonyl-2-methylpiperazine (H7) or chelerythrine chloride (CHC) or the PKC activator phorbol 12-myristate 13-acetate (PMA). Calcium transient amplitudes and cell shortening were measured simultaneously in single, enzymatically dissociated ventricular myocytes loaded with fura2-AM. Exposure of myocytes to H2O2, 25 microM or 75 microM, for 15 min caused a time- and concentration-dependent increase in calcium transient amplitude, cell shortening and the diastolic 340/380 fluorescence ratio. Significant increases in calcium transient amplitude were observed from 7 to 15 min of superfusion with 25 microM H2O2 and the transient amplitude remained elevated throughout the 10 min washout period. In the presence of 75 microM H2O2, transient amplitude was elevated following 2 min and remained elevated for the remainder of the experiment. Significant increases in cell shortening were also observed from 7 to 15 min in the presence of either 25 or 75 microM H2O2. This effect was reversed upon washout of the lower concentration of H2O2 but persisted during the initial 5 min of washout at the higher concentration. The diastolic 340/380 fluorescence ratio was unaltered in the presence of 25 microM of H2O2, however this parameter was significantly increased from 7 to 15 min following exposure to 75 microM H2O2 and remained elevated throughout the washout period. The H2O2-induced increases in calcium transient amplitude and cell shortening were significantly attenuated in myocytes which were pretreated with either H7 or CHC.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of sodium-hydrogen exchange activity in cardiac myocytes during acidosis and realkalinisation: effects on calcium, pHi, and cell shortening.

OBJECTIVE: The aim was to examine the effects of the Na+/H+ exchange inhibitor methylisobutylamiloride (MIA) as well as protein kinase C, a putative regulator of Na+/H+ exchange, on intracellular calcium, intracellular pH, and unloaded cell shortening in isolated guinea pig cardiac myocytes subjected to lactic acid induced acidosis followed by realkalinisation. METHODS: Calcium transient amplitude and cell shortening were measured simultaneously in single isolated myocytes loaded with fura2-AM. Intracellular pH was measured in cells loaded with BCECF-AM. RESULTS: Exposure of cells to 5 min of lactate (20 mM) acidosis (pH 6.8) caused an increase in calcium transient amplitude and a decrease in cell shortening and intracellular pH. During realkalinisation (pH 7.3), the calcium transient gradually decreased while intracellular pH became more alkaline than pre-acidosis values. The cells underwent transient hypercontractility as evidenced by a marked increase in systolic cell shortening and a decrease in diastolic cell length. Inhibition of sodium/hydrogen exchange with MIA (1 microM) caused a significant attenuation of the increase in calcium transient amplitude during acidosis and further depressed cell shortening as well as intracellular pH. In addition, MIA significantly attenuated hypercontractility and abolished cell contracture upon realkalinisation. In contrast, phorbol 12-myristate 13-acetate (10(-12) M) exerted no effects on the response to acidosis; however, this treatment exacerbated cell hypercontractility and reduced functional recovery upon realkalinisation. CONCLUSIONS: Inhibition of Na+/H+ exchange activity during acidosis/realkalinisation enhances recovery of cell function.

Adenosine-sensitive alpha 1-adrenoceptor effects on reperfused ischaemic hearts: comparison with phorbol ester.

1. We have examined the effects of the alpha 1-adrenoceptor agonists, phenylephrine or methoxamine, on contractility in rat and rabbit isolated hearts as well as their effects on postischaemic ventricular recovery. We compared these effects to those of 12-phorbol 13-myristate acetate (PMA), a direct activator of protein kinase C (PKC). 2. The positive inotropic effect of alpha 1-receptor agonists was significantly attenuated in the presence of the Na/H exchange inhibitor, methylisobutyl amiloride (MIA, 1 microM), whereas the positive inotropic effect of PMA was unaffected. 3. Reperfusion of rat hearts subjected to either 30 or 60 min of zero-flow ischaemia, resulted in recovery of contractility to 91 +/- 2% and 57 +/- 7% of the preischaemic values, respectively which was unaffected by phenylephrine. In contrast, PMA at a concentration (10 pM) devoid of direct depressant effects, significantly decreased recovery following 60 min of ischaemia to 31 +/- 4% of pre-ischaemic value (P < 0.05 from control); an effect which was completely prevented by the PKC inhibitor, bisindolylmaleimide. A similar inhibitory effect of PMA and lack of effect of phenylephrine were seen in reperfused rabbit hearts. 4. As alpha 1-receptor activation has been shown previously to stimulate cardiac adenosine production, we assessed whether blockade of adenosine A1 receptors with the specific antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.5 microM) would unmask the actions of phenylephrine in hearts subjected to 30 min ischaemia and reperfusion. In the presence of DPCPX, phenylephrine reduced recovery to 44 +/- 9% compared to 82 +/- 10% recovery in the absence of phenylephrine (P < 0.05). Identical results were observed in rabbit hearts treated with DPCPX in which recovery was reduced from 57.1 +/- 11.2% to 17.8 +/- 6.8% by phenylephrine (P < 0.05). Another A1 receptor antagonist, (+/-)-N6-endonorbornan-2-yl-9-methyladenine (N-0861, 0.5 microM) produced virtually identical results to those observed with DPCPX. 5. MIA failed to modulate the inhibition of postischaemic recovery by phenylephrine. Bisindolylmaleimide, on the other hand, partially prevented the effects of phenylephrine on postischaemic contractile dysfunction. The inhibitory effect of either PMA or phenylephrine on postischaemic recovery of both rat and rabbit hearts was generally dissociated from alterations in energy metabolism, although in the case of rat hearts, inhibition by phenylephrine was associated with diminished high energy phosphate content. 6. Our results demonstrate that both alpha 1-receptor activation as well as direct activation of PKC with phorbol ester can attenuate post-ischaemic ventricular recovery. Moreover, our results strongly suggest that endogenous adenosine protects the heart against the deleterious effects of alpha 1-receptor activation during ischaemia and reperfusion.

Protective effects of the potent Na/H exchange inhibitor methylisobutyl amiloride against post-ischemic contractile dysfunction in rat and guinea-pig hearts.

We studied the effects of the potent Na/H exchange inhibitor methylisobutyl amiloride (MIA, 1 microM) on post-ischemic ventricular recovery and energy metabolic status in spontaneously contracting, isolated rat and guinea-pig hearts subjected to 45 min zero-flow ischemia followed by reperfusion. For both species, MIA was added either 15 min prior to ischemia and was present throughout reperfusion or was added at the time of reperfusion only. In control rat hearts, force recovery after 30 min of reperfusion was 25.6 +/- 6.0% of the pre-ischemic value whereas in hearts pre-treated with MIA recovery was enhanced to 55.4 +/- 9% (P < 0.05). Elevation of resting tension during the first 20 min of reperfusion was also significantly reduced by MIA pre-treatment. When MIA was added at the time of reperfusion only, recovery was generally lower than that seen with MIA pre-treatment although significantly higher values were seen through much of the reperfusion period. In rat hearts, MIA reduced the time required for return to sustained contractile recovery particularly in those hearts where the drug was added prior to ischemia (control, 11.4 +/- 2.7 min; MIA, 2.6 +/- 0.5 min, P < 0.05). Similar effects of MIA pre-treatment were seen in guinea-pig hearts in terms of contractile recovery, time to recovery and reduction in resting tension although MIA addition at the time of reperfusion was without beneficial effect either on the magnitude of contractile recovery or time required for restoration of function. In guinea-pig hearts, recovery of function was accompanied by substantial bradycardia. However, maintenance of ventricular rate through electrical pacing exerted no significant influence on the protective effects of MIA pre-treatment. There was no effect of MIA on energy metabolites in reperfused rat hearts or paced guinea-pig hearts, although in spontaneously contracting guinea-pig hearts improved recovery of function was associated with significantly higher levels of high energy phosphates. No effects of tissue metabolites were seen in ischemic non-reperfused hearts irrespective of treatment. The protective effects of MIA were not related to diminished release of creatine kinase during reperfusion. Our results demonstrate marked protective effects of MIA, on the reperfused rat and guinea-pig myocardium. These studies also demonstrate, for the first time, that the effects of amiloride analogues are not species specific and further support the concept that Na/H exchange inhibition may represent an effective therapeutic approach for the protection of reperfused cardiac tissue.

Role of Na+/H+ exchange in cardiac physiology and pathophysiology: mediation of myocardial reperfusion injury by the pH paradox.

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.

Effects of epoxyeicosatrienoic acids on isolated hearts and ventricular myocytes.

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.

Age-related alterations in the phosphorylation of sarcoplasmic reticulum and myofibrillar proteins and diminished contractile response to isoproterenol in intact rat ventricle.

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.

Positive and negative inotropic effects of phorbol 12-myristate 13-acetate: relationship to PKC-dependence and changes in [Ca2+]i.

The present study examined the concentration-dependent effects of phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, on contractile force and [Ca2+]i in guinea-pig hearts and isolated cardiac myocytes, respectively. Contractile force was measured using isolated Langendorff-perfused hearts while [Ca2+]i was measured independently in isolated cardiac myocytes loaded with fura2-AM. Phorbol 12-myristate 13-acetate, as well as another PKC-activating phorbol, phorbol dibutyrate (PDBu), and two non-PKC-activating phorbols, alpha-phorbol didecanoate (alpha PDD) and 4 alpha-phorbol, exerted time- and concentration-dependent effects on contractility. A significant positive inotropic response was observed with either PMA (10(-12) M; 5-15 min of perfusion) or PDBu (10(-12) M; 5 min of perfusion). In contrast, 10(-10) M PMA caused a significant negative inotropic effect following 30 min of perfusion while 10(-8) M PMA produced a significant negative inotropic effect which occurred earlier (10 min) and was sustained throughout the 30 min perfusion period. A similar negative inotropic effect was seen with 10(-8) M of either PDBu or alpha PDD. In addition, 4 alpha-phorbol (10(-8) M) exerted a modest, but significant negative inotropic effect following 25 and 30 min of perfusion. Both concentration-dependent increases and decreases of +dF/dt and -dF/dt were observed in the presence of PMA. In addition, both PMA and PDBu caused a concentration-dependent increase in coronary perfusion pressure. The positive inotropic responses and coronary perfusion pressure effects elicited by PMA and PDBu were largely prevented by the addition of the PKC inhibitors H7 (6 nM) or HAG (10 nM); however, these drugs were without effect on the negative inotropic response to higher concentrations of both PKC-activating (PMA, PDBu) and non-PKC-activating (alpha PDD, 4 alpha-phorbol) phorbol compounds. The lowest concentration of either PMA or PDBu (10(-12) M) increased the 340/380 fluorescence ratio of isolated cardiac myocytes loaded with fura2-AM on a time scale similar to that at which the positive inotropic response was seen in the whole heart. However, in contrast to results in the isolated heart, PDBu elicited a greater and sustained increase in the fluorescence ratio measured in isolated cardiac myocytes. The higher concentration of either PMA or PDBu (10(-8) M), resulted in a decrease in the 340/380 ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Potential cellular mechanisms of hydrogen peroxide-induced cardiac arrhythmias.

The electrophysiologic effects of hydrogen peroxide on the isolated guinea pig right ventricular free wall were studied using simultaneous recordings of action potentials from the epicardium and the endocardium. Exposure to hydrogen peroxide caused a time- and concentration-dependent change in action potential characteristics. Action potential durations at 50 and 90% of repolarization (APD50 and APD90, respectively) were significantly prolonged by hydrogen peroxide in both the epicardium and the endocardium. Although prolongation occurred at lower concentrations (0.5 mM) in the epicardium, increases in APD in response to higher concentrations of hydrogen peroxide (1 or 4 mM) were maintained for a longer period of time in the endocardium. In addition, hydrogen peroxide (1 or 4 mM) caused significant depolarization in the epicardium after 10 min, although this effect was observed only in the endocardium exposed to 4 mM hydrogen peroxide. Ventricular arrhythmias were observed in 5 of 7, 6 of 7, and 7 of 7 preparations exposed to 0.5, 1, and 4 mM hydrogen peroxide, respectively. The most frequently observed electrophysiologic abnormalities were associated with increased automaticity. Coupled beats, including clearly identifiable early and delayed depolarizations, were also observed. Verapamil (2 microM) and amiloride (0.1 mM) reduced both the incidence and the duration of hydrogen peroxide-induced arrhythmias but did not influence the effects on APD. This study is the first demonstration of hydrogen peroxide-mediated transmural dispersion in APD that could play an important role in the development of ventricular arrhythmias. In addition, our results demonstrate that hydrogen peroxide can induce ventricular arrhythmias through several cellular mechanisms, including increased automaticity, coupled beats, and triggered activity.

Protective effects of D,L-carnitine against arrhythmias induced by lysophosphatidylcholine or reperfusion.

Electrophysiological effects of lysophosphatidylcholine (50 or 100 microM) and D,L-carnitine (100 microM) were studied under control conditions and in response to simulated ischaemia and reperfusion using the superfused right ventricular free wall preparation from the guinea pig heart. Lysophosphatidylcholine, 100 microM, induced a significant depolarization of the maximum diastolic potential (MDP) in the epicardium, as well as the development of ventricular premature beats, salvos and ventricular tachycardia. Both coupled beats and abnormal automaticity were observed in lysophosphatidylcholine (100 microM)-treated preparations. Carnitine (100 microM) alone had no effect on preparations superfused with normal Tyrode solution. However, it delayed the time to onset and reduced the cumulative duration of lysophosphatidylcholine-induced arrhythmias (P less than 0.05). The incidence of lysophosphatidylcholine-induced abnormal automaticity and salvos was also significantly decreased in the presence of carnitine. Twenty minutes of simulated ischaemia caused depolarization of MDP as well as prolongation followed by block of transmural conduction. Lysophosphatidylcholine (100 microM) did not alter this response however, carnitine significantly reduced ischaemia-induced depolarization in the epicardium. All control preparations developed arrhythmic activity during 30 min of reperfusion. Carnitine accelerated recovery of MDP in the epicardium upon reperfusion, prolonged the time to onset of arrhythmic activity and reduced both its cumulative duration and incidence. In contrast, reperfusion in the presence of lysophosphatidylcholine (100 microM) significantly increased the incidence of arrhythmic activity. Carnitine exerted only minimal antiarrhythmic action when preparations were exposed to reperfusion in the presence of lysophosphatidylcholine. In conclusion, this study demonstrates that carnitine can modify various cellular mechanisms of arrhythmia induced by lysophosphatidylcholine or by reperfusion but is much less effective when lysophosphatidylcholine and reperfusion are combined.

Positive inotropic effects of low concentrations of leukotrienes C4 and D4 in rat heart.

We examined the effects of leukotrienes (LT) B4, C4, D4, and E4 (0.010-2.5 ng/ml) on contractile and coronary function in isolated rat hearts. Concentration-dependent effects were examined either by the cumulative addition of LTs or by addition of specific concentrations to individual preparations. Neither LTB4 nor LTE4 produced myocardial or coronary effects at any concentration, irrespective of addition protocol. At 0.010 ng/ml, both LTC4 and LTD4 produced an increase in force that was associated with a 30% elevation in coronary pressure. Further cumulative addition of either leukotriene resulted in a negative inotropic effect and a further increase in coronary pressure. In contrast, following single additions of LTC4 or LTD4 (0.01-0.50 ng/ml) a positive inotropic effect and an increased coronary pressure were observed. LTC4 or LTD4 at 0.5 ng/ml produced a negative inotropic effect in hearts pretreated with 0.01 ng/ml of LTD4 or LTC4, respectively. Reversal of this addition protocol resulted in a negative inotropic effect of either 0.01 ng/ml LTD4 or LTC4. Verapamil and nifedipine significantly attenuated the positive inotropic and coronary constricting effect of 0.5 ng/ml LTC4 and LTD4. The addition of either LT following BAY K 8644 resulted in a negative inotropic effect, in contrast to the positive inotropic influence seen with leukotriene alone. Our results demonstrate a positive inotropic effect of low concentrations of LTC4 and LTD4 concomitant with coronary artery constriction, a phenomenon determined by leukotriene addition protocols and suggestive of LTC4/LTD4 receptor interaction. The effects of calcium channel antagonists and BAY K 8644 on the inotropic response suggest a leukotriene-mediated activation of the calcium channel resulting in increased intracellular calcium concentrations.

Protective effects of phosphatidylcholine against mechanisms of ischemia and reperfusion-induced arrhythmias in isolated guinea pig ventricular tissues.

The effects of exogenous phosphatidylcholine (PC) on some potential mechanisms of ischemia and reperfusion-induced arrhythmias were tested using the superfused right ventricular free wall of the guinea pig. Exposure of the preparation to simulated "ischemia" (hypoxia, acidosis, glucose deprivation and hyperkalemia) resulted in several electrophysiological derangements, including a marked depolarization of the maximum diastolic potential (MDP) in both endocardium and epicardium, shortening of the action potential duration (APD), and prolongation of the transmural conduction time followed by transmural conduction block. In a few preparations, coupled beats were also observed. Reperfusion was associated with arrhythmic activity in all preparations. Both the characteristics and the severity of reperfusion-associated arrhythmias were dependent upon the duration of the preceding "ischemia". In hearts exposed to ischemic conditions for 40 min, transmural conduction block persisted until 45 min of reperfusion and no electrical activity was present in the epicardium during this time. However, both coupled beats as well as abnormal automaticity were observed in the endocardium. When the period of "ischemia" was reduced to 20 min, recovery from transmural conduction block occurred sooner and coupled beats and abnormal automaticity were detected in both epicardial and endocardial layers. Superfusion with PC during both "ischemia" and reperfusion (PC1 group), or during reperfusion only (PC2 group), significantly altered the response of the preparations to reperfusion. Following 40 min "ischemia", preparations treated with PC recovered from transmural conduction block more rapidly (PC1 group, 4 min, P less than 0.05; PC2 group, 23 min, ns), compared to control.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms for cardiac depression induced by phorbol myristate acetate in working rat hearts.

1. 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. 2. 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 4 alpha-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. 3. The protein kinase C (PKC) inhibitor (+/-)-1-O-hexadecyl-2-O-acylglycerol significantly attenuated the loss in AFR and CFR following addition of PMA. 4. 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. 5. Nifedipine, a dihydropyridine calcium channel blocker reduced PMA toxicity to a similar degree as Na+/N+ exchange inhibition whereas the calcium channel agonist Bay K 8644 was without effect. 6. Tissue content of energy metabolites including high energy phosphates, total adenine nucleotides or lactate were not significantly affected by PMA perfusion. 7. 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 [Ca21]i. Pharmacological manipulation of any of these steps prevents PMA toxicity by virtue of a reduction in the accumulation of [Ca21]i. PMA effects or their prevention are unrelated to any changes in energy metabolism.