Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis.

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.

Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes.

OBJECTIVES: The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes. BACKGROUND: Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells. However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility. METHODS: Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent. RESULTS: Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner. The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate). These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters. The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes. Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes. KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects. CONCLUSIONS: These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.

Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes.

BACKGROUND: Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury. METHODS AND RESULTS: Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine. CONCLUSIONS: The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.

Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts.

OBJECTIVES: The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND: The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS: Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS: In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS: The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.

Myocardial stretch induced by increased left ventricular diastolic pressure preconditions isolated perfused hearts of normotensive and spontaneously hypertensive rats.

OBJECTIVE: The aim of our study was to determine whether myocardial stretch (non-ischemic stress) could precondition isolated perfused hearts of both normotensive Wister-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: The perfused hearts in Langendorff mode were subjected to 30 min of global no-flow ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) were measured. In the control group, LVEDP was set at 10 mmHg. In the stretch group, LVEDP was increased to 30 or 60 mmHg for 5 min before 30 min of ischemia. In the ischemic preconditioning group, the hearts were exposed to two cycles of a 5-min period of ischemia before 30 min of ischemia. Myocardial lactate contents were measured at the baseline and at the end of the 60 mmHg stretch. RESULTS: Hemodynamic parameters of LVDP and LVEDP at 30 min of reperfusion improved in the stretch group (LVEDP of 60 mmHg) and the ischemic preconditioning group. Coronary flow did not decrease during the stretch. Recovery of the coronary flow during reperfusion was better in the stretch and ischemic preconditioning groups. Postischemic contractile function was better in WKY rats than in SHR. Myocardial lactate contents at the end of 60 mmHg stretch were negligible. CONCLUSIONS: Myocardial stretch induced by increasing LVEDP preconditioned isolated perfused hearts of both WKY rats and SHR, via mechanisms not involving myocardial ischemia during stretch.

Estimation of coronary flow reserve with the instantaneous coronary flow velocity versus pressure relation: a new index of coronary flow reserve independent of perfusion pressure.

Our goal was to develop a new method to evaluate coronary flow reserve independent of perfusion pressure by equipping dogs with a Doppler flow probe in the left anterior descending artery. The slope of the linear portion of the velocity-pressure loop in the late diastolic phase was defined as VP slope; the hyperemic:basal ratio of the VP slope was defined as VP slope ratio, and the hyperemic:basal ratio of the mean coronary blood flow velocity was defined as coronary flow reserve (CFR). We measured VP slope ratio and CFR, altering aortic pressure by aortic banding and inferior vena cava occlusion. VP slope ratio was independent of aortic pressure, but CFR increased with increments in aortic pressure. The VP slope ratio and CFR decreased in the presence of coronary stenoses. In conclusion, VP slope ratio is considered to be a physiologic index of the severity of coronary stenosis, which is independent of perfusion pressure. This measure is easily applicable in clinical practice.

Effects of glibenclamide and nicorandil in post-ischaemic contractile dysfunction of perfused hearts in normotensive and spontaneously hypertensive rats.

OBJECTIVE: We have demonstrated previously that nicorandil, an ATP-sensitive potassium channel opener, improved post-ischaemic contractile dysfunction of perfused hearts in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats dose-dependently. This study aimed to characterize the effect of glibenclamide, an ATP-sensitive potassium channel blocker, and nicorandil in post-ischaemic contractile dysfunction of SHR and WKY rats. METHODS: The perfused hearts were subjected to 30 min of global ischaemia and then 30 min of reperfusion. Administration of 10 or 50 mumol/l glibenclamide or of a combination of glibenclamide and 300 mumol/l nicorandil was performed for 10 min before the ischaemia. The left ventricular developed pressure and end-diastolic pressure were measured. RESULTS: Postischaemic contractile function was better in WKY rats than it was in SHR. Neither glibenclamide nor a combination of glibenclamide and nicorandil influenced the postischaemic contractile function or increased the incidence of reperfusion arrhythmias. The recoveries of coronary flow and heart rate after reperfusion were poor and the incidence of reperfusion arrhythmias was low in SHR. CONCLUSIONS: These results suggest that nicorandil improves postischaemic contractile dysfunction via a mechanism involving ATP-sensitive potassium channel opening both in SHR and in WKY rats. The hypertensive hearts were more susceptible to cardiac reperfusion dysfunction, compared with normal hearts.

Effect of ischemic preconditioning on mitochondrial oxidative phosphorylation and high energy phosphates in rat hearts.

The ability of ischemic preconditioning (IP) to protect the myocardium against prolonged ischemia may derive from improved energy balance. We therefore examined myocardial energy metabolism and mitochondrial oxidative phosphorylation in isolated perfused rat hearts which were either subjected (IP group), or not subjected (control group), to preconditioning prior to 30 min sustained ischemia and 30 min reperfusion. Preconditioning was achieved with two cycles of 5 min ischemia followed by 5 min reperfusion. Recovery of myocardial function was significantly greater, and creatine kinase release was significantly lower, in the IP group. Although ATP hydrolysis during the sustained ischemia remained unchanged in both groups, greater preservation of high energy phosphate (eg. ATP and CP) was observed in the IP group after reperfusion. CP content immediately after preconditioning greatly exceeded pre-ischemic values. Lactate production during the sustained ischemia was significantly lower in the IP group, suggesting a decrease in anaerobic glycolysis and a probable attenuation of intracellular acidosis. Oligomycin-sensitive mitochondrial ATPase activity in the control group was significantly decreased both after the sustained ischemia and the reperfusion, but in the IP group it did not change after the preconditioning, sustained ischemia, or reperfusion. Although atractyloside-inhibitable adenine nucleotide translocase activity was markedly decreased during sustained ischemia in both groups, its activity was significantly higher after reperfusion in the IP group. These data suggest that (1) mitochondrial ATPase contributes only slightly to ATP depletion during sustained ischemia, (2) both the CP overshoot phenomenon and the decrease in anaerobic glycolysis can be attributable to cardioprotection during the sustained ischemia, and (3) the preservation of ATPase and adenine nucleotide translocase activities may be a possible explanation for the restoration of high energy phosphates after sustained ischemia-reperfusion injury in the preconditioned hearts of rats.

Effects of disopyramide on the atrial fibrillation threshold in the human atrium.

The effects of disopyramide on the atrial fibrillation threshold (AFT) in the human atrium were investigated. To evaluate atrial vulnerability, the following electrophysiologic parameters were measured before and after the administration of disopyramide (2 mg/kg) in 12 patients with paroxysmal atrial fibrillation: The right atrial effective refractory period (ERP) and percentage maximum atrial fragmentation (%MAF) were measured by atrial premature stimulation based on a cycle length of 500 ms. The inter-atrial conduction time (ACT) was measured by burst pacing (120/min) for 30 s. AFT was measured by applying a high-frequency (50 Hz) stimulation for 1 s given at the right atrial appendage. AFT was defined as the lowest intensity of electrical current that could induce atrial fibrillation lasting for more than 30 s. Disopyramide significantly reduced %MAF, and prolonged ERP and ACT. AFT was measured in all patients and the mean AFT was 3.1 +/- 1.7 mA. After the administration of disopyramide, AFT significantly increased to 6.1 +/- 3.6 mA. There was a positive correlation between ERP and AFT, and a negative correlation between %MAF and AFT. No correlation was detected between ACT and AFT. In conclusion, disopyramide increased AFT in the human atrium.

Effect of doxorubicin on postrest contraction in isolated rat hearts.

Clinical use of doxorubicin is limited by its cardiotoxicity. In doxorubicin-induced cardiomyopathy, vacuolization of the sarcoplasmic reticulum (SR) has been reported. We investigated whether doxorubicin had a direct action on the sarcoplasmic reticulum (SR) in isolated perfused rat hearts. The left and right atria were trimmed to maintain heart rate (HR) <200 beats/min. Postrest contractions, which are believed to be due primarily to Ca2+ release from the SR, were evoked with a programmable stimulator after variable rest intervals. The amplitude of the postrest contractions increased markedly as the rest interval increased. Doxorubicin (0.1 mM) significantly suppressed this potentiation of the postrest contractions. Furthermore, doxorubicin slowly induced aftercontractions and an increase in left ventricular diastolic pressure (LVDP), phenomena that are usually associated with ouabain intoxication. We conclude that doxorubicin-induced cardiomyopathy may be due to SR dysfunction, leading to intracellular Ca2+ overload.

Kinetics of frequency-dependent conduction delay by class I antiarrhythmic drugs in human atrium.

We investigated use-dependent prolongation of interatrial conduction time (IACT) by class I antiarrhythmic drugs in 16 patients. Changes in IACT at the initiation of atrial pacing were used to evaluate the onset kinetics. We examined recovery kinetics by giving a single extra stimulus with a varying coupling interval after discontinuing train stimulation. Time constants of the onset kinetics were 1.52 +/- 0.15/n(fast) and 0.087 +/- 0.031/n(slow) for mexiletine, 0.075 +/- 0.015/n for aprindine, 0.078 +/- 0.019/n for disopyramide, and 0.050 +/- 0.006/n for pilsicainide. The recovery time constants were 203 +/- 66 ms for mexiletine, 1,021 +/- 162 ms for aprindine, 993 +/- 101 ms for disopyramide, and 2,930 +/- 569 ms for pilsicainide. Class I antiarrhythmic drugs produced use-dependent IACT prolongation in humans, with characteristic kinetics for each agent similar to that of depression of the maximum upstroke velocity of cardiac action potential (Vmax) reported in in vitro studies.

Effect of nicorandil on cardiac dysfunction during reperfusion in normotensive and spontaneously hypertensive rats.

The cardioprotective effect of nicorandil, an opener of ATP-sensitive potassium channels, was studied in the isolated perfused hearts of the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat. The hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. Controls received no drug. In the nicorandil group, the hearts were treated with 0.03 to 0.3 mmol/L nicorandil for 15 min before ischemia. Left ventricular developed pressure (LVDP) and end diastolic pressure (LVEDP) at 30 min of reperfusion were significantly lower and larger, respectively, in SHR than in WKY rats. Nicorandil improved LVDP and decreased LVEDP at 30 min of reperfusion in both SHR and WKY rats dose-dependently. The hypertensive heart in the early stage is already susceptible to reperfusion-cardiac dysfunction. Nicorandil has a beneficial effect on the post-ischemic dysfunction in both SHR and WKY rats.

Effects of E-4031 on atrial fibrillation threshold in guinea pig atria: comparative study with class I antiarrhythmic drugs.

The effects of E-4031, a new class III antiarrhythmic agent, on atrial fibrillation threshold (AFT), atrial effective refractory period (ERP), and interatrial conduction time (ACT) were investigated in Langendorff-perfused guinea pig hearts; the results were then compared with those of the class I agents disopyramide, procainamide, lidocaine, and flecainide. Whole guinea pig hearts were perfused with Tyrode's solution containing acetylcholine (ACh 3 x 10(-7) M). The three indexes were measured before and after administration of the test drugs, using right atrial extrastimulus and 50-Hz continuous stimulation. Disopyramide, procainamide, and flecainide (> or = 10(-6) M) significantly increased AFT. Although E-4031 (> or = 3 x 10(-6) M) also increased AFT, this effect was less potent than that observed with the other drugs. E-4031 (> or = 10(-6) M) significantly prolonged ERP, and this prolongation was less pronounced than that observed with disopyramide but similar to that observed with procainamide or flecainide. E-4031 did not affect ACT, and the greatest prolongation of ACT was observed with flecainide. Lidocaine had no effect on any of the indexes. These findings suggest that in guinea pig hearts E-4031 exerts an antifibrillatory effect by prolonging atrial ERP alone, but this effect is less pronounced than that observed with class I drugs, because AFT measured by 50-Hz continuous stimulation is influenced by both ERP and ACT.

Effects of ischemic preconditioning on the release of cardiac troponin T in isolated rat hearts.

The aim of this study was to examine the effect of ischemic preconditioning on the releases of cardiac troponin T (TnT) during reperfusion in isolated rat hearts. Experiments were done on 22 rat hearts, which were perfused according to the method of Langendorff and were divided into the control group (n = 14) and the preconditioning group (n = 8). Double 5 min of ischemia each followed by 5 min reflow were applied as ischemic preconditioning. After 20 min of global ischemia, the releases of TnT, creatine kinase (CK), and lactate dehydrogenase (LD) in coronary effluent and the left ventricular developed pressure (LVP) were measured during 60 min of reperfusion. Ischemic preconditioning significantly suppressed the amounts of TnT released during reperfusion, as with those of CK and LD, and also improved contractile dysfunction (nine hearts in which ventricular fibrillation was sustained were excluded from the evaluation for hemodynamics), though the release kinetics of TnT was different from that of CK and LD. There were good inverse relationships between the LVP and the total amounts of TnT released during reperfusion period (sigma TnT) or TnT levels at 60 min of reperfusion. Cardiac TnT can be used as a useful biochemical marker for hemodynamics and myocardial damage after reperfusion.

Effects of repeated ischemia on release kinetics of troponin T, creatine kinase, and lactate dehydrogenase in coronary effluent from isolated rat hearts.

We studied the release kinetics of cardiac troponin T (TnT) from coronary effluent in a re-stenosis model of 13 isolated rat hearts. After a 20-min period of global ischemia, we reperfused the hearts for 60 min according to the method of Langendorff. A second period of global ischemia was then induced for 5 min (protocol A) or 20 min (protocol B), followed by a second 60-min period of reperfusion. Coronary flow was measured by a timed collection of the coronary effluent. Levels of TnT in the effluent were compared to those of creatine kinase (CK) and lactate dehydrogenase (LD). Levels of TnT increased after the second global ischemia, but no differences were found in the released levels of TnT between protocols A and B. However, the amounts of CK and LD released in protocol B were much greater than those released in protocol A. These studies indicate that the release kinetics of TnT are different from that of CK and LD during reperfusion. It appears that after the initial ischemic damage to TnT, subsequent ischemia causes damage to TnT regardless of the duration of the insult, whereas the damage to sarcolemma is dependent on the duration of the ischemia.

[Frequency dependent prolongation of inter-atrial conduction time by mexiletine in human atrium].

To investigate frequency dependent conduction slowing by mexiletine in the human atrium, we examined inter-atrial conduction time (IACT) at different stimulation frequencies (100/min to 220/min) in 13 patients with paroxysmal atrial fibrillation (Paf) and 7 patients without Paf. IACT was prolonged as the stimulation frequency was increased, and either before or after mexiletine administration IACT was longer in the Paf group (max 114 +/- 9 msec, p < 0.01, mean +/- SD) at any stimulation frequency than in the non-Paf group (max 100 +/- 8 msec). The change in IACT induced by mexiletine administration (% delta IACT) was larger in the Paf group (max 14.8 +/- 5.4%, p < 0.05) than in the non-Paf group (max 5.5 +/- 2.2%) at stimulation frequency over 140/min. Effective refractory period measured at the high right atrium was slightly decreased due to mexiletine in both the non-Paf and the Paf group. In conclusion, mexiletine showed frequency dependent suppression of conduction in the human atrial myocardium especially in patients with Paf.

[Effect of pilsicainide hydrochloride on atrial fibrillation threshold].

To inventigate the electrophysiologic effects of pilsicainide hydrochloride on atrial fibrillation, we compared atrial fibrillation threshold (AFT), right atrial effective refractory period (RAERP), and inter-atrial conduction time (Inter-ACT) before and after the administration of pilsicainide in 12 patients with lone paroxysmal atrial fibrillation. The following electrophysiologic study was performed before and after the administration of the drug as the paced cycle length of 500msec. First, RAERP was measured. Secondly, Inter-ACT from the stimulating artifact to the initial deflection in the elecrocardiograms of the coronary sinus was measured. Thirdly, high-frequency (50Hz) stimulation was given at right atrial appendage continuously for one second just after the eighth basic paced beat. The stimulation current was increased by 1mA in a stepwise fashion from 2mA until atrial fibrillation ensued. AFT was defined as the lowest intensity of the current that induced atrial fibrillation or flutter of more than 30 seconds. Pilsicanide significantly increased AFT and Inter-ACT, but did not change RAERP. In conclusion, it is suggested that pilsicainide might decrease atrial vulnerability mainly by its effect on inter-atrial conduction delay and by the resulting increase in AFT.