Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the 'working' mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 +/- 0.3 ml/min and 10.5 +/- 0.3 kPa to 22.2 +/- 0.3 ml/min (p < 0.05) and 14.3 +/- 0.5 kPa (p < 0.05), 19.3 +/- 0.3 ml/min (p < 0.05) and 12.3 +/- 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.     

Effects of L-carnitine and its acetyl and propionyl esters on ATP and PCr levels of isolated rat hearts perfused without fatty acids and investigated by means of 31P-NMR spectroscopy

³¹P-NMR in vivo spectroscopy is a non-invasive and non-hazardous technique which investigates chemical composition and metabolism of living objects, for example by determining phosphocreatine (PCr) and ATP concentrations. In the present study we investigated the influence of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine on the energetic state of the Langendorff rat heart subjected to an ischemic period of 20 min followed by a reperfusion period of 60 min. To avoid an overlapping of the effects of fatty acids and glucose, the hearts were perfused with a Tyrode solution containing no fatty acids. Ischemia causes a rapid decrease in the PCr signal, followed by a decrease in the ATP signal after a prolonged period of ischemia. At the same time, a drastic increase in the P_i signal was observed. A partial recovery of the ATP and PCr signals was observed in the reperfusion period. With L-carnitine a markedly improved recovery of the high energy phosphates (e.g. increased PCr/P_i ratios) was found. With acetyl-L-carnitine this effect was enhanced in the first postischemic phase. It was followed, however, by a more rapid decrease in the PCr/P_i ratio in the late reperfusion period. The effect of propionyl-L-carnitine was not significantly improved in the first minutes of the reperfusion period, but during the whole reperfusion phase a stabilization of the PCr/P_i ratio was observed. Intracellular pH can be calculated from determination of the P_i-chemical shift. This shows that L-carnitine and its derivatives have a protective effect against intracellular pH decrease during ischemia. L-carnitine improves the energetic state of the heart, which leads to increased ischemia tolerance. Hearts under L-carnitine were able to tolerate up to four ischemia-reperfusion periods in succession, whereas the controls were not able to do so. These NMR results confirm the hypothesis that L-carnitine and its esters have a protective effect in the reperfusion period of the ischemic rat heart. This could be of importance for the treatment of ischemic cardiac diseases.     

Propionyl-L-carnitine effects on postischemic recovery of heart function and substrate oxidation in the diabetic rat

Previous studies have shown that propionyl-L-carnitine (PLC) can exert cardiac antiischemic effects in models of diabetes. In the nonischemic diabetic rat heart, PLC improves ventricular function secondary to stimulation in the oxidation of glucose and palmitate. Whether this increase in the oxidation of these substrates can explain the beneficial effects of PLC in the ischemic reperfused diabetic rat heart has yet to be determined. Diabetes was induced in male Sprague-Dawley rats by an intravenous injection of streptozotocin (60 mg/kg). Treatment was initiated by supplementing the drinking water with propionyl-L-carnitine at the concentration of 1 g/L. After a 6-week treatment period, exogenous substrate oxidation and recovery of mechanical function following ischemia were determined in isolated working hearts. In aerobically perfused diabetic hearts, compared with those of controls, rates of glucose oxidation were lower, but those of palmitate oxidation were similar. Diabetes was also characterized by a pronounced decrease in heart function. Following treatment with by propionyl-L-carnitine, however, there was a marked increase in rates at which glucose and palmitate were oxidized by diabetic hearts and a significant improvement in heart performance. Postischemic recovery of function in diabetic hearts was also improved with PLC. This improvement in contractile function was accompanied by an increase in both glucose and palmitate oxidation. Our findings show that postischemic diabetic rat heart can be improved following chronic PLC treatment. This beneficial effect of propionyl-L-carnitine can be explained, in part, by an improvement in the oxidation of glucose and palmitate.     

Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts

The effects of kernel extract obtained from sour cherry (Prunus cerasus) seed on the postischemic cardiac recovery were studied in isolated working rat hearts. Rats were treated with various daily doses of the extract for 14 days, and hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. The incidence of ventricular fibrillation (VF) and tachycardia (VT) fell from their control values of 92% and 100% to 50% (not significant) and 58% (not significant), 17% (P<0.05), and 25% (P<0.05) with the doses of 10 mg/kg and 30 mg/kg of the extract, respectively. Lower concentrations of the extract (1 and 5 mg/kg) failed to significantly reduce the incidence of VF and VT during reperfusion. Sour cherry seed kernel extract (10 and 30 mg/kg) significantly improved the postischemic recovery of cardiac function (coronary flow, aortic flow, and left ventricular developed pressure) during reperfusion. We have also demonstrated that the extract-induced protection in cardiac function significantly reflected in a reduction of infarct size. Immunohistochemistry indicates that a reduction in caspase-3 activity and apoptotic cells by the extract, beside other potential action mechanisms of proanthocyanidin, trans-resveratrol, and flavonoid components of the extract, could be responsible for the cardioprotection in ischemic-reperfused myocardium.     

Antiradical effects in L-propionyl carnitine protection of the heart against ischemia-reperfusion injury: the possible role of iron chelation.

L-Propionyl carnitine has been shown to improve the heart's mechanical recovery and other metabolic parameters after ischemia-reperfusion. However, the mechanism of protection is unknown. The two dominating hypotheses are: (i) L-propionyl carnitine can serve as an energy source for heart muscle cells by being enzymatically converted to propionyl-CoA and subsequently utilized in the Krebs cycle (a metabolic hypothesis), and (ii) it can act as an antiradical agent, protecting myocardial cells from oxidative damage (a free radical hypothesis). To test the two possible pathways, we compared the protection afforded to the ischemia-reperfused hearts by L-propionyl carnitine and its optical isomer, D-propionyl carnitine. The latter cannot be enzymatically utilized as an energy source. The Langendorff perfusion technique was used and the hearts were subjected to 40 min of ischemia and 20 min of reperfusion. In analysis of ischemia-reperfused hearts, a strong correlation was found between the recovery of mechanical function and the presence of protein oxidation products (protein carbonyls). Both propionyl carnitines efficiently prevented protein oxidation but L-propionyl carnitine-perfused hearts had two times greater left ventricular developed pressure. The results indicate that both metabolic and antiradical pathway are involved in the protective mechanism of L-propionyl carnitine. To obtain a better insight of the antiradical mechanism of L-propionyl carnitine, we compared the ability of L- and D-propionyl carnitines, L-carnitine, and deferoxamine to interact with: (i) peroxyl radicals, (ii) oxygen radicals, and (iii) iron. We found that none of the carnitine derivatives were able to scavenge peroxyl radicals or superoxide radicals. L- and D-propionyl carnitine and deferoxamine (not L-carnitine) suppressed hydroxyl radical production in the Fenton system, probably by chelating the iron required for the generation of hydroxyl radicals. We suggest that L-propionyl carnitine protects the heart by a dual mechanism: it is an efficient fuel source and an antiradical agent.     

Distinct effects of acute pretreatment with lipophilic and hydrophilic statins on myocardial stunning, arrhythmias and lethal injury in the rat heart subjected to ischemia/reperfusion

Although both lipophilic and more hydrophilic statins share the same pathway of the inhibition of HMG-CoA reductase, their pleiotropic cardioprotective effects associated with the ability to cross cellular membranes, including membranes of heart cells, may differ. To test this hypothesis, isolated rat hearts were Langendorff-perfused either with simvastatin (S, 10 micromol/l) or pravastatin (P, 30 micromol/l), 15 min prior to ischemia. Control untreated hearts (C) were perfused with perfusion medium only. Postischemic contractile dysfunction, reperfusion-induced ventricular arrhythmias and infarct size were investigated after exposure of the hearts to 30-min global ischemia and 2-h reperfusion. Both lipophilic S and hydrophilic P reduced the severity of ventricular arrhythmias (arrhythmia score) from 4.3 +/- 0.2 in C to 3.0 +/- 0 and 2.7 +/- 0.2 in S and P, respectively, (both P < 0.05), decreased the duration of ventricular tachycardia and suppressed ventricular fibrillation. Likewise, the extent of lethal injury (infarct size) determined by tetrazolium staining and expressed in percentage of risk area, was significantly lower in both treated groups, moreover, the effect of P was more pronounced (27 +/- 2 % and 10 +/- 2 % in S and P groups, respectively, vs. 42 +/- 1 % in C; P < 0.05). In contrast, only S, but not P, was able to improve postischemic recovery of left ventricular developed pressure (LVDP; 48 +/- 12 % of preischemic values vs. 25 +/- 4 % in C and 21 +/ -7 % in P groups; P < 0.05). Our results suggest that differences in water solubility of statins indicating a different ability to cross cardiac membranes may underlie their distinct cardioprotective effects on myocardial stunning and lethal injury induced by ischemia/reperfusion.     

Effects of L-carnitine on mechanical recovery of isolated rat hearts in relation to the perfusion with glucose and palmitate

The purpose of this study was to investigate the effects of L-carnitine on the hemodynamic parameters of Langendorff hearts. Isolated rat hearts were perfused with various solutions containing high or low concentrations of fatty acids, additional glucose or no glucose, and L-carnitine or no L-carnitine. The most interesting part of the experiments was the behaviour of the hearts in the reperfusion period after no-flow ischemia of 20 min. The results were: (1) With glucose and high fatty acid concentrations the hearts showed an improved recovery of the left ventricular functions in the reperfusion period compared with low fatty acid concentrations. Without glucose the left ventricular pressure is much lower in the reperfusion period. (2) Addition of L-carnitine improved the recovery of the ischemically damaged hearts. This improvement is especially impressive at low fatty acid concentrations. L-carnitine addition at high fatty acid concentrations but without glucose strongly improved reperfusion behaviour. (3) The coronary flow is increased by 2 experimental conditions: (i) perfusion at low levels of fatty acids, carnitine and with glucose and (ii) high levels of fatty acids and carnitine but without glucose. These findings suggest that supplementation of L-carnitine has a beneficial effect on the isolated heart under various conditions, and possibly on specific human heart diseases.     

Low-pressure reperfusion alters mitochondrial permeability transition

We hypothesized that low-pressure reperfusion may limit myocardial necrosis and attenuate postischemic contractile dysfunction by inhibiting mitochondrial permeability transition pore (mPTP) opening. Male Wistar rat hearts (n = 36) were perfused according to the Langendorff technique, exposed to 40 min of ischemia, and assigned to one of the following groups: 1) reperfusion with normal pressure (NP = 100 cmH(2)O) or 2) reperfusion with low pressure (LP = 70 cmH(2)O). Creatine kinase release and tetraphenyltetrazolium chloride staining were used to evaluate infarct size. Modifications of cardiac function were assessed by changes in coronary flow, heart rate (HR), left ventricular developed pressure (LVDP), the first derivate of the pressure curve (dP/dt), and the rate-pressure product (RPP = LVDP x HR). Mitochondria were isolated from the reperfused myocardium, and the Ca(2+)-induced mPTP opening was measured using a potentiometric approach. Lipid peroxidation was assessed by measuring malondialdehyde production. Infarct size was significantly reduced in the LP group, averaging 17 +/- 3 vs. 33 +/- 3% of the left ventricular weight in NP hearts. At the end of reperfusion, functional recovery was significantly improved in LP hearts, with RPP averaging 10,392 +/- 876 vs. 3,969 +/- 534 mmHg/min in NP hearts (P < 0.001). The Ca(2+) load required to induce mPTP opening averaged 232 +/- 10 and 128 +/- 16 microM in LP and NP hearts, respectively (P < 0.001). Myocardial malondialdehyde was significantly lower in LP than in NP hearts (P < 0.05). These results suggest that the protection afforded by low-pressure reperfusion involves an inhibition of the opening of the mPTP, possibly via reduction of reactive oxygen species production.     

The role of NO in ischemia/reperfusion injury in isolated rat heart

Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dt(max) (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 +/- 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 +/- 5.7 %, p < 0.05). Recovery of both CF and dP/dt(max) after TI was also significantly higher in the adapted hearts (101.5 +/- 5.8 % and 83.64 +/- 3.92 % ) as compared with the controls (71.9 +/- 6.3 % and 35.7 +/- 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 +/- 3.1 %; dP/dt(max) 67.5 +/- 5.92 %) and increased CF to 82.4 +/- 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 +/- 3.1 %; CF 70.3 +/- 3.4 % and dP/dt(max) 43.25 +/- 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) - 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). In conclusion: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.     

The role of heme oxygenase-related carbon monoxide and ventricular fibrillation in ischemic/reperfused hearts

Reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO)-related carbon monoxide (CO) production in isolated ischemic/reperfused rat hearts were studied by gas chromatography. Hearts were subjected to 30 min ischemia followed by 2 h reperfusion, and the expression of HO-1 mRNA (about 4-fold) was observed in ischemic/reperfused-nonfibrillated hearts. In fibrillated hearts, the reduction (about 75%) in HO-1 mRNA expression was detected. These changes in HO-1 mRNA expression were reflected in tissue CO production. Thus, in the absence of VF, CO production was increased about 3.5-fold, while in the presence of VF, CO production was under the detectable level in comparison with the control group. Our results suggest that the stimulation of HO-1 mRNA expression may lead to the prevention of reperfusion VF via an increase in endogenous CO production. To prove this, hearts were treated with 1 microM of N-tert-butyl-alpha-phenylnitrone (PBN) as an inducer of HO-1. PBN treatment resulted in about 20 times increase in HO-1 mRNA expression, and even a higher production rate in endogenous CO. HO protein level and enzyme activity followed the same pattern, as it was observed in HO-1 mRNA expression, in fibrillated and nonfibrillated myocardium. Five mM/l of zinc-protoporphyrin IX (ZnPPIX) significantly blocked HO enzyme activity and increased the incidence of VF, therefore the application of ZnPPIX led to a significant reduction in HO-1 mRNA and protein expression. Our data provide direct evidence of an inverse relationship between the development of reperfusion-induced VF and endogenous CO production. Thus, interventions that are able to increase tissue CO content may prevent the development of reperfusion-induced VF.     

In vivo and in vitro intervention with L-carnitine prevents abnormal energy metabolism in isolated diabetic rat heart: chemical and phosphorus-31 NMR evidence

The beneficial effects of in vivo injections (200 mg/kg, twice daily) or in vitro perfusion (5.0 mM) of L-carnitine on an intrinsic abnormality in energy metabolism was investigated in isolated, perfused diabetic rat heart. Hearts were aerobically perfused for 60 min with elevated fatty acid substrate to simulate diabetic conditions. Phosphorus-31 nuclear magnetic resonance spectroscopy revealed a temporal decline in myocardial ATP levels (to approx 82%) during perfusion of diabetic hearts, but not in control hearts. This reduction was prevented by prior treatment in vivo with L-carnitine or by providing L-carnitine acutely in the perfusion medium. Chemical analysis of tissue extracts indicated that L-carnitine injections were effective in replenishing the decrease in total myocardial carnitine content which was present in diabetic hearts and in preventing the accumulation of long chain fatty acyl CoA. Perfusion with L-carnitine also attenuated the elevation of long chain fatty acyl CoA in diabetic hearts. This study gives additional support to the hypothesis that decreases in ATP which occur in the isolated, perfused diabetic heart are correlated with a concomitant elevation in long chain fatty acyl CoA, a known inhibitor of adenine nucleotide translocase. In the presence of elevated exogenous fatty acids, a primary deficiency in the total myocardial carnitine pool would result in elevations in tissue concentrations of long chain fatty acyl CoA since carnitine is a required carrier for transport of fatty acids into mitochondria. Replenishment of the carnitine in vivo was shown to be sufficient to prevent subsequent alteration in long chain fatty acyl CoA and ATP in isolated perfused diabetic hearts despite the burden of elevated fatty acid substrates.     

Sevoflurane postconditioning attenuates reperfusion-induced ventricular arrhythmias in isolated rat hearts exposed to ischemia/reperfusion injury

Sevoflurane postconditioning has been proven to protect the hearts against ischemia/reperfusion injury, manifested mainly by improved cardiac function, reduced myocardial specific biomarker release, and decreased infarct size. This study is to observe the effects of sevoflurane postconditioning on reperfusion-induced ventricular arrhythmias and reactive oxygen species generation in Langendorff perfused rat hearts. Compared with the unprotected hearts subjected to 25 min of global ischemia followed by 30 min of reperfusion, exposure of 3% sevoflurane during the first 15 min of reperfusion significantly improved cardiac function, reduced cardiac troponin I release, decreased infarct size and attenuated reperfusion-induced ventricular arrhythmia. Further analysis on arrhythmia during the 30 min of reperfusion showed that, sevoflurane postconditioning decreased both the duration and incidence of ventricular tachycardia and ventricular fibrillation. In the meantime, intracellular malondialdehyde and reactive oxygen species levels were also reduced. These above results demonstrate that sevoflurane postconditioning protects the hearts against ischemia/reperfusion injury and attenuates reperfusion-induced arrhythmia, which may be associated with the regulation of lipid peroxidation and reactive oxygen species generation.     

Carnitine transport and exogenous palmitate oxidation in chronically volume-overloaded rat hearts

L-Carnitine transport and free fatty acid oxidation have been studied in hearts of rats with 3-month-old aorto-caval fistula. For carnitine transport experiments, the hearts were perfused via the ascending aorta with a bicarbonate buffer containing 11 mM glucose and variable concentrations L-[14C]carnitine (10-200 microM). In some experiments, the active component of carnitine transport was suppressed by the adjunction of 0.05 mM mersalyl acid. The subtraction of passive from total transport allowed reconstruction of the saturation curves of the carrier-mediated transport of L-carnitine. Our data suggest that at a physiological carnitine concentration (50 microM), the rate of [14C]carnitine accumulation was significantly depressed in mechanically overloaded hearts. In addition, according to Lineweaver-Burk analysis, the affinity of the membrane carrier for L-carnitine was considerably diminished (Km carnitine 125 instead of 83 microM, Vmax unchanged). The above alterations of L-carnitine transport did not result from a decrease of the transmembrane gradient of sodium, since the intracellular Na+ content of the hypertrophied hearts was quite similar to that of control hearts. The ability of atrially perfused, working hearts to oxidize the exogenous free fatty acids was assessed from 14CO2 production obtained in the presence of [U-14C]palmitate or [1-14C]octanoate. The total 14CO2 production, expressed per min per g dry weight, was significantly diminished in hearts from rats with the aorto-caval fistula if 1.2 mM palmitate was used. On the other hand, in the presence of 2.4 mM octanoate, a substrate which circumvents the carnitine-acylcarnitine translocase, no such reduction of the 14CO2 production could be detected. Our results suggest that the decrease of L-carnitine transport, resulting in a significant depression of tissue carnitine, may impair long-chain fatty acid activation and/or translocation into mitochondria. In contrast, the oxidation of short-chain fatty acids, the activation of which takes place directly in mitochondrial matrix, is not limited in volume-overloaded hearts.     

Attenuation of neutrophil-mediated myocardial ischemia-reperfusion injury by a calpain inhibitor

Calpains are ubiquitous neutral cysteine proteases. Although their physiological role has yet to be clarified, calpains seem to be involved in the expression of cell adhesion molecules. Therefore, we hypothesized that a selective calpain inhibitor could attenuate polymorphonuclear (PMN) leukocyte-induced myocardial ischemia-reperfusion (I/R) injury. We examined the effects of the calpain inhibitor Z-Leu-Leu-CHO in isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. Z-Leu-Leu-CHO (10 and 20 microM, respectively) significantly improved left ventricular developed pressure (LVDP) (P < 0.01) and the maximal rate of development of LVDP (P < 0.01) compared with I/R hearts perfused without Z-Leu-Leu-CHO. In addition, Z-Leu-Leu-CHO significantly reduced PMN adherence to the vascular endothelium and subsequent infiltration into the postischemic myocardium (P < 0.01). Moreover, Z-Leu-Leu-CHO significantly inhibited expression of P-selectin on the rat coronary microvascular endothelium (P < 0.01). These results provide evidence that Z-Leu-Leu-CHO significantly attenuates PMN-mediated I/R injury in the isolated perfused rat heart to a significant extent via downregulation of P-selectin expression.     

The effect of high buffer cardioplegia and secondary cardioplegia on cardiac preservation and postischemic functional recovery: a 31P NMR and functional study in Langendorff perfused pig hearts.

High buffer cardioplegia may provide protection against ischemic damage by reducing the extent of intracellular acidosis. Secondary cardioplegia may improve postischemic recovery by restoration of high energy phosphates, ionic gradients, and intracellular pH. To test these hypotheses, pig hearts were arrested with high buffer (150 mM MOPS) cardioplegia or modified St. Thomas' solution II and then kept ischemic at 12 degrees C for 8 h. High energy phosphates and intracellular pH were followed during the period of ischemia, using 31P nuclear magnetic resonance spectroscopy, and functional recovery was followed during reperfusion. The hearts arrested by high buffer cardioplegia showed significantly higher intracellular pH than hearts preserved with St. Thomas' solution, but there were no significant differences in high energy phosphates. There were no significant differences in functional recovery. We found, however, that secondary cardioplegia abolished ventricular fibrillation, and resulted in improved functional recovery after 8 h of ischemic preservation compared with the hearts reperfused with Krebs-Henseleit solution alone. Our results suggest that despite attenuating the decreases in intracellular pH, high buffer cardioplegia does not improve recovery following 8 h of preservation at 12 degrees C. Secondary cardioplegia reduces the incidence of ventricular fibrillation and improves postischemic functional recovery of the myocardium.     

Protection of the reperfused heart by L-propionylcarnitine.

The effects of L-propionylcarnitine on mechanical function, creatine phosphate and ATP content, and lactate dehydrogenase leakage were studied in isolated perfused rat hearts exposed to global no-flow ischemia for 30 min followed by reperfusion for 20 min. Five and 10 mM L-propionylcarnitine resulted in a 100% recovery of left ventricular-developed pressure, whereas the recovery was only 40% in the hearts perfused without this agent. Ischemia-reperfusion caused a 85% loss of creatine phosphate and a 77% loss of ATP, which was prevented by 10 mM L-propionylcarnitine. Five millimolar L-propionylcarnitine protected the heart from the loss of creatine phosphate but not from the loss of ATP. Ten millimolar L-propionylcarnitine failed to improve the postischemic left ventricular-developed pressure, when it was added to the perfusate only after ischemia. L-propionylcarnitine alleviated the decrease of coronary flow in the reperfused hearts. Lactate dehydrogenase leakage was aggravated in the beginning of the reperfusion period by 10 mM L-propionylcarnitine. This adverse effect was, however, transient. L-Propionylcarnitine provides protection for the postischemic reperfused heart in a dose-dependent manner. The optimal time for administration is before the ischemic insult. High doses of this compound may perturb cell membrane integrity. Moreover, the present data point to an intracellular, metabolic, and perhaps anaplerotic mechanism of action of L-propionylcarnitine in cardiac ischemia-reperfusion injury.     

Erythropoietin pretreatment protects against acute chemotherapy toxicity in isolated rat hearts

The use of chemotherapeutic agents, such as anthracycline or trastuzumab, in oncology is limited by their cardiac toxicity. Recent experimental studies suggest that recombinant human erythropoietin (rhEPO) can be considered as a protective agent because its administration protects against cardiac ischemic injury, improving functional recovery, and reducing cell death. The aim of this study was to investigate whether pretreatment by rhEPO protects against acute cardiotoxicity induced by doxorubicin and trastuzumab, using the isolated rat heart model. Rats were treated with rhEPO (5000 IU/kg, intraperitoneally [i.p.]) or vehicle. One hour later, hearts were isolated and retrogradely perfused at constant flow. Following 20 mins of stabilization, hearts were perfused for 60 mins with modified-Krebs solution containing 6 mg/l doxorubicin or 10 mg/l trastuzumab. Hearts receiving doxorubicin were paced; those receiving trastuzumab were unpaced. Control hearts were perfused with modified-Krebs solution only. Doxorubicin exposure decreased left ventricular developed pressure (LVDP; approximately -40% of baseline) and increased end diastolic pressure (EDP; approximately +390% of baseline) and coronary perfusion pressure (CPP; approximately +70% of baseline). Incidence of ventricular tachycardia or fibrillation (VT/VF) was also significantly enhanced (86% vs. 0% in control group). Trastuzumab exposure increased CPP and EDP (approximately +70% of baseline for the both) without affecting LVDP. Prior rhEPO treatment significantly prevented doxorubicin-induced deleterious effects on LVDP, EDP, and VT/VF incidence. rhEPO administration also prevented trastuzumab-induced deleterious effects on CPP and EDP. This study shows that pretreatment by rhEPO protects myocardium against functional damage and electrophysiologic injury induced by acute doxorubicin or trastuzumab exposure. Further investigations are required to elucidate the precise mechanisms involved.     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.     

Effect of cicletanine on reperfusion-induced arrhythmias and its relation to 6-keto-PGF1 alpha and thromboxane B2 release

Isolated hearts, excised from spontaneously hypertensive male rats treated orally with cicletanine, a new furopyridine anti-hypertensive drug, were subjected to 30 min of global ischemia followed by 10 min of reperfusion. The effect of cicletanine on reperfusion-induced arrhythmias in relation to 6-keto-PGF1 alpha and thromboxane (TXB2) release was studied. After 30 min of global ischemia, the incidence (total) of ventricular fibrillation (VF) and ventricular tachycardia (VT) was reduced by 2-week pretreatment of the rats with 30 and 100 mg/kg of cicletanine (VF, 33% at 30 mg/kg and 25% at 100 mg/kg vs. 91% in untreated rats; VT, 42% at 30 mg/kg and 42% at 100 mg/kg vs. 100% in untreated rats), while lower doses of cicletanine (3 and 10 mg/kg) failed to reduce the incidence of reperfusion-induced rhythm disturbances. Reperfusion of the ischemic myocardium resulted in a fivefold increase of 6-keto-PGF1 alpha and TXB2 release in the perfusion effluent of fibrillated hearts but not in the perfusion effluent of nonfibrillated hearts. Cicletanine failed to influence the reperfusion-stimulated release of 6-keto-PGF1 alpha and TXB2. These results indicate that the anti-arrhythmic effect of cicletanine in the reperfused myocardium is not related to PGI2 and thromboxane A2 release.