Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

Effects of L-carnitine and its derivatives on postischemic cardiac function,ventricular fibrillation and necrotic and apoptotic cardiomyocyte death in isolated rat hearts

The study aimed to examine whether L-carnitine and its derivatives, acetyl-L-carnitine and propionyl-L-carnitine, were equally effective and able to improve postischemic cardiac function, reduce the incidence of reperfusion-induced ventricular fibrillation, infarct size, and apoptotic cell death in ischemic/reperfused isolated rat hearts. There are several studies indicating that L-carnitine, a naturally occurring amino acid and an essential cofactor, can improve mechanical function and substrate metabolism not only in hypertrophied or failing myocardium but also in ischemic/reperfused hearts. The effects of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine, on the recovery of heart function, incidence of reperfusion-induced ventricular fibrillation (VF), infarct size, and apoptotic cell death after 30 min ischemia followed by 120 min reperfusion were studied in isolated working rat hearts. Hearts were perfused with various concentrations of L-carnitine (0.5 and 5 mM), acetyl-L-carnitine (0.5 and 5 mM), and propionyl-L-carnitine (0.05, 0.5, and 5 mM), respectively, for 10 min before the induction of ischemia. Postischemic recovery of CF, AF, and LVDP was significantly improved in all groups perfused with 5 mM of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine. Significant postischemic ventricular recovery was noticed in the hearts perfused with 0.5 mM of propionyl-L-carnitine, but not with the same concentration of L-carnitine or L-acetyl carnitine. The incidence of reperfusion VF was reduced from its control value of 90 to 10% (p < 0.05) in hearts perfused with 5 mM of propionyl-L-carnitine only. Other doses of various carnitines failed to reduce the incidence of VF. The protection in CF, AF, LVDP, and VF reflected in a reduction in infarct size and apoptotic cell death in hearts treated with various concentrations of carnitine derivatives. The difference between effectiveness of various carnitines on the recovery of postischemic myocardium may be explained by different membrane permeability properties of carnitine and its derivatives.     

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.     

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

The role of inhibition of NO formation in the metabolic recovery of ischemic rat heart by apelin-12

The effects of apelin-12, a 12 amino acid peptide (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A-12), on recovery of energy metabolism and cardiac function have been studied in isolated working rat hearts perfused with Krebs buffer (KB) containing 11 mM glucose and subjected to global ischemia and reperfusion. Infusion of 140 μM A-12 before ischemia enhanced myocardial ATP, the total pool of adenine nucleotides (ΣAN = ATP+ADP+AMP) and the energy charge of cardiomyocytes ((ATP + 0.5ADP)/ΣAN) at the end of reperfusion compared with control (KB infusion) and decreased lactate content and lactate/pyruvate ratio in the reperfused myocardium up to the initial values. This was accompanied by improved recovery of coronary flow and cardiac function. Co-administration of A-12 and 100 μM L-NAME (an inhibitor of NO synthases) significantly attenuated the A-12 effects on metabolic and functional recovery of reperfused hearts. These results indicate involvement of NO in mechanisms of cardioprotection that are tightly associated with recovery of energy metabolism in the postischemic heart.     

Relationship between oxidative phosphorylation and adenine nucleotide translocase activity of two populations of cardiac mitochondria and mechanical recovery of ischemic hearts following reperfusion

The possible relationship of the atractyloside-sensitive adenine nucleotide translocase activity, oxidative phosphorylation, and the recovery of ventricular contractility following reperfusion of the ischemic isolated rat heart was studied. Five minutes of total global ischemia without reperfusion produced a significant depression in adenine nucleotide translocase in subsarcolemmal mitochondria (SLM), whereas a minimum of 10 min ischemia was required to observe a significant depression in interfibrillar mitochondria (IFM). Increasing durations of ischemia resulted in a progressively larger depression in translocase activity, with a maximum depression of approximately 75% seen in both populations following 20 min ischemia. In contrast, oxidative phosphorylation was totally unaffected in either mitochondrial population following up to 20 min of ischemia. We assessed whether translocase activity or oxidative phosphorylation were related to contractile recovery in hearts reperfused following various durations of ischemia. In SLM, translocase activity was further depressed following reperfusion compared with pre-reperfusion ischemic values, whereas with IFM only reperfusion following 5 min ischemia produced a further depression in translocase values. Oxidative phosphorylation rates of SLM and IFM were significantly depressed following reperfusion of ischemic hearts, although SLM exhibited a generally higher sensitivity in this regard. In reperfused hearts, an overall significant relationship was found between oxidative phosphorylation rate and adenine translocase activity as well as between translocase activity and post-reperfusion contractile recovery. These data show that ischemia can produce a significant depression in translocase activity in the absence of any change in oxidative phosphorylation. The results also suggest that the depression in mitochondrial ADP/ATP translocase and subsequent inhibition of oxidative phosphorylation in the reperfused heart may represent one of the important contributory mechanisms involved in cardiac failure and injury during acute ischemia and reperfusion.     

VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/- mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/- I/R, and (4) Flt-1+/- + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/- mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/- compared to wild-type I/R. Flt-1+/- KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/- mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.     

Preischemic administration of ribose to delay the onset of irreversible ischemic injury and improve function: studies in normal and hypertrophied hearts

Compared with normal hearts, those with pathology (hypertrophy) are less tolerant of metabolic stresses such as ischemia. Pharmacologic intervention administered prior to such stress could provide significant protection. This study determined, firstly, whether the pentose sugar ribose, previously shown to improve postischemic recovery of energy stores and function, protects against ischemia when administered as a pretreatment. Secondly, the efficacy of this same pretreatment protocol was determined in hearts with pathology (hypertrophy). For study 1, Sprague-Dawley rats received equal volumes of either vehicle (bolus i.v. saline) or ribose (100 mg/kg) before global myocardial ischemia. In study 2, spontaneously hypertensive rats (SHR; blood pressure approximately 200/130) with myocardial hypertrophy underwent the same treatment protocol and assessments. In vivo left ventricular function was measured and myocardial metabolites and tolerance to ischemia were assessed. In normal hearts, ribose pretreatment significantly elevated the heart's energy stores (glycogen), and delayed the onset of irreversible ischemic injury by 25%. However, in vivo ventricular relaxation was reduced by 41% in the ribose group. In SHR, ribose pretreatment did not produce significant elevations in the heart's energy or improvements in tolerance to global ischemia, but significantly improved ventricular function (maximal rate of pressure rise (+dP/dt(max)), 25%; normalized contractility ((+dP/dt)/P), 13%) despite no change in hemodynamics. Thus, administration of ribose in advance of global myocardial ischemia does provide metabolic benefit in normal hearts. However, in hypertrophied hearts, ribose did not affect ischemic tolerance but improved ventricular function.     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Impaired insulin-signaling in hypertrophied hearts contributes to ischemic injury

Despite increased glucose utilization by hypertrophied myocardium, these hearts exhibit a slower rate of glucose uptake (GU). We hypothesized that, in hypertrophied myocardium, a defect of the insulin-responsive glucose transporter is responsible for impaired GU and metabolism during ischemia, contributing to post-ischemic myocardial dysfunction. In a rabbit model of pressure-overload hypertrophy, GU ((31)P NMR spectroscopy) and total/phosphorylated insulin-signaling intermediates were assayed: insulin-receptor, insulin-receptor-substrate-1 (IRS-1), phosphatidylinositol-3-kinase (PI3-k), GLUT-4 translocation and contractile function in an isolated heart ischemia/reperfusion model. Total protein was not different between hypertrophied and control hearts. Phosphorylation of IRS-1 and PI3-k activity was significantly lower in hypertrophy during ischemia. GU was impaired pre-ischemia in hypertrophy, remained lower during early reperfusion, and was associated with impaired recovery of contractile function. In conclusion, a defect in IRS-1 phosphorylation and PI3-k activation in hypertrophied hearts restricts insulin-mediated GLUT-4 translocation and ischemia, a known stimulus of GLUT-4 translocation, does not compensate for this defect.     

Energy metabolism and mechanical recovery after cardioplegia in moderately hypertrophiedrats

It is well established that severe hypertrophy induces metabolic and structural changes in the heart which result in enhanced susceptibility to ischemic damage during cardioplegic arrest while much less is known about the effect of cardioplegic arrest on moderately hypertrophied hearts. The aim of this study was to elucidate the differences in myocardial high energy phosphate metabolism and in functional recovery after cardioplegic arrest and ischemia in mildly hypertrophied hearts, before any metabolic alterations could be shown under baseline conditions.Cardiac hypertrophy was induced in rats by constriction of the abdominal aorta resulting in 20% increase in heart weight/body weight ratio (hypertrophy group) while sham operated animals served as control. In both groups, isolated hearts were perfused under normoxic conditions for 40 min followed by infusion of St.Thomas' Hospital No. 1 cardioplegia and 90 min ischemia at 25øC with infusions of cardioplegia every 30 min. The changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) were followed by³¹ P nuclear magnetic resonance (NMR) spectroscopy. Systolic and diastolic function was assessed with an intraventricular balloon before and after ischemia.Baseline concentrations of PCr, ATP and Pi as well as coronary flow and cardiac function were not different between the two groups. However, after cardioplegic arrest PCr concentration increased to 61.8 ± 4.9 mol/g dry wt in the control group and to 46.3 ± 2.8 mol/g in hypertrophied hearts. Subsequently PCr, pH and ATP decreased gradually, concomitant with an accumulation of Pi in both groups. PCr was transiently restored during each infusion of cardioplegic solution while Pi decreased. PCr decreased faster after cardioplegic infusions in hypertrophied hearts. The most significant difference was observed during reperfusion: PCr recovered to its pre-ischemic levels within 2 min following restoration of coronary flow in the control group while similar recovery was observed after 4 min in the hypertrophied hearts. A greater deterioration of diastolic function was observed in hypertrophied hearts.Moderate hypertrophy, despite absence of metabolic changes under baseline conditions could lead to enhanced functional deterioration after cardioplegic arrest and ischemia. Impaired energy metabolism resulting in accelerated high energy phosphate depletion during ischemia and delayed recovery of energy equilibrium after cardioplegic arrest observed in hypertrophied hearts could be one of the underlying mechanisms.     

Ischemia/reperfusion in rat heart induces leptin and leptin receptor gene expression

Concentrations of leptin, an adipocyte-derived hormone, are elevated in obesity. Recently, leptin has been shown to participate in multiple biological actions including inflammation, reproduction, and angiogenesis. Leptin has also been documented as a critical component in the process of wound healing; however, leptin involvement in cardiovascular disease is poorly understood. We examined the expression of leptin (ob) and leptin receptor (ob-R) genes in the rat heart following ischemia/reperfusion, which was induced by coronary artery ligation, and mRNA was obtained from hearts 0.5 to 36 h after initiating reperfusion. Expressions of ob and ob-R mRNA were examined by real-time quantitative RT-PCR and immunohistochemistry. The ob and ob-Ra mRNA and protein expressions were significantly increased (p<0.01) and ob-Rb mRNA was significantly decreased (p<0.01) in hearts after 8 h of reperfusion. Furthermore, ob and ob-R proteins were expressed in injured myocytes where inflammatory cells infiltrated. In contrast, those expressions were not influenced in hearts after 8 h of ischemia stress only. To determine the functional effects of leptin on the ischemic/reperfused heart, rats were treated with anti-leptin antibodies prior to ischemia/reperfusion; however, this treatment did not affect the elevation of mRNA expression levels of inflammatory markers such as TNF-alpha and IL-1beta in ischemic hearts. Our results demonstrated for the first time that ischemia/reperfusion induced leptin and leptin receptor gene expression in the rat heart. This study helps to elucidate the mechanisms behind the onset and development of ischemic heart disease concomitant with obesity.     

Injection of isolated mitochondria during early reperfusion for cardioprotection

Previously, we demonstrated that ischemia induces mitochondrial damage and dysfunction that persist throughout reperfusion and impact negatively on postischemic functional recovery and cellular viability. We hypothesized that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, would enhance postischemic functional recovery and limit infarct size. New Zealand White rabbits (n = 52) were subjected to 30 min of equilibrium and 30 min of regional ischemia (RI) induced by snaring the left anterior descending coronary artery. At 29 min of RI, the RI zone was injected with vehicle (sham control and RI vehicle) or vehicle containing mitochondria (7.7 x 10(6) +/- 1.5 x 10(6)/ml) isolated from donor rabbit left ventricular tissue (RI-Mito). The snare was released at 30 min of RI, and the hearts were reperfused for 120 min. Our results show that left ventricular peak developed pressure and systolic shortening in RI-Mito hearts were significantly enhanced (P < 0.05 vs. RI-vehicle) to 75% and 83% of equilibrium value, respectively, at 120 min of reperfusion compared with 57% and 62%, respectively, in RI-vehicle hearts. Creatine kinase-MB, cardiac troponin I, and infarct size relative to area at risk were significantly decreased in RI-Mito compared with RI-vehicle hearts (P < 0.05). Confocal microscopy showed that injected mitochondria were present and viable after 120 min of reperfusion and were distributed from the epicardium to the subendocardium. These results demonstrate that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, significantly enhance postischemic functional recovery and cellular viability.     

Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts

Little is known concerning the effect of oxidative stress on the expression of antioxidative enzymes in the decompensated cardiac hypertrophy of spontaneously hypertensive rats (SHR), considered as a model of dilative cardiomyopathy in man. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) were characterized in isolated perfused hearts of 18 month old SHR and the age-matched normotensive control Wistar-Kyoto (WKY) rats, before and after 30 min infusion of 25 microM H(2)O(2). After infusion of H(2)O(2), aortic flow decreased in WKY from 26.2 +/- 2.2 to 16.0 +/- 0.8 ml/min (p <.05) but not in SHR (18.2 +/- 1.9 vs. 20.7 +/- 2.2 ml/min). This protection was related to the higher myocardial activities of GPx, MnSOD and CuZnSOD in SHR, compared with those of the WKY group. Although total SOD activity in the SHR fell after H(2)O(2) exposure (to 1.81 +/- 0.13 from 3.56 +/- 0.49 U/mg of protein), catalase activity increased (to 2.46 +/- 0.34 from 1.56 +/- 0.29 k min(-1)mg(-1)protein), compared with the pre-infusion period (p <.05 in each case). In additional studies, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The results obtained in ischemic/reperfused hearts show the same changes in enzyme activities measured as it was observed in H(2)O(2) perfused hearts, indicating that oxidative stress is independent of the way it was induced. The higher catalase activity derived from elevated mRNA synthesis. The antioxidative system in dilative cardiomyopathic hearts of SHR is induced, probably due to episodes of oxidative stress, during the process of decompensation. This conditioning of the antioxidative potential may help overcome acute stress situations caused by reactive oxygen species in the failing myocardium.     

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.