Ken-ichi Yabe

Preconditioning preserves mitochondrial function and glycolytic flux during an early period of reperfusion in perfused rat hearts

OBJECTIVE The purpose of the present study was to examine the effects of preconditioning on glycolysis and oxidative phosphorylation during reperfusion in perfused rat hearts. METHODS Preconditioning was induced by 5 min of ischemia and 5 min of reperfusion before 40 min of sustained ischemia and subsequent 30 min of reperfusion. Tissue energy metabolite levels, mitochondrial oxygen consumption capacity and adenine nucleotide translocator content of the perfused hearts were assessed at 40 min of ischemia, 5 and 30 min of reperfusion. RESULTS Preconditioning improved the postischemic recovery of rate x pressure product (92.5 +/- 8.7 vs. 24.9 +/- 1.2% for non-preconditioned group) and high-energy phosphate content (ATP and CrP; 39.5 +/- 2.0 and 96.2 +/- 4.9% of initial vs. 24.1 +/- 0.9 and 56.1 +/- 4.3% of initial for the non-preconditioned group). The mitochondrial oxygen consumption capacity and the adenine nucleotide translocator content of the non-preconditioned heart were decreased by sustained ischemia and remained decreased throughout reperfusion. Preconditioning prevented these decreases. The tissue lactate level of the non-preconditioned heart was high throughout reperfusion (16.5-fold vs. basal), whereas in the preconditioned heart it returned to the basal level within a few minutes of reperfusion. Furthermore, the ratios of [fructose 1,6-bisphosphate]/([glucose 6-phosphate] + [fructose 6-phosphate]) at 5-min reperfusion were higher (2.2-fold) than those of the non-preconditioned heart. CONCLUSIONS The results suggest that preconditioning preserves the capacity for normal mitochondrial function and the facilitation of glycolysis during reperfusion, which may play an important role in the improvement of postischemic contractile function and high-energy phosphate content.

Beta-adrenoceptor stimulation-mediated preconditioning-like cardioprotection in perfused rat hearts

To determine whether adrenergic stimulation induces preconditioning-like cardioprotection, rat hearts were perfused for 2 min with either norepinephrine, phenylephrine, or isoproterenol followed by 10-min drug-free perfusion. Then the hearts were subjected to 40-min ischemia and 30-min reperfusion. Little recovery of left ventricular developed pressure (LVDP) and loss of the myocardial creatine kinase (CK) during reperfusion were observed in the drug-untreated heart. Preperfusion with norepinephrine (0.25 microM) or isoproterenol (0.25 microM), but not phenylephrine (10 microM), resulted in a better recovery of LVDP in the postischemic reperfused heart and a reduction in CK release during reperfusion. A similar improvement of postischemic cardiac contractile dysfunction and CK loss was seen in the heart subjected to 5-min ischemia followed by 5-min reperfusion (ischemic preconditioning) before the prolonged period of ischemia/reperfusion. Pretreatment with timolol, a beta-adrenoceptor blocker, abolished the protective effect of norepinephrine, whereas pretreatment with bunazosin, an alpha 1-adrenoceptor blocker, did not affect the protective effect of isoproterenol. The results suggest that a brief period of stimulation of cardiac beta-adrenoceptor exerts the preconditioning-mimetic protective effect against postischemic contractile dysfunction in perfused rat hearts.

Pharmacologic preconditioning induced by beta-adrenergic stimulation is mediated by activation of protein kinase C.

Ischemic preconditioning (I-PC) occurs via activation of protein kinase C (PKC). This study was undertaken to determine whether pharmacologic preconditioning by beta-adrenergic stimulation (beta-PC) is mediated by PKC activation. Isolated rat hearts were subjected to 40-min ischemia and 30-min reperfusion. Beta-PC was induced by 0.25 microM isoproterenol pretreatment for 2 min followed by 10-min normoxic perfusion. Beta-PC enhanced the recovery of rate-pressure product of the ischemic/reperfused heart (79.1 +/- 8.4% vs. 12.4 +/- 1.6% of initial for Non-PC group, n = 6) and attenuated the release of creatine kinase during 30-min reperfusion (30.2 +/- 2.2 vs. 59.8 +/- 6.1 nmol/min/g wet wt for Non-PC group, n = 6), similar to an I-PC stimulus of 5-min ischemia and 5-min reperfusion. Treatment with 50 microM polymyxin B, a PKC inhibitor, abolished the cardioprotection of both beta-PC and I-PC. Furthermore, similar changes in subcellular distribution of PKC were induced by both beta-PC and I-PC. The changes in subcellular distribution of PKC-delta suggested its translocation from cytosol to membrane fraction, a marker of PKC activation. These results suggest that the cardioprotection induced by beta-PC, like I-PC, is mediated by PKC activation.

A role of PKC in the improvement of energy metabolism in preconditioned heart.

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function.

EFFECTS OF LONG-TERM TREATMENT WITH EICOSAPENTAENOIC ACID ON THE HEART SUBJECTED TO ISCHEMIA/REPERFUSION AND HYPOXIA/REOXYGENATION IN RATS

The effects of eicosapentaenoic acid (EPA) and long-term treatment with EPA-ethylester (EPA-E) were examined in perfused rat hearts subjected to ischemia/reperfusion and adult rat cardiomyocytes subjected to hypoxia/reoxygenation. EPA (0.1 μM) improved postischmic contractile dysfunction of the ischemic/reperfused heart. EPA (10 μM) attenuated hypoxia/reoxygenation-induced morphological deterioration of cardiomyocytes. The results suggest the presence of direct cardioprotective effects of EPA. Rats were orally treated for 4 weeks with 1 g/kg/day of EPA-E to elucidate ex vivo effects of EPA, and the fatty acid composition of cardiac phospholipids was determined. The percent ratio of EPA in total fatty acids of cardiac phospholipids increased whereas that of arachidonic acid decreased. The percent ratio of n-3/n-6 fatty acid did not increase. Treatment with EPA-E did not improve the post-ischemic contractile function, but attenuated the ischemia/reperfusion-induced release of prostaglandins during reperfusion. Treatment with EPA-E preserved a better morphological appearance of the cardiomyocytes subjected to hypoxia/reoxygenation. The results suggest that the mechanisms responsible for cytoprotective effects of hypoxic/reoxygeanted cardiomyocytes or inhibition of metabolic alterations of the ischemic/reperfused heart by long-term EPA-E treatment did not contribute substantially to recovery of post-ischemic contractile dysfunction. The direct in vitro effects of EPA may play a role in the protection of the heart from ischemia/reperfusion or hypoxia/reoxygenation injury.

Role of cardiac renin-angiotensin system in sarcoplasmic reticulum function and gene expression in the ischemic-reperfused heart

The aim of this study was to explore the possible participation of cardiac renin-angiotensin system (RAS) in the ischemiareperfusion induced changes in heart function as well as Ca2+-handling activities and gene expression of cardiac sarcoplasmic reticulum (SR) proteins. The isolated rat hearts, treated for 10 min without and with 30 captopril or 100 µM losartan, were subjected to 30 min ischemia followed by reperfusion for 60 min and processed for the measurement of SR function and gene expression. Attenuated recovery of the left ventricular developed pressure (LVDP) upon reperfusion of the ischemic heart was accompanied by a marked reduction in SR Ca2+-pump ATPase, Ca2+-uptake and Ca2+-release activities. Northern blot analysis revealed that mRNA levels for SR Ca2+-handling proteins such as Ca2+-pump ATPase (SERCA2a), ryanodine receptor, calsequestrin and phospholamban were decreased in the ischemia-reperfused heart as compared with the non-ischemic control. Treatment with captopril improved the recovery of LVDP as well as SR Ca2+-pump ATPase and Ca2+-uptake activities in the postischemic hearts but had no effect on changes in Ca2+-release activity due to ischemic-reperfusion. Losartan neither affected the changes in contractile function nor modified alterations in SR Ca2+-handling activities. The ischemia-reperfusion induced decrease in mRNA levels for SR Ca2+-handling proteins were not affected by treatment with captopril or losartan. The results suggest that the improvement of cardiac function in the ischemic-reperfused heart by captopril is associated with the preservation of SR Ca2+-pump activities; however, it is unlikely that this action of captopril is mediated through the modification of cardiac RAS. Furthermore, cardiac RAS does not appear to contribute towards the ischemia-reperfusion induced changes in gene expression for SR Ca2+-handling proteins. (Mol Cell Biochem 212: 227–235, 2000)

Hypoxic preconditioning in isolated rat hearts: Non-Involvement of activation of adenosine A1 receptor, Gi protein, and ATP-sensitive K+ channel

SummaryActivation of the adenosine A1(A1) receptor, Gi protein, and ATP-sensitive K+ (Katp)-channel system has been shown to play an important role in the cardioprotective effects of ischemic preconditioning in dogs. The present study was undertaken to elucidate the possible involvement of this system in hypoxic preconditioning, which ameliorates injury induced by prolonged ischemia and subsequent reperfusion in perfused rat hearts. Ten minutes of hypoxic preconditioning resulted in an appreciable improvement of post-ischemic cardiac contractile recovery. This was associated with a significant reduction in the release of creatine kinase (CK) from reperfused hearts. Hypoxic preconditioning shortened the time to ischemic contracture onset and prevented a further rise in left ventricular end-diastolic pressure (LVEDP) during reperfusion. Neither the selective A1 receptor antagonist, 8-cyclopentyltheophylline (CPT) nor theKatp channel blocker, glibenclamide, altered the beneficial effects of hypoxic preconditioning. In vivo pretreatment with an inhibitor of Gi protein, pertussis toxin (PTX), also did not diminish the preconditioning effect. The results suggest that, although hypoxic preperfusion ameliorates postischemic contractile dysfunction, neither the activation of the A1 receptor, nor the opening of theKatp-channel, nor transduction through Gi protein are involved in the post-ischemic functional recovery of hypoxic preconditioning in the perfused rat heart.

Does glycogen depletion play an important role in ischemic preconditioning

SummaryThe present study was undertaken to determine whether or not tissue glycogen depletion prior to ischemia, and subsequent attenuation of tissue lactate accumulation during ischemia, correlates with postischemic functional recovery of the preconditioned heart. Isolated rat hearts were subjected to 40-min ischemia and 30-min reperfusion. Preconditioning with 5-min ischemia and 5-min reperfusion reduced the preischemic glycogen and postischemic lactate levels of the heart to 60.5±5.6% and 66.9±7.7% respectively, of values in non-preconditioned hearts (n=6), and improved the recovery of the rate-pressure product (RPP) of the ischemic/reperfused heart (87.0±5.8% versus 25.2±4.5% of the initial value for the non-preconditioned group,n=8). Treatment with polymyxin B (50µM) abolished the preconditioning-induced postischemic recovery of the RPP. Treatment of the non-preconditioned heart with phorbol 12-myristate 13-acetate (15 pmol/5min) resulted in an improvement in the postischemic recovery of RPP. Neither of these treatments affected the preischemic glycogen and postischemic lactate levels. The results suggest that preischemic glycogen depletion and subsequent attenuation of ischemic lactate accumulation do not play a major role in the preconditioning-induced protection against postischemic contractile dysfunction in perfused rat hearts.

Hypoxic Preconditioning of Isolated Cardiomyocytes of Adult Rat

The present study was undertaken to examine whether or not cytoprotective effects of hypoxic preconditioning were detectable in isolated, quiescent cardiomyocytes of adult rats. The cardiomyocytes were incubated for 120 minutes under hypoxic condtions (sustained hypoxia), followed by 15-minute reoxygenation. Sustained hypoxia decreased the number of viable cells (from 99% to 70% of the initial cell), which consisted of rod- and square-shaped cardiomyocytes. It also decreased the number of rod-shaped cardiomyocytes (from 90% to 40% of the initial cell) and simultaneously increased the number of square-shaped cells (from 10% to 30% of the initial cell). Fifteen-minute reoxygenation resulted in a further decrease in the numbers of viable cells (less than 50% of the initial cell) and square-shaped cells (10% of the initial cell), whereas it did not change the number of rod-shaped cells. Hypoxia-reoxygenation also induced a release of purine nucleosides and bases (ATP metabolites) into the incubation medium. When the cardomyocytes were subjected to 20 minutes of hypoxic incubation, followed by 30 minutes of normoxic incubation (hypoxic preconditioning), sustained hypoxia-induced decreases in the numbers of viable cells and rod-shaped cells were attenuated (80% and 60% of the initial cell, respectively). The intervention also attenuated sustained hypoxia-induced increase in the number of square-shaped cells (18% of the initial cell). The number of rod-shaped cells subjected to hypoxic preconditioning at the end of 15-minute reoxygenation was similar to that at the end of sustained hypoxia, whereas the number of square-shaped cells decreased to 10% of the initial cells, which was similar to that of square-shaped cells without hypoxic preconditioning. The intervention also suppressed the release of ATP metabolites during hypoxia-reoxygenation.