Temporary blockade of contractility during reperfusion elicits a cardioprotective effect of the p38 MAP kinase inhibitor SB-203580

p38 MAP kinase activation is known to be deleterious not only to mitochondria but also to contractile function. Therefore, p38 MAP kinase inhibition therapy represents a promising approach in preventing reperfusion injury in the heart. However, reversal of p38 MAP kinase-mediated contractile dysfunction may disrupt the fragile sarcolemma of ischemic-reperfused myocytes. We, therefore, hypothesized that the beneficial effect of p38 MAP kinase inhibition during reperfusion can be enhanced when contractility is simultaneously blocked. Isolated and perfused rat hearts were paced at 330 rpm and subjected to 20 min of ischemia followed by reperfusion. p38 MAP kinase was activated after ischemia and early during reperfusion (<30 min). Treatment with the p38 MAP kinase inhibitor SB-203580 (10 microM) for 30 min during reperfusion, but not the c-Jun NH(2)-terminal kinase inhibitor SP-600125 (10 microM), improved contractility but increased creatine kinase release and infarct size. Cotreatment with SB-203580 and the contractile blocker 2,3-butanedione monoxime (BDM, 20 mM) or the ultra-short-acting beta-blocker esmorol (0.15 mM) for the first 30 min during reperfusion significantly reduced creatine kinase release and infarct size. In vitro mitochondrial ATP generation and myocardial ATP content were significantly increased in the heart cotreated with SB-203580 and BDM during reperfusion. Dystrophin was translocated from the sarcolemma during ischemia and reperfusion. SB-203580 increased accumulation of Evans blue dye in myocytes depleted of sarcolemmal dystrophin during reperfusion, whereas cotreatment with BDM facilitated restoration of sarcolemmal dystrophin and mitigated sarcolemmal damage after withdrawal of BDM. These results suggest that treatment with SB-203580 during reperfusion aggravates myocyte necrosis but concomitant blockade of contractile force unmasks cardioprotective effects of SB-203580.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

Ischemic preconditioning does not protect via blockade of electron transport.

Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles of 5-min 37 degrees C global ischemia and 5-min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally expressed complex I activity measured as rotenone-sensitive NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge on mitochondria as effectors of cardioprotection; however, each modulates mitochondrial metabolism during ischemia by different mechanisms to achieve cardioprotection.     

Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca(2+) responses in intact hearts

Ca(+) loading during reperfusion after myocardial ischemia is linked to reduced cardiac function. Like ischemic preconditioning (IPC), a volatile anesthetic given briefly before ischemia can reduce reperfusion injury. We determined whether IPC and sevoflurane preconditioning (SPC) before ischemia equivalently improve mechanical and metabolic function, reduce cytosolic Ca(2+) loading, and improve myocardial Ca(2+) responsiveness. Four groups of guinea pig isolated hearts were perfused: no ischemia, no treatment before 30-min global ischemia and 60-min reperfusion (control), IPC (two 2-min occlusions) before ischemia, and SPC (3.5 vol%, two 2-min exposures) before ischemia. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured at the left ventricular (LV) free wall with the fluorescent probe indo 1. Ca(2+) responsiveness was assessed by changing extracellular [Ca(2+)]. In control hearts, initial reperfusion increased diastolic [Ca(2+)] and diastolic LV pressure (LVP), and the maximal and minimal derivatives of LVP (dLVP/dt(max) and dLVP/dt(min), respectively), O(2) consumption, and cardiac efficiency (CE). Throughout reperfusion, IPC and SPC similarly reduced ischemic contracture, ventricular fibrillation, and enzyme release, attenuated rises in systolic and diastolic [Ca(2+)], improved contractile and relaxation indexes, O(2) consumption, and CE, and reduced infarct size. Diastolic [Ca(2+)] at 50% dLVP/dt(min) was right shifted by 32-53 +/- 8 nM after 30-min reperfusion for all groups. Phasic [Ca(2+)] at 50% dLVP/dt(max) was not altered in control but was left shifted by -235 +/- 40 nM [Ca(2+)] after IPC and by -135 +/- 20 nM [Ca(2+)] after SPC. Both SPC and IPC similarly reduce Ca(2+) loading, while augmenting contractile responsiveness to Ca(2+), improving postischemia cardiac function and attenuating permanent damage.     

Participation of protein kinase C in the activation of Nrf2 signaling by ischemic preconditioning in the isolated rabbit heart

Activation of protein kinase C (PKC) is a critical intracellular signaling triggered by ischemic preconditioning (IPC), but the precise mechanisms underlying the actions of PKC in IPC-mediated cardioprotection remain unclear. Here, we investigated the role of PKC activation on the antioxidant activity by IPC in rabbit hearts. Isolated rabbit hearts were subjected to 60?min of global ischemia by cold cardioplegic arrest (4?°C) and 60?min of reperfusion (37?°C). IPC was induced by three cycles of 2-min ischemia following 3?min of reperfusion (37?°C) before cardioplegic arrest. IPC resulted in a better recovery of mechanical function, increased tissue reduced glutathione-to-oxidized glutathione ratio (GSH/GSSG), superoxide dismutase and catalase content, and decreased tissue malondialdehyde (MDA) content compared to control hearts subjected to 60?min of cardioplegic ischemia and 60?min of reperfusion. IPC also significantly induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the inductions of antioxidant genes heme oxygenase-1 (HO-1) and manganese superoxide dismutase (MnSOD). Injection of phorbol 12-myristate 13 acetate, an activator of PKC, before cardioplegic ischemia induced translocation of PKC-?? and -?? isoforms to membrane fraction, nuclear accumulation of Nrf2, and conferred cardioprotection similar to IPC. Polymyxin B, an inhibitor of PKC, blocked the membrane translocation of PKC-?? and -?? during IPC, inhibited Nrf2 nuclear accumulation, and significantly diminished the IPC-induced cardioprotection when administrated before IPC. These results indicate that the activation of PKC induces the translocation of Nrf2 and the enhancement of endogenous antioxidant defenses in the IPC hearts and suggest that PKC may target Nrf2 to confer cardioprotection.     

Simulated ischemia-induced preconditioning of isolated ventricular myocytes from young adult and aged Fischer-344 rat hearts

The impact of ischemic preconditioning (IPC) on contraction, Ca(2+) homeostasis, and cell survival was compared in isolated ventricular myocytes from young adult ( approximately 3 mo) and aged ( approximately 24 mo) male Fischer-344 rats. Myocytes were field stimulated at 4 Hz (37 degrees C). Contraction (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously. Viability was assessed with trypan blue. All cells were exposed to 30 min of simulated ischemia followed by reperfusion. Some cells were preconditioned by exposure to 5 min of simulated ischemia before prolonged ischemia. Pretreatment with IPC abolished postischemic contractile depression, inhibited diastolic contracture, and increased Ca(2+) transient amplitudes in reperfusion in young adult and aged cells. IPC did not affect the modest rise in diastolic Ca(2+) in ischemia in young adult myocytes. However, IPC abolished the marked rise in diastolic Ca(2+) observed in ischemia and early reperfusion in aged myocytes. IPC also suppressed mechanical alternans in ischemia in aged cells, but younger myocytes showed little evidence of mechanical alternans whether or not cells were preconditioned. IPC markedly improved cell viability in reperfusion in young adult but not aged cells. These results suggest that IPC augments the recovery of contractile function in reperfusion by increasing Ca(2+) transient amplitudes in ventricular myocytes from young adult and aged rats. IPC reduced diastolic Ca(2+) accumulation in ischemia in aged myocytes, which may diminish the severity of mechanical alternans in aged cells. Nonetheless, the efficacy of IPC is compromised in aging, as IPC did not improve survival of aged myocytes exposed to ischemia and reperfusion.     

Acetaminophen-mediated cardioprotection via inhibition of the mitochondrial permeability transition pore-induced apoptotic pathway

Our laboratory has previously reported that acetaminophen confers functional cardioprotection following cardiac insult, including ischemia/reperfusion, hypoxia/reoxygenation, and exogenous peroxynitrite administration. In the present study, we further examined the mechanism of acetaminophen-mediated cardioprotection following ischemia/reperfusion injury. Langendorff-perfused guinea pig hearts were exposed to acute treatment with acetaminophen (0.35 mM) or vehicle beginning at 15 min of a 30-min baseline stabilization period. Low-flow global myocardial ischemia was subsequently induced for 30 min followed by 60 min of reperfusion. At the completion of reperfusion, hearts were homogenized and separated into cytosolic and mitochondrial fractions. Mitochondrial swelling and mitochondrial cytochromec release were assessed and found to be significantly and completely reduced in acetaminophen- vs. vehicle-treated hearts following reperfusion. In a separate group of hearts, ventricular myocytes were isolated and subjected to fluorescence-activated cell sorting. Acetaminophen-treated hearts showed a significant decrease in late stage apoptotic myocytes compared with vehicle-treated hearts following injury (58 +/- 1 vs. 81 +/- 5%, respectively). These data, together with electron micrograph analysis, suggest that acetaminophen mediates cardioprotection, in part, via inhibition of the mitochondrial permeability transition pore and subsequent apoptotic pathway.     

Transgenic MMP-2 expression induces latent cardiac mitochondrial dysfunction

Matrix metalloproteinases (MMPs) are central to the development and progression of dysfunctional ventricular remodeling after tissue injury. We studied 6 month old heterozygous mice with cardiac-specific transgenic expression of active MMP-2 (MMP-2 Tg). MMP-2 Tg hearts showed no substantial gross alteration of cardiac phenotype compared to age-matched wild-type littermates. However, buffer perfused MMP-2 Tg hearts subjected to 30 min of global ischemia followed by 30 min of reperfusion had a larger infarct size and greater depression in contractile performance compared to wild-type hearts. Importantly, cardioprotection mediated by ischemic preconditioning (IPC) was completely abolished in MMP-2 Tg hearts, as shown by abnormalities in mitochondrial ultrastructure and impaired respiration, increased lipid peroxidation, cell necrosis and persistently reduced recovery of contractile performance during post-ischemic reperfusion. We conclude that MMP-2 functions not only as a proteolytic enzyme but also as a previously unrecognized active negative regulator of mitochondrial function during superimposed oxidative stress.     

Suppression of ischemic and reperfusion ventricular arrhythmias by inhalational anesthetic-induced preconditioning in the rat heart

Inhalational anesthetic-induced preconditioning (APC) has been shown to reduce infarct size and attenuate contractile dysfunction caused by myocardial ischemia. Only a few studies have reported the effects of APC on arrhythmias during myocardial ischemia-reperfusion injury, focusing exclusively on reperfusion. Accordingly, the aim of the present study was to examine the influence of APC on ventricular arrhythmias evoked by regional no-flow ischemia. APC was induced in adult male Wistar rats by 12-min exposures to two different concentrations (0.5 and 1.0 MAC) of isoflurane followed by 30-min wash-out periods. Ventricular arrhythmias were assessed in the isolated perfused hearts during a 45-min regional ischemia and a subsequent 15-min reperfusion. Myocardial infarct size was determined after an additional 45 min of reperfusion. The incidence, severity and duration of ventricular arrhythmias during ischemia were markedly reduced by APC. The higher concentration of isoflurane had a larger effect on the incidence of ventricular fibrillation than the lower concentration. The incidence of ventricular tachycardia and reversible ventricular fibrillation during reperfusion was also significantly reduced by APC; the same was true for myocardial infarct size. In conclusion, we have shown that preconditioning with isoflurane confers profound protection against myocardial ischemia- and reperfusion-induced arrhythmias and lethal myocardial injury.     

Warm ischemic preconditioning improves mitochondrial redox balance during and after mild hypothermic ischemia in guinea pig isolated hearts

Ischemic preconditioning (IPC) induces distinctive changes in mitochondrial bioenergetics during warm (37 degrees C) ischemia and improves function and tissue viability on reperfusion. We examined whether IPC before 2 h of hypothermic (27 degrees C) ischemia affords additive cardioprotection and improves mitochondrial redox balance assessed by mitochondrial NADH and flavin adenine dinucleotide (FAD) autofluorescence in intact hearts. A mediating role of ATP-sensitive K(+) (K(ATP)) channel opening was investigated. NADH and FAD fluorescence was measured in the left ventricular wall of guinea pig isolated hearts assigned to five groups of eight animals each: hypothermia alone, hypothermia with ischemia, IPC with cold ischemia, 5-hydroxydecanoic acid (5-HD) alone, and 5-HD with IPC and cold ischemia. IPC consisted of two 5-min periods of warm global ischemia spaced 5 min apart and 15 min of reperfusion before 2 h of ischemia at 27 degrees C and 2 h of warm reperfusion. The K(ATP) channel inhibitor 5-HD was perfused from 5 min before until 5 min after IPC. IPC before 2 h of ischemia at 27 degrees C led to better recovery of function and less tissue damage on reperfusion than did 27 degrees C ischemia alone. These improvements were preceded by attenuated increases in NADH and decreases in FAD during cold ischemia and the reverse changes during warm reperfusion. 5-HD blocked each of these changes induced by IPC. This study indicates that IPC induces additive cardioprotection with mild hypothermic ischemia by improving mitochondrial bioenergetics during and after ischemia. Because effects of IPC on subsequent changes in NADH and FAD were inhibited by 5-HD, this suggests that mitochondrial K(ATP) channel opening plays a substantial role in improving mitochondrial bioenergetics throughout mild hypothermic ischemia and reperfusion.     

Intermittent hypoxia attenuates ischemia／reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2／Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemiaJreperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts comparedwith normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reducemyocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.     

The effect of myosin ATPase inhibition on metabolic and functional recovery of isolated rat heart after global ischemia

The effect of myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM) used in the range of concentrations 1.25–10.0 mM), on recovery of functions of isolated rat heart subjected to normothermic (37 °C) total ischemia for 35 min has been investigated. BDM perfusion was performed at a flow rate of 4 ml/min during 5 min before ischemia (BDM-I) or before 25-min reperfusion (BDM-R). Control hearts were perfused with Krebs solution at the same flow rate. The highest functional recovery of heart and coronary vessels was observed during infusion of 2.5 mM BDM before ischemia. At the end of reperfusion ATP and phosphocreatine (PCr) content in hearts of this group was significantly higher whereas the level of lactate was two times lower than in control; total creatine content (ΣCr) did not differ from the initial level. Similar but less pronounced changes in the improvement of aerobic metabolism and maintenance of ΣCr after reperfusion were also observed in the case of infusion of 2.5 mM BDM before reperfusion. They were consistent with reduced recovery of functions of heart and coronary flow compared with these parameters observed in the BDM-I group. 2.5 mM BDM caused almost 2-fold decrease in release of cardiac lactate dehydrogenase into myocardial perfusate in the BDM-I and BDM-R groups (compared with control); this suggests lower damage of cell membranes. These results suggest that improvement of energy supply of postischemic cardiomyocytes may be a key factor determining cardioprotector effectiveness of short-term administration of BDM before ischemia.     

AMPK-Regulated and Akt-Dependent Enhancement of Glucose Uptake Is Essential in Ischemic Preconditioning-Alleviated Reperfusion Injury

Aims

Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury.

Methods

Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by ¹⁸F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay.

Results

IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05).

Conclusions

The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.     

Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion

Reactive oxygen species (ROS) are believed to be involved in triggering cardiac ischemic preconditioning (IPC). Decreased formation of ROS on reperfusion after prolonged ischemia may in part underlie protection by IPC. In heart models, these contentions have been based either on the effect of ROS scavengers to abrogate IPC-induced preservation or on a measurement of oxidation products on reperfusion. Using spectrophotofluorometry at the left ventricular wall and the fluorescent probe dihydroethidium (DHE), we measured intracellular ROS superoxide (O(2)(-).) continuously in isolated guinea pig heart and tested the effect of IPC and the O(2)(-). scavenger manganese(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) on O(2)(-). formation throughout the phases of preconditioning (PC), 30-min ischemia and 60-min reperfusion (I/R). IPC was evidenced by improved contractile function and reduced infarction; MnTBAP abrogated these effects. Brief PC pulses increased O(2)(-). during the ischemic but not the reperfusion phase. O(2)(-). increased by 35% within 1 min of ischemia, increased further to 95% after 20 min of ischemia, and decreased slowly on reperfusion. In the IPC group, O(2)(-). was not elevated over 35% during index ischemia and was not increased at all on reperfusion; these effects were abrogated by MnTBAP. Our results directly demonstrate how intracellular ROS increase in intact hearts during IPC and I/R and clarify the role of ROS in triggering and mediating IPC.     

Effects of pharmacological preconditioning by emodin/oleanolic acid treatment and/or ischemic preconditioning on mitochondrial antioxidant components as well as the susceptibility to ischemia-reperfusion injury in rat hearts

Using an ex vivo rat heart model of ischemia-reperfusion (I-R) injury, we examined the effect of pharmacological preconditioning by chronic treatment with emodin (EMD)/oleanolic acid (OA) at low dose (25 μ mol/kg/day × 15) and/or ischemic preconditioning (IPC) (4 cycles of 5 min ischemia followed by 5 min of reperfusion) on myocardial I-R injury. The results indicated that EMD/OA pretreatment, IPC, or their combinations (EMD+IPC and OA+IPC) protected against myocardial I-R injury, as assessed by lactate dehydrogenase leakage and contractile force recovery. The cardioprotection was associated with a differential enhancement in mitochondrial antioxidant components. The combined EMD/OA and IPC pretreatment produced cardioprotective action in a semi-additive manner. This suggested that EMD/OA pretreatment and IPC protected against myocardial I-R injury via a similar but not identical biochemical mechanism.     

Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors

Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P(2y) purinoceptors. This study examined whether ATP-stimulated P(2y) purinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 microM 8-(p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 microM) or reactive blue (RB) (10 microM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 microM), ATP (30 microM), or UTP (50 microM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM). Although the protein kinase C inhibitor Ro318425 (0.3 microM) or tyrosine kinase inhibitor genistein (50 microM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.     

Post-conditioning protects rat cardiomyocytes via PKCepsilon-mediated calcium-sensing receptors

Protein kinase C (PKC) plays a role in cardioprotection through reduction of intracellular Ca(2+) concentration [Ca(2+)](i) during ischemic preconditioning (IPC). Cardioprotection against ischemic post-conditioning (PC) could be associated with reduced [Ca(2+)](i) through PKC. The calcium-sensing receptor (CaR), G protein-coupled receptor, causes accumulation of inositol phosphate (IP) to increase the release of intracellular Ca(2+). However, this phenomenon can be negatively regulated by PKC through phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that the prevention of cardiomyocyte damage by PC is associated with [Ca(2+)](i) reduction through an interaction of PKC with the CaR. Isolated rat hearts were subjected to 40min of ischemia followed by 90min of reperfusion. The hearts were post-conditioned after the 40min of ischemia by three cycles of 30s of reperfusion and 30s of re-ischemia applied before the 90min of reperfusion. Immunolocalization of PKCepsilon in the cell membrane was observed with IPC and PC, and in hearts exposed to GdCl(3) during PC. CaR was expressed in cardiac cell membrane and interacted with PKC in IPC, PC, and exposure to GdCl(3) during PC groups. On laser confocal microscopy, intracellular Ca(2+) was significantly decreased with IPC, PC, and exposure to GdCl(3) during PC compared with the I/R and PKC inhibitor groups, and cell structure was better preserved and promoted the recovery of cardiac function after reperfusion in the same groups. These results suggested that PKC is involved in cardioprotection against PC through negative feedback of a CaR-mediated reduction in [Ca(2+)](i).     

Atractyloside and 5-hydroxydecanoate block the protective effect of puerarin in isolated rat heart

The aim of the present study was to determine whether the clinically effective cardioprotection conferred by puerarin (Pue) against ischemia and reperfusion is mediated by mitochondrial transmembrane pores and/or channels. Hearts isolated from male Sprague-Dawley rats were perfused on a Langendorff apparatus and subjected to 30 min of global ischemia followed by 120 min of reperfusion. The production of formazan, which provides an index of myocardial viability, was measured by absorbance at 550 nm, and the level of lactate dehydrogenase (LDH) in the coronary effluent was determined. In this model, Pue (0.0024-2.4 mmol/l) had a dose-dependent, negatively inotropic effect. Pretreatment with Pue at 0.24 mmol/l for 5 min before ischemia increased myocardial formazan content, reduced LDH release, improved recovery of left ventricular end-diastolic pressure and rate-pressure product (left ventricular developed pressure multiplied by heart rate) during reperfusion. Administration of atractyloside (20 micromol/l), an opener of the mitochondrial permeability transition pore, for the first 20 min of reperfusion, and 5-hydroxydecanoate (100 micromol/l), the mitochondrial-specific ATP-sensitive potassium channel blocker, for 20 min before ischemia, attenuated the protective effects of Pue. In mitochondria isolated from hearts pretreated with 0.24 mmol/l Pue for 5 min, a significant inhibition of Ca(2+)-induced swelling was observed, and this inhibition was attenuated by 5-hydroxydecanoate. In isolated ventricular myocytes, pretreatment with Pue prevented ischemia-induced cell death and depolarization of the mitochondrial membrane, and atractyloside and 5-hydroxydecanoate attenuated the effects of Pue. These findings indicate that puerarin protects the myocardium against ischemia and reperfusion injury via inhibiting mitochondrial permeability transition pore opening and activating the mitochondrial ATP-sensitive potassium channel.     

Involvement of the mitochondrial calcium uniporter in cardioprotection by ischemic preconditioning

The objective of the present study was to determine whether the mitochondrial calcium uniporter plays a role in the cardioprotection induced by ischemic preconditioning (IPC). Isolated rat hearts were subjected to 30 min of regional ischemia by ligation of the left anterior descending artery followed by 120 min of reperfusion. IPC was achieved by two 5-min periods of global ischemia separated by 5 min of reperfusion. IPC reduced the infarct size and lactate dehydrogenase release in coronary effluent, which was associated with improved recovery of left ventricular contractility. Treatment with ruthenium red (RR, 5 μM), an inhibitor of the uniporter, or with Ru360 (10 μM), a highly specific uniporter inhibitor, provided cardioprotective effects like those of IPC. The cardioprotection induced by IPC was abolished by spermine (20 μM), an activator of the uniporter. Cyclosporin A (CsA, 0.2 μM), an inhibitor of the mitochondrial permeability transition pore, reversed the effects caused by spermine. In mitochondria isolated from untreated hearts, both Ru360 (10 μM) and RR (1 μM) decreased pore opening, while spermine (20 μM) increased pore opening which was blocked by CsA (0.2 μM). In mitochondria from preconditioned hearts, the opening of the pore was inhibited, but this inhibition did not occur in the mitochondria from hearts treated with IPC plus spermine. These results indicate that the mitochondrial calcium uniporter is involved in the cardioprotection conferred by ischemic preconditioning.