Differential role of sarcolemmal and mitochondrial K(ATP) channels in adenosine-enhanced ischemic preconditioning

Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-HD-I and 5-HD-R had no effect on APC functional recovery. HMR-IR significantly decreased postischemic functional recovery (P < 0.05 vs. APC) but had no effect on infarct size. These data indicate that APC infarct size reduction is modulated by mitoK(ATP) channels primarily during ischemia and suggest that functional recovery is modulated by sarcK(ATP) channels during ischemia and reperfusion.     

Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts

NADH increases during ischemia because O(2) shortage limits NADH oxidation at the electron transport chain. Ischemic (IPC) and anesthetic preconditioning (APC) attenuate cardiac reperfusion injury. We examined whether IPC and APC similarly alter NADH, i.e., mitochondrial metabolism. NADH fluorescence was measured at the left ventricular wall of 40 Langendorff-prepared guinea pig hearts. IPC was achieved by two 5-min periods of ischemia and APC by exposure to 0.5 or 1.3 mM sevoflurane for 15 min, each ending 30 min before 30 min of global ischemia. During ischemia, NADH initially increased in nonpreconditioned control hearts and then gradually declined below baseline levels. This increase in NADH was lower after APC but not after IPC. The subsequent decline was slower after IPC and APC. On reperfusion, NADH was less decreased after IPC or APC, mechanical and metabolic functions were improved, and infarct size was lower compared with controls. Our results indicate that IPC and APC cause distinctive changes in mitochondrial metabolism during ischemia and thus lead to improved function and tissue viability on 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.     

Overexpression of A(3) adenosine receptors decreases heart rate,preserves energetics,and protects ischemic hearts

To determine whether A(3) adenosine receptor (A(3)AR) signaling modulates myocardial function, energetics, and cardioprotection, hearts from wild-type and A(3)AR-overexpressor mice were subjected to 20-min ischemia and 40-min reperfusion while (31)P NMR spectra were acquired. Basal heart rate and left ventricular developed pressure (LVDP) were lower in A(3)AR-overexpressor hearts than wild-type hearts. Ischemic ATP depletion was delayed and postischemic recoveries of contractile function, ATP, and phosphocreatine were greater in A(3)AR-hearts. To determine the role of depressed heart rate and to confirm A(3)AR-specific signaling, hearts were paced at 480 beats/min with or without 60 nmol/l MRS-1220 (A(3)AR-specific inhibitor) and then subjected to ischemia-reperfusion. LVDP was similar in paced A(3)AR-overexpressor and paced wild-type hearts. Differences in ischemic ATP depletion and postischemic contractile and energetic dysfunction remained in paced A(3)AR-overexpressor hearts versus paced wild-type hearts but were abolished by MRS-1220. In summary, A(3)AR overexpression decreased basal heart rate and contractility, preserved ischemic ATP, and decreased postischemic dysfunction. Pacing abolished the decreased contractility but not the ATP preservation or cardioprotection. Therefore, A(3)AR overexpression results in cardioprotection via a specific A(3)AR effect, possibly involving preservation of ATP during ischemia.     

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.     

Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart

Although adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (SPT; 100 microM) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 microM) or reactive blue (RB; 10 microM) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (50 microM) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene adenosine diphosphate (AMP-CP; 100 microM), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively.     

Ischemic preconditioning requires opening of pannexin-1/P2X7 channels not only during preconditioning but again after index ischemia at full reperfusion

Protection of the ex vivo rat heart from ischemia/reperfusion injury can be provided by ischemic preconditioning (IPC). Previous studies revealed that a complex of pannexin-1 with the P2X? receptor forms a channel during IPC that results in the release of cardioprotectants such as adenosine and sphingosine 1-phosphate (S1P) that bind to G-protein-coupled cell surface receptors triggering cardioprotective cell signaling pathways. Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X? receptors (brilliant blue G) are known to block IPC when administered at the time of preconditioning (Vessey et al. J Cardiovasc Pharmacol Ther 15:190, 2010). We now demonstrate that these same antagonists also block the cardioprotective effects of IPC when added after the index ischemia during full reperfusion. Likewise, addition at full reperfusion of binding antagonists to the endogenous cardioprotectants S1P (VPC) or adenosine (8-SPT) reduced the effectiveness of IPC. These data suggest that IPC has a component that requires the release of cardioprotectants via pannexin-1/P2X? channels not only during preconditioning phase but again during the early stages of reperfusion following the index ischemia. It was found that the level of cardioprotectant release required at reperfusion to achieve cardioprotection was lower when hearts had been preconditioned. Further, pharmacologic preconditioning with S1P or adenosine was also blocked at reperfusion by antagonists of the pannexin-1/P2X? channels indicating that pharmacologic preconditioning also requires opening of the channel at full reperfusion. In untreated hearts, key components of the PI3 kinase/Akt signaling pathway were revealed by western analysis to be lost during ischemia. This correlates with an inability to generate phospho-Akt at reperfusion. IPC prevents this loss and thereby primes the cell for response to cardioprotectants released at full reperfusion.     

Endogenous adenosine protects preconditioned heart during early minutes of reperfusion by activating Akt

Ischemic preconditioning (IPC) is thought to protect by activating survival kinases during reperfusion. We tested whether binding of adenosine receptors is also required during reperfusion and, if so, how long these receptors must be populated. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. IPC reduced infarct size from 32.1 +/- 4.6% of the risk zone in control hearts to 7.3 +/- 3.6%. IPC protection was blocked by a 20-min pulse of the nonselective adenosine receptor blocker 8-(p-sulfophenyl)-theophylline when started either 5 min before or 10 min after the onset of reperfusion but not when started after 30 min of reperfusion. Protection was also blocked by either 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1-selective receptor antagonist, or MRS1754, an A2B-selective antagonist, but not by 8-(3-chlorostyryl)caffeine, an A2A-selective antagonist. Blockade of phosphatidylinositol 3-OH kinase (PI3K) with a 20-min pulse of wortmannin also aborted protection when started either 5 min before or 10 or 30 min after the onset of reperfusion but failed when started after 60 min of reflow. U-0126, an antagonist of MEK1/2 and therefore of ERK1/2, blocked protection when started 5 min before reperfusion but not when started after only 10 min of reperfusion. These studies reveal that A1 and/or A2B receptors initiate the protective signal transduction cascade during reperfusion. Although PI3K activity must continue long into the reperfusion phase, adenosine receptor occupancy is no longer needed by 30 min of reperfusion, and ERK activity is only required in the first few minutes of reperfusion.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

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.     

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.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α₂-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α₂-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α₂-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α₂-adrenergic receptor stimulation.     

Preconditioning and postconditioning: innate cardioprotection from ischemia-reperfusion injury.

Reperfusion is the definitive treatment to salvage ischemic myocardium from infarction. A primary determinant of infarct size is the duration of ischemia. In myocardium that has not been irreversibly injured by ischemia, reperfusion induces additional injury in the area at risk. The heart has potent innate cardioprotective mechanisms against ischemia-reperfusion that reduce infarct size and other presentations of postischemic injury. Ischemic preconditioning (IPC) applied before the prolonged ischemia exerts the most potent protection observed among known strategies. It has been assumed that IPC exerts protection during ischemia. However, recent data suggest that cardioprotection is also exerted during reperfusion. Postconditioning (PoC), defined as brief intermittent cycles of ischemia alternating with reperfusion applied after the ischemic event, has been shown to reduce infarct size, in some cases equivalent to that observed with IPC. Although there are similarities in mechanisms of cardioprotection by these two interventions, there are key differences that go beyond simply exerting these mechanisms before or after ischemia. A significant limitation of IPC has been the inability to apply this maneuver clinically except in situations where the ischemic event can be predicted. On the other hand, PoC is applied at the point of service in the hospital (cath-lab for percutaneous coronary intervention, coronary artery bypass grafting, and other cardiac surgery) where and when reperfusion is initiated. Initial clinical studies are in agreement with the success and extent to which PoC reduces infarct size and myocardial injury, even in the presence of multiple comorbidities.     

Sarcolemmal KATP channel triggers delayed ischemic preconditioning in rats

Previous work from our laboratory has shown that the sarcolemmal K(ATP) channel (sK(ATP)) is required as a trigger for delayed cardioprotection upon exogenous opioid administration. We also established that the mitochondrial K(ATP) (mK(ATP)) channel is not required for triggering delayed delta-opioid-induced infarct size reduction. Because mechanistic differences have been found among delta-opioids and that due to ischemic preconditioning (IPC), we determined whether the triggering mechanism of delayed IPC-induced infarct size reduction involves either the sK(ATP) or mK(ATP). Male Sprague-Dawley rats received either sham surgery or IPC (3- to 5-min cycles of ischemia and reperfusion) 24 h before being subjected to 30 min of ischemia and 2 h of reperfusion. Infarct size was determined and expressed as a percentage of the area at risk, with significance compared with sham reported at P 相似文献   

Akt and Erk1/2 activate the ornithine decarboxylase/polyamine system in cardioprotective ischemic preconditioning in rats: the role of mitochondrial permeability transition pores

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in polyamine biosynthesis, which is essential for cell survival. We hypothesized that the ODC/polyamine system is involved in ischemic preconditioning (IPC)-mediated cardioprotection through the activation of Erk1/2 and Akt and through the inhibition of the mitochondrial permeability transition (mPT). Isolated rat hearts were subjected to 40 min of ischemia either with or without IPC (3 cycles of 5-min global ischemia), and ODC protein expression, polyamine content, and Akt and Erk1/2 phosphorylation were evaluated after 30 min of reperfusion. IPC significantly upregulated the ODC/polyamine pathway, promoted Erk1/2 and Akt phosphorylation, and reduced the infarct size and heart dysfunction after reperfusion. An inhibitor of ODC, α-difluoromethylornithine (DFMO), abolished the IPC-induced cardioprotection. Moreover, the inhibition of the IPC-induced activation of Erk1/2 and Akt using PD98059 or wortmannin downregulated the ODC/polyamine system. In separate studies, the Ca²⁺ load required to open the mPT pore was significantly lower in DFMO-treated cardiac mitochondria than in mitochondria from IPC hearts. Furthermore, spermine or spermidine significantly inhibited the mPT induced by CaCl₂. These results suggest that IPC upregulates the ODC/polyamine system and mediates preconditioning cardioprotection, which may depend on the phosphorylation/activation of Erk1/2 and Akt and on the inhibition of the mPT during reperfusion.     

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.     

Essential role of EGFR in cardioprotection and signaling responses to A1 adenosine receptors and ischemic preconditioning

Transactivation of epidermal growth factor receptor (EGFR) may contribute to specific protective responses (e.g. mediated by δ-opioid, bradykinin, or muscarinic receptors). No studies have assessed EGFR involvement in cardioprotection mediated by adenosine receptors (ARs), and the role of EGFR in ischemic preconditioning (IPC) is unclear. We tested EGFR, matrix metalloproteinase (MMP), and heparin-binding EGF (HB-EGF) dependencies of functional protection via A(1)AR agonism or IPC. Pretreatment of mouse hearts with 100 nM of A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) or IPC (3 × 1.5-min ischemia/2-min reperfusion) substantially improved recovery from 25-min ischemia, reducing left ventricular diastolic dysfunction up to 50% and nearly doubling pressure development and positive change in pressure over time (+dP/dt). Benefit with both CCPA and IPC was eliminated by inhibitors of EGFR tyrosine kinase (0.3 μM AG1478), MMP (0.3 μM GM6001), or HB-EGF ligand (0.3 ng/ml CRM197), none of which independently altered postischemic outcome. Phosphorylation of myocardial EGFR, Erk1/2, and Akt increased two- to threefold during A(1)AR agonism, with responses blocked by AG1478, GM6001, and CRM197. Studies in HL-1 myocytes confirm A(1)AR-dependent Erk1/2 phosphorylation is negated by AG1478 or GM6001, and reduced with CRM197 (as was Akt activation). These data collectively reveal that A(1)AR- and IPC-mediated functional protection is entirely EGFR and MMP dependent, potentially involving the HB-EGF ligand. Myocardial survival kinase activation (Erk1/2, Akt) by A(1)AR agonism is similarly MMP/HB-EGF/EGFR dependent. Thus MMP-mediated EGFR activation appears essential to cardiac protection and signaling via A(1)ARs and preconditioning.     

Measurements of 1,2-diacylglycerol and ceramide in hearts subjected to ischemic preconditioning

An accumulation of recent evidence suggests that the mechanism in ischemic preconditioning (IPC) may involve the activation of protein kinase C (PKC) regulatory pathway. In this study, we examined whether the content of 1,2-diacylglycerol (1,2-DAG) and ceramide, which are intracellular second messengers regulating PKC activity, change during IPC in isolated perfused rat hearts, and whether the observed change in 1,2-DAG is accompanied with alteration in its fatty acid composition. Hearts subjected to IPC, consisting of 5-min transient global ischemia followed by 5-min reperfusion, presented a significant functional recovery during subsequent 40-min reperfusion following 40-min global ischemia compared with non-preconditioned hearts. An increase in 1,2-DAG content was observed in hearts subjected to 5-min transient ischemia compared with non-ischemic control hearts, however this was not seen in hearts harvested after 5-min reperfusion following 5-min ischemia. While fatty acid composition in 1,2-DAG was virtually unchanged in hearts subjected to 5-min ischemia, saturated 1,2-DAG decreased and monounsaturated/polyunsaturated 1,2-DAG increased in hearts reperfused for 5-min following 5-min ischemia compared with the non-ischemic control hearts. Ceramide mass did not change significantly, suggesting that the contribution of ceramide may be small in IPC. These data are in concert with the hypothesis that 1,2-DAG is a second messenger in IPC and the changes in fatty acid composition of 1,2-DAG may add new insight concerning signal transduction pathway in IPC.     

Extracellular Adenosine Formation by Ecto-5’-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning

Background

Adenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73^-/- and wild type (WT) mice.

Methods and Results

3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73^-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73^-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73^-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73^-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73^-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73^-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.

Conclusion

The infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.     

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.