Urocortin (Ucn) protects hearts against ischemia and reperfusion injury whether given prior to ischemia or at reperfusion. Here we investigate the roles of protein kinase C, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute Ucn treatment improved hemodynamic recovery during reperfusion after 30 min global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of [3H]-2-deoxyglucose). Ucn pre-treatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that Ucn decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 hr) treatment with Ucn reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells which had been pre-treated with Ucn suggesting that this is the mechanism by which Ucn desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKCε or PKCα translocate to the mitochondria following acute Ucn treatment, inhibition of PKC with chelerythrine eliminated the effect of Ucn on oxidative stress. Our data suggests that acute Ucn treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.
reperfusion; mitochondria; oxygen radicals; peptide hormones
Ranolazine (Ran), an anti-anginal drug, is a late Na+ channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if Ran's protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with Ran just before 30 min ischemia and then isolated cardiac mitochondria at the end of 30 min ischemia and/or 30 min ischemia followed by 10 min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe-S clusters, ELISA for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ran treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ran treatment also led to more normalized electron transfer via Fe-S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with Ran were associated with improved cardiac function after IR. However, these protective effects of Ran are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I.
Complex I; mitochondria; IR injury; ranolazine; EPR; heart
The mechanisms by which ischemic preconditioning (IP) inhibits mitochondrial permeability transition pore opening and, hence, ischemia–reperfusion injury remain unclear. Here we investigate whether and how mitochondria‐bound hexokinase 2 (mtHK2) may exert part of the cardioprotective effects of IP.
Methods and Results
Control and IP Langendorff‐perfused rat hearts were subject to ischemia and reperfusion with measurement of hemodynamic function and infarct size. Outer mitochondrial membrane (OMM) permeabilization after ischemia was determined by measuring rates of respiration and H2O2 production in the presence and absence of added cytochrome c in isolated mitochondria and permeabilized fibers. IP prevented OMM permeabilization during ischemia and reduced the loss of mtHK2, but not Bcl‐xL, observed in control ischemic hearts. By contrast, treatment of permeabilized fibers with glucose‐6‐phosphate at pH 6.3 induced mtHK2 loss without OMM permeabilization. However, metabolic pretreatments of the perfused heart chosen to modulate glucose‐6‐phosphate and intracellular pHi revealed a strong inverse correlation between end‐ischemic mtHK2 content and infarct size after reperfusion. Loss of mtHK2 was also associated with reduced rates of creatine phosphate generation during the early phase of reperfusion. This could be mimicked in permeabilized fibers after mtHK2 dissociation.
We propose that loss of mtHK2 during ischemia destabilizes mitochondrial contact sites, which, when accompanied by degradation of Bcl‐xL, induces OMM permeabilization and cytochrome c loss. This stimulates reactive oxygen species production and mitochondrial permeability transition pore opening on reperfusion, leading to infarction. Consequently, inhibition of mtHK2 loss during ischemia could be an important mechanism responsible for the cardioprotection mediated by IP and other pretreatments.
hexokinase; ischemia/reperfusion injury; mitochondria; permeability transition pore; reactive oxygen species
Heart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms.
Hearts were perfused with 50 μM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively.
30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor.
The results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.
Recovery of the mitochondrial inner membrane potential (ΔΨm) is a key determinant of postischemic functional recovery of the heart. Mitochondrial ROS-induced ROS release causes the collapse of ΔΨm and the destabilization of the action potential (AP) through a mechanism involving a mitochondrial inner membrane anion channel (IMAC) modulated by the mitochondrial benzodiazepine receptor (mBzR). Here, we test the hypothesis that this mechanism contributes to spatiotemporal heterogeneity of ΔΨm during ischemia-reperfusion (IR), thereby promoting abnormal electrical activation and arrhythmias in the whole heart. High-resolution optical AP mapping was performed in perfused guinea pig hearts subjected to 30 minutes of global ischemia followed by reperfusion. Typical electrophysiological responses, including progressive AP shortening followed by membrane inexcitablity in ischemia and ventricular fibrillation upon reperfusion, were observed in control hearts. These responses were reduced or eliminated by treatment with the mBzR antagonist 4′-chlorodiazepam (4′-Cl-DZP), which blocks depolarization of ΔΨm. When applied throughout the IR protocol, 4′-Cl-DZP blunted AP shortening and prevented reperfusion arrhythmias. Inhibition of ventricular fibrillation was also achieved by bolus infusion of 4′-Cl-DZP just before reperfusion. Conversely, treatment with an agonist of the mBzR that promotes ΔΨm depolarization exacerbated IR-induced electrophysiological changes and failed to prevent arrhythmias. The effects of these compounds were consistent with their actions on IMAC and ΔΨm. These findings directly link instability of ΔΨm to the heterogeneous electrophysiological substrate of the postischemic heart and highlight the mitochondrial membrane as a new therapeutic target for arrhythmia prevention in ischemic heart disease.
Reduction of nitrite to nitric oxide during ischemia protects the heart against injury from ischemia/reperfusion. However the optimal dose of nitrite and the mechanisms underlying nitrite-induced cardioprotection are not known. We determined the ability of nitrite and nitrate to confer protection against myocardial infarction in two rat models of ischemia/reperfusion injury and the role of xanthine oxidoreductase, NADPH oxidase, nitric oxide synthase and KATP channels in mediating nitrite-induced cardioprotection. In vivo and in vitro rat models of myocardial ischemia/reperfusion injury were used to cause infarction. Hearts (n=6/group) were treated with nitrite or nitrate for 15 min prior to 30 min regional ischemia and 180 min reperfusion. Xanthine oxidoreductase activity was measured after 15 min aerobic perfusion and 30 min ischemia. Nitrite reduced myocardial necrosis and decline in ventricular function following ischemia/reperfusion in the intact and isolated rat heart in a dose or concentration-dependent manner with an optimal dose of 4 mg/kg in vivo and concentration of 10 μM in vitro. Nitrate had no effect on protection. Reduction in infarction by nitrite was abolished by inhibition of flavoprotein reductases and the molybdenum site of xanthine oxidoreductase, and was associated with an increase in activity of xanthine dehydrogenase and xanthine oxidase during ischemia. Inhibition of nitric oxide synthase had no effect on nitrite-induced cardioprotection. Inhibition of NADPH oxidase and KATP channels abolished nitrite-induced cardioprotection. Nitrite but not nitrate protects against infarction by a mechanism involving xanthine oxidoreductase, NADPH oxidase and KATP channels.
nitrite; xanthine oxidoreductase; NADPH oxidase; KATP channels; infarction
Remote preconditioning is a phenomenon in which brief episodes of ischemia and reperfusion to remote organs protect the target organ against sustained ischemia/reperfusion (I/R)-induced injury. Protective effects of remote aortic preconditioning (RAPC) are well established in the heart, but their mechanisms still remain to be elucidated.
This study has been designed to investigate the possible involvement of α-1-adrenergic receptor (AR) and KATP channels in cardio-protective effect of RAPC in isolated rat heart.
Materials and Methods:
Four episodes of ischemia and reperfusion, each comprising of 5 min occlusion and 5 min reperfusion, were used to produce RAPC. Isolated perfused rat heart was subjected to global ischemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analyzed for LDH and CK-MB release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining.
Phenylephrine (20 μ/kg i.p.), as α-1-AR agonist, was noted to produce RAPC-like cardio-protection. However, administration of glibenclamide concomitantly or prior to phenylephrine abolished cardioprotection. Moreover, prazocin (1 mg/kg. i.p), as α-1-AR antagonist and glibenclamide (1 mg/kg i.p), a KATP channel blocker, abolished the cardioprotective effect of RAPC.
These data provide the evidence that α-1-AR activation involved in cardioprotective effect of RAPC-mediated trough opening of KATP channels.
Cardio-protection; ischemic preconditioning; ischemia / reperfusion injury; remote aortic preconditioning
The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function.
Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca2+ induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K+ transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K+ channels (mitoKATP). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoKATP activation.
We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoKATP activation.
Ranolazine is a clinically approved drug for treating cardiac ventricular dysrhythmias and angina. Its mechanism(s) of protection is not clearly understood but evidence points to blocking the late Na+ current that arises during ischemia, blocking mitochondrial complex I activity, or modulating mitochondrial metabolism. Here we tested the effect of ranolazine treatment before ischemia at the mitochondrial level in intact isolated hearts and in mitochondria isolated from hearts at different times of reperfusion. Left ventricular (LV) pressure (LVP), coronary flow (CF), and O2 metabolism were measured in guinea pig isolated hearts perfused with Krebs-Ringer’s solution; mitochondrial (m) O2•−, Ca2+, NADH/FAD (redox state), and cytosolic (c) Ca2+ were assessed on-line in the LV free wall by fluorescence spectrophotometry. Ranolazine (5 µM), infused for 1 min just before 30 min of global ischemia, itself did not change O2•−, cCa2+, mCa2+ or redox state. During late ischemia and reperfusion (IR) O2•− emission and m[Ca2+] increased less in the ranolazine group vs. the control group. Ranolazine decreased c[Ca2+] only during ischemia while NADH and FAD were not different during IR in the ranolazine vs. control groups. Throughout reperfusion LVP and CF were higher, and ventricular fibrillation was less frequent. Infarct size was smaller in the ranolazine group than the control group. Mitochondria isolated from ranolazine-treated hearts had mild resistance to permeability transition pore (mPTP) opening and less cytochrome c release than control hearts. Ranolazine may provide functional protection of the heart during IR injury by reducing cCa2+ and mCa2+ loading secondary to its effect to block the late Na+ current. Subsequently it indirectly reduces O2•− emission, preserves bioenergetics, delays mPTP opening, and restricts loss of cytochrome c, thereby reducing necrosis and apoptosis.
mitochondrial Ca2+; oxidative stress; permeability transition pore; ranolazine
Myocardial ischemia–reperfusion (I/R) causes severe cardiac damage. Although the primary function of oxymyoglobin (Mb) has been considered to be cellular O2 storage and supply, previous research has suggested that Mb is a potentially protective element against I/R injury. However, the mechanism of its protective action is still largely unknown. With a real-time fluorescent technique, we observed that at the onset of ischemia, there was a small burst of superoxide (O2•–) release, as visualized in an isolated rat heart. Thus, we hypothesize that the formation of O2•– correlates to Mb due to a decrease in oxygen tension in the myocardium. Measurement of O2•– production in a Langendorff apparatus was performed using surface fluorometry. An increase in fluorescence was observed during the onset of ischemia in hearts perfused with a solution of hydroethidine, a fluorescent dye sensitive to intracellular O2•–. The increase of fluorescence in the ischemic heart was abolished by a superoxide dismutase mimic, carbon monoxide, or by Mb-knockout gene technology. Furthermore, we identified that O2•– was not generated from the intracellular endothelium but from the myocytes, which are a rich source of Mb. These results suggest that during the onset of ischemia, Mb is responsible for generating O2•–. This novel mechanism may shed light on the protective role of Mb in I/R injury.
ischemia; confocal; rat; oxymyoglobin
Myocardial injury is increased in the aged heart following ischemia and reperfusion (I-R) in both humans and experimental models. Hearts from aged 24 mo. old Fischer 344 rats sustain greater cell death and decreased contractile recovery after I-R compared to 6 mo. adult controls. Cardiac mitochondria incur damage during I-R contributing to cell death. Aged rats have a defect in complex III of the mitochondrial electron transport chain (ETC) localized to the interfibrillar population of cardiac mitochondria (IFM), situated in the interior of the cardiomyocyte among the myofibrils. The defect involves the quinol oxidation site (Qo) and increases the production of reactive oxygen species (ROS) in the baseline state. Ischemia further decreases complex III activity via functional inactivation of the iron-sulfur subunit. We studied the contribution of ischemia-induced defects in complex III to the increased cardiac injury in the aged heart. The reversible blockade of the ETC proximal to complex III during ischemia using amobarbital protects mitochondria against ischemic damage, removing the ischemia component of mitochondrial dysfunction. Reperfusion of the aged heart in the absence of ischemic mitochondrial damage decreases net ROS production from mitochondria and reduces cell death. Thus, even despite the persistence of the age-related defects in electron transport, protection against ischemic damage to mitochondria can reduce injury in the aged heart. The direct therapeutic targeting of mitochondria protects against ischemic damage and decreases cardiac injury during reperfusion in the high risk elderly heart.
ischemia; cytochrome c; myocardial infarction; aging; electron transport chain; reactive oxygen species
Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia-reperfusion injury, and we recently found that cytokines link TLR4 to postischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia-reperfusion is unknown. In the present study, we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia-reperfusion; 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function; and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia-reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery after ischemia-reperfusion. Recombinant HSC70 induced NF-κB activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore, this study demonstrates for the first time that 1) the myocardium releases HSC70 during ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism, and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function after ischemia-reperfusion.
Toll-like receptor 4; cytokines; nuclear factor-κB; messenger ribonucleic acid
Repetitive cycles of reflow/reocclusion in the initial 2 min following release of a prolonged coronary occlusion, i.e., ischemic postconditioning (IPoC), salvages ischemic myocardium. We have proposed that the intermittent ischemia prevents formation of mitochondrial permeability transition pores (MPTP) by maintaining an acidic myocardial pH for several minutes until survival kinases can be activated. To determine other requisites of IPoC, isolated rabbit hearts were subjected to 30 min of regional myocardial ischemia and 120 min of reperfusion. Infarct size was determined by staining with triphenyltetrazolium chloride. During the first 2 min of reperfusion the perfusate was either at pH7.4 following equilibration with 95% O2/5% CO2, pH6.9 following equilibration with 80% N2/20% CO2, or pH7.8 following equilibration with 100% O2. Whereas acidic, oxygenated perfusate for the first 2 min of reperfusion was cardioprotective, protection was lost when acidic perfusate was hypoxic. However, the acidic, hypoxic hearts could be rescued by addition of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, to the perfusate. Therefore, both low pH and restoration of oxygenation are necessary for protection, and the signaling step requiring combined oxygen and H+ must be upstream of PKC. To gain further insight into the mechanism of IPoC, the latter was effected with 6 cycles of 10-sec reperfusion/10-sec reocclusion. Its protective effect was abrogated by either making the oxygenated perfusate alkaline during the reperfusion phases or making the reperfusion buffer hypoxic. Presumably the repeated coronary occlusions during IPoC keep myocardial pH low while the resupply of oxygen during the intermittent reperfusion provides fuel for the redox signaling that acts to prevent MPTP formation even after restoration of normal myocardial pH. Hearts treated simultaneously with IPoC and alkaline perfusate could not be rescued by addition to the perfusate of either PMA or SB216763 which inhibits GSK-3β, the putative last cytoplasmic signaling step in the signal transduction cascade leading to MPTP inhibition. Yet cyclosporin A which also inhibits MPTP formation does rescue hearts made alkaline during IPoC. In view of prior studies in which the ROS scavenger N-2-mercaptopropionyl glycine aborts IPoC's protection, our data reveal that IPoC's reperfusion periods are needed to support redox signaling rather than improve metabolism. The low pH, on the other hand, is equally necessary and seems to suppress MPTP directly rather than through upstream signaling.
One or more brief periods of ischemia, termed preconditioning, dramatically limits infarct size and reduces intracellular acidosis during subsequent ischemia, potentially via enhanced sarcolemmal proton efflux mechanisms. To test the hypothesis that preconditioning increases the functional activity of sodium-dependent proton efflux pathways, isolated rat hearts were subjected to 30 min of global ischemia with or without preconditioning. Intracellular sodium (Nai) was assessed using 23Na magnetic resonance spectroscopy, and the activity of the Na-H exchanger and Na-K-2Cl cotransporter was measured by transiently exposing the hearts to an acid load (NH4Cl washout). Creatine kinase release was reduced by greater than 60% in the preconditioned hearts (P < 0.05) and was associated with improved functional recovery on reperfusion. Preconditioning increased Nai by 6.24 +/- 2.04 U, resulting in a significantly higher level of Nai before ischemia than in the control hearts. Nai increased significantly at the onset of ischemia (8.48 +/- 1.21 vs. 2.57 +/- 0.81 U, preconditioned vs. control hearts; P < 0.01). Preconditioning did not reduce Nai accumulation during ischemia, but the decline in Nai during the first 5 min of reperfusion was significantly greater in the preconditioned than in the control hearts (13.48 +/- 1.73 vs. 2.54 +/- 0.41 U; P < 0.001). Exposure of preconditioned hearts to ethylisopropylamiloride or bumetanide in the last reperfusion period limited in the increase in Nai during ischemia and reduced the beneficial effects of preconditioning. After the NH4Cl prepulse, preconditioned hearts acidified significantly more than control hearts and had significantly more rapid recovery of pH (preconditioned, delta pH = 0.35 +/- 0.04 U over 5 min; control, delta pH = 0.15 +/- 0.02 U over 5 min). This rapid pH recovery was not affected by inhibition of the Na-K-2Cl cotransporter but was abolished by inhibition of the Na-H exchanger. These results demonstrate that preconditioning alters the kinetics of Nai accumulation during global ischemia as well as proton transport after NH4Cl washout. These observations are consistent with stimulation of the Na-K-2Cl cotransporter and Na-H exchanger by preconditioning.
The aged heart sustains greater injury during ischemia (ISC) and reperfusion (REP) compared to the adult heart. In the Fischer 344 (F344) rat, aging decreases oxidative phosphorylation and complex III activity increasing the production of reactive oxygen species in interfibrillar mitochondria (IFM) located among the myofibrils. In the isolated, perfused 24 month old elderly F344 rat heart 25 min. of stop-flow ISC causes additional damage to complex III, further decreasing the rate of OXPHOS. We did not observe further progressive mitochondrial damage during REP. We next asked if ISC or REP increased oxidative damage within mitochondria of the aged heart. Cardiolipin (CL) is a phospholipid unique to mitochondria consisting predominantly of four linoleic acid residues (C18:2). Following ISC and REP in the aged heart, there is a new CL species containing three oxygen atoms added to one linoleic residue. ISC alone was sufficient to generate this new oxidized molecular species of CL. Based upon oxidative damage to CL, complex III activity, and oxidative phosphorylation, mitochondrial damage thus occurs in the aged heart mainly during ISC, rather than during REP. Mitochondrial damage during ischemia sets the stage for mitochondrial-driven cardiomyocyte injury during reperfusion in the aged heart.
ischemia; reperfusion; heart; ubiquinone:cytochrome c reductase (complex III); cardiolipin; mitochondria; aging
The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.
Chromogranin A; Cardioprotection; Ischemia; Post-conditioning; Reperfusion injury
Although Toll-like receptor 4 (TLR4) has been implicated in the myocardial injury caused by regional ischemia/reperfusion, its role in the myocardial inflammatory response and in contractile dysfunction after global ischemia/reperfusion is unclear. Cytokines, particularly tumor necrosis factor-α (TNF-α), contribute to the mechanism of myocardial dysfunction after global ischemia/reperfusion. We hypothesized that a TLR4-mediated cytokine cascade modulates myocardial contractile function after global ischemia/reperfusion. This study examined whether TLR4 regulates TNF-α and interleukin (IL)-1β peptide production during global ischemia/reperfusion and whether TLR4 signaling influences postischemic cardiac function through TNF-α and IL-1β.
Isolated hearts from wild-type mice, two strains of TLR4 mutants, TNF-α knockouts, and IL-1β knockouts underwent global ischemia/reperfusion. Cardiac contractile function was analyzed, and myocardial nuclear factor-κB activity and TNF-α and IL-1β levels were measured.
In wild-type hearts, global ischemia/reperfusion induced nuclear factor-κB activation and the production of TNF-α and IL-1β peptides. In TLR4-mutant hearts, these changes were significantly reduced and postischemic functional recovery was improved. Application of TNF-α and IL-1β to TLR4-mutant hearts abrogated this improvement in postischemic functional recovery. Postischemic functional recovery also improved in TNF-α knockout and IL-1β knockout hearts, as well as in wild-type hearts treated with TNF-binding protein or IL-1 receptor antagonist.
This study demonstrates that TLR4 signaling contributes to cardiac dysfunction after global ischemia/reperfusion. TLR4 signaling mediates the production of TNF-α and IL-1β peptides, and these two cytokines link TLR4 signaling to postischemic cardiac dysfunction.
The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.
Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.
Resveratrol pretreatment can protect the heart by inducing pharmacological preconditioning. Whether resveratrol protects the heart when applied at reperfusion remains unknown. We examined the effect of resveratrol on myocardial infarct size when given at reperfusion and investigated the mechanism underlying the effect. Isolated rat hearts were subjected to 30 min ischemia followed by 2 h of reperfusion, and myocardial samples were collected from the risk zone for Western blot analysis. Mitochondrial swelling was spectrophotometrically measured as a decrease in absorbance at 520 nm (A520). Resveratrol reduced infarct size and prevented cardiac mitochondrial swelling. Resveratrol enhanced GSK-3β phosphorylation upon reperfusion, an effect that was mediated by the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway. Resveratrol translocated GSK-3β from cytosol to mitochondria via the cGMP/PKG pathway. Further studies showed that mitochondrial GSK-3β was co-immunoprecipitated with cyclophilin D but not with VDAC (voltage dependent anion channel) or ANT (adenine nucleotide translocator). These data suggest that resveratrol prevents myocardial reperfusion injury presumably by targeting the mPTP through translocation of GSK-3β from cytosol to mitochondria. Translocated GSK-3β may ultimately interact with cyclophilin D to modulate the mPTP opening.
Resveratrol; Reperfusion injury; Glycogen synthase kinase 3β; Mitochondrial permeability transition pore
Inhibition of GSK-3 reduces ischemia-reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to explore possible molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors prior to ischemia significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and treated with uncoupler or cyanide, or made anoxic. GSK inhibition slowed ATP consumption under these conditions, which could be due to inhibition of ATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0-ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not due to opening of the mitochondrial permeability transition pore. Since GSK is a kinase, we assessed whether protein phosphorylation might be involved. Therefore, we performed western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. LC/MS analysis revealed one of these proteins to be VDAC2. Taken together, we found that GSK mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and suggest that VDAC may be an important regulatory site in ischemia-reperfusion injury.
Mitochondria; GSK-3; VDAC; adenine nucleotide transport
Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex.
Methods and Results
In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 μM followed by an 8-minute washout before 30 minutes of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cγ1/protein kinase Cε (PLC-γ1/PKCε) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-γ1/PKCε pathway, but also cardiac protection. This protection was also blocked by a PKCε translocation inhibitor peptide (PKCε TIP).
Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-γ1 and PKCε.
Inhibition of glucose oxidation during initial reperfusion confers protection against ischemia-reperfusion (IR) injury in the heart. Mitochondrial metabolism is altered with progression of type 2 diabetes (T2DM). We hypothesized that the metabolic alterations present at onset of T2DM induce cardioprotection by metabolic shutdown during IR, and that chronic alterations seen in late T2DM cause increased IR injury.
Isolated perfused hearts from 6 (prediabetic), 12 (onset of T2DM) and 24 (late T2DM) weeks old male Zucker diabetic fatty rats (ZDF) and their age-matched heterozygote controls were subjected to 40 min ischemia/120 min reperfusion. IR injury was assessed by TTC-staining. Myocardial glucose metabolism was evaluated by glucose tracer kinetics (glucose uptake-, glycolysis- and glucose oxidation rates), myocardial microdialysis (metabolomics) and tissue glycogen measurements.
T2DM altered the development in sensitivity towards IR injury compared to controls. At late diabetes ZDF hearts suffered increased damage, while injury was decreased at onset of T2DM. Coincident with cardioprotection, oxidation of exogenous glucose was decreased during the initial and normalized after 5 minutes of reperfusion. Metabolomic analysis of citric acid cycle intermediates demonstrated that cardioprotection was associated with a reversible shutdown of mitochondrial glucose metabolism during ischemia and early reperfusion at onset of but not at late type 2 diabetes.
The metabolic alterations of type 2 diabetes are associated with protection against IR injury at onset but detrimental effects in late diabetes mellitus consistent with progressive dysfunction of glucose oxidation. These findings may explain the variable efficacy of cardioprotective interventions in individuals with type 2 diabetes.
Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia.
myosin light chain; phosphorylation; matrix metalloproteinase; ischemia-reperfusion; ML-7; doxycycline
The cardioprotective potential of human recombinant erythropoietin (alpha) (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of this study was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.
Materials and Methods:
The heart was mounted on a Langendorff apparatus. After 10 min of stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120 min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically using the triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release of lactate dehydrogenase and creatine kinase-MB in the coronary effluent.
The present study demonstrates that Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, or Chelerythrine (1 µM), a protein kinase-C (PKC) inhibitor, or AG490 (5 µM), a JAK-2 inhibitor, significantly attenuated the cardioprotective effects of Epo-induced preconditioning.
Our result suggest that the cardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.
Epo preconditioning; ischemic preconditioning; JAK-2; PI-3K; PKC