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ABSTRACT: The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion.
Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS.
These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.
Journal of the American Heart Association. 01/2013; 2(1):e005975.
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ABSTRACT: Loss of calcium (Ca(2+)) homeostasis contributes through different mechanisms to cell death occurring during the first minutes of reperfusion. One of them is an unregulated activation of a variety of Ca(2+)-dependent enzymes, including the non-lysosomal cysteine proteases known as calpains. This review analyses the involvement of the calpain family in reperfusion-induced cardiomyocyte death. Calpains remain inactive before reperfusion due to the acidic pHi and increased ionic strength in the ischaemic myocardium. However, inappropriate calpain activation occurs during myocardial reperfusion, and subsequent proteolysis of a wide variety of proteins contributes to the development of contractile dysfunction and necrotic cell death by different mechanisms, including increased membrane fragility, further impairment of Na(+) and Ca(2+) handling, and mitochondrial dysfunction. Recent studies demonstrating that calpain inhibition contributes to the cardioprotective effects of preconditioning and postconditioning, and the beneficial effects obtained with new and more selective calpain inhibitors added at the onset of reperfusion, point to the potential cardioprotective value of therapeutic strategies designed to prevent calpain activation.
Cardiovascular research 07/2012; 96(1):23-31. · 5.80 Impact Factor
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ABSTRACT: Mitochondrial permeability transition (MPT) is critical in cardiomyocyte death during reperfusion but it is not the only mechanism responsible for cell injury. The objectives of the study is to investigate the role of the duration of myocardial ischemia on mitochondrial integrity and cardiomyocyte death. Mitochondrial membrane potential (ΔΨm, JC-1) and MPT (calcein) were studied in cardiomyocytes from wild-type and cyclophilin D (CyD) KO mice refractory to MPT, submitted to simulated ischemia and 10 min reperfusion. Reperfusion after 15 min simulated ischemia induced a rapid recovery of ΔΨm, extreme cell shortening (contracture) and mitochondrial calcein release, and CyD ablation did not affect these changes or cell death. However, when reperfusion was performed after 25 min simulated ischemia, CyD ablation improved ΔΨm recovery and reduced calcein release and cell death (57.8 ± 4.9% vs. 77.3 ± 4.8%, P < 0.01). In a Langendorff system, CyD ablation increased infarct size after 30 min of ischemia (61.3 ± 6.4% vs. 45.3 ± 4.0%, P = 0.02) but reduced it when ischemia was prolonged to 60 min (52.8 ± 8.1% vs. 87.6 ± 3.7%, P < 0.01). NMR spectroscopy in rat hearts showed a rapid recovery of phosphocreatine after 30 min ischemia followed by a marked decay associated with contracture and LDH release, that were preventable with contractile blockade but not with cyclosporine A. In contrast, after 50 min ischemia, phosphocreatine recovery was impaired even with contractile blockade (65.2 ± 4% at 2 min), and cyclosporine A reduced contracture, LDH release and infarct size (52.1 ± 4.2% vs. 82.8 ± 3.6%, P < 0.01). In conclusion, the duration of ischemia critically determines the importance of MPT on reperfusion injury. Mechanisms other than MPT may play an important role in cell death after less severe ischemia.
Archiv für Kreislaufforschung 09/2011; 106(6):1259-68. · 7.35 Impact Factor
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ABSTRACT: Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague-Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by (31)P-NMR spectroscopy (3.6±0.2min and 3.5±0.2min respectively vs. 1.4±0.2min in control group, P<0.01) and caused equivalent cardioprotection (48% and 41% of infarct reduction respectively, P<0.01), but only postconditioning increased myocardial cGMP levels (P=0.02) and activated PKG. Blockade of cGMP/PKG pathway by the addition of the guanylyl cyclase inhibitor ODQ or the PKG inhibitor KT5823 during reperfusion accelerated pHi recovery and abolished cardioprotection in postconditioned hearts, but had no effect in hearts subjected to acidic reperfusion suggesting that PKG signaling was upstream of delayed pHi normalization in postconditioned hearts. In isolated cardiomyocytes the cGMP analog 8-pCPT-cGMP delayed Na(+)/H(+)-exchange mediated pHi normalization after acidification induced by a NH(4)Cl pulse. These results demonstrate that the cGMP/PKG pathway contributes to postconditioning protection at least in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na(+)/H(+)-exchanger.
Journal of Molecular and Cellular Cardiology 02/2011; 50(5):903-9. · 5.17 Impact Factor
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ABSTRACT: Platelets activated during experimental acute myocardial infarction (AMI) contribute to myocardial injury. This study aimed to investigate whether platelets from patients with AMI increase myocardial injury after ischaemia and reperfusion in isolated rat hearts and the modification of this effect by the P2Y12 receptor antagonist cangrelor and the glycoprotein IIb/IIIa receptor blocker abciximab. Isolated rat hearts were subjected to 40 minutes of global ischaemia and 60 minutes of reperfusion. Hearts (four simultaneous experiments per patient) were infused with platelets from nine AMI patients (seven thrombolysed, all on aspirin), untreated or incubated with 10 microM cangrelor or 5 microg/ml abciximab. Control experiments were performed using platelets from healthy volunteers and platelet-poor plasma. P-selectin expression on isolated platelets was higher in AMI patients than in controls and was not modified by the treatments. Control platelets or platelet-poor plasma did mild or no harm. In contrast, platelets from AMI patients significantly augmented myocardial injury, as demonstrated by worse left ventricular (LV) developed pressure, higher maximal LV end-diastolic pressure and coronary resistance, and greater lactate dehydrogenase release and infarct size. Both cangrelor and abciximab greatly attenuated these effects. In conclusion, activated platelets from AMI patients increase myocardial injury after ischaemia and reperfusion, and cangrelor and abciximab attenuate this effect. The results support the notion that very early antiplatelet treatment might reduce infarct size by direct effects on reperfused myocardium in these patients.
Thrombosis and Haemostasis 07/2010; 104(1):128-35. · 5.04 Impact Factor
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ABSTRACT: We previously demonstrated that ischemic preconditioning (IPC) attenuates calpain activation during reperfusion. Herein, we tested the hypothesis that enhancement of Na+/K+-ATPase activity during early reperfusion as a result of calpain inhibition is involved in the protection afforded by myocardial IPC.
Intracellular Na+ concentration ([Na+]i) measured using 23Na-magnetic resonance spectroscopy, Na+/K+-ATPase activity, detachment of Na+/K+-ATPase alpha subunits from the membrane cytoskeleton, degradation of fodrin and ankyrin, and calpain activation were analysed in isolated rat hearts reperfused after 60 min of ischemia with or without previous IPC and different treatments aimed to mimic or blunt the effects of IPC.
In non-treated hearts subjected to ischemia (control hearts), reperfusion for 5 min severely reduced Na+/K+-ATPase activity and dissociated alpha1 and alpha2 subunits of Na+/K+-ATPase from the membrane-cytoskeleton complex in parallel with proteolysis of alpha-fodrin and ankyrin-B and calpain activation. IPC accelerated the recovery of [Na+]i, increased Na+/K+-ATPase activity, and prevented dissociation of Na+/K+-ATPase from the membrane-cytoskeleton complex. IPC also prevented alpha-fodrin and ankyrin-B loss and calpain activation, effects that were associated with attenuated lactate dehydrogenase (LDH) release and infarct size and improved contractile recovery. These effects of IPC were reproduced by perfusing the hearts with the calpain inhibitor MDL-28170 and by transient stimulation of cAMP-dependent protein kinase (PKA) with CPT-cAMP, and they were reverted by perfusing with the PKA inhibitor H89.
The results of the present study are consistent with the hypothesis that enhanced recovery of Na+/K+-ATPase activity during reperfusion as a result of attenuated calpain-mediated detachment of the protein from the membrane-cytoskeleton complex contributes to the protection afforded by IPC.
Cardiovascular Research 06/2006; 70(2):364-73. · 6.06 Impact Factor
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ABSTRACT: Na+ overload and secondary Ca2+ influx via Na+/Ca2+ exchanger are key mechanisms in cardiomyocyte contracture and necrosis during reperfusion. Impaired Na+/K+-ATPase activity contributes to Na+ overload, but the mechanism has not been established. Because Na+/K+-ATPase is connected to the cytoskeleton protein fodrin through ankyrin, which are substrates of calpains, we tested the hypothesis that calpain mediates Na+/K+-ATPase impairment in reperfused cardiomyocytes. In isolated rat hearts reperfused for 5 minutes after 60 minutes of ischemia, Na+/K+-ATPase activity was reduced by 80%, in parallel with loss of alpha-fodrin and ankyrin-B and detachment of alpha1 and alpha2 subunits of Na+/K+-ATPase from the membrane-cytoskeleton complex. Calpain inhibition with MDL-7943 during reperfusion prevented the loss of these proteins, increased Na+/K+-ATPase activity, attenuated lactate dehydrogenase release, and improved contractile recovery, and these beneficial effects of MDL-7943 were reverted by ouabain. The impairment of Na+/K+-ATPase was not a mere consequence of cell death because it was not altered in hearts in which contracture and cell death had been prevented by contractile blockade with 2,3-butanedione monoxime. In these hearts, concomitant calpain inhibition preserved Na+/K+-ATPase content and function and attenuated cell death occurring on withdrawal of 2,3-butanedione monoxime. In vitro assay showed no detectable degradation of Na+/K+-ATPase subunits after 10 minutes of incubation with activated calpain. Thus, we conclude that calpain activation contributes to the impairment of Na+/K+-ATPase during early reperfusion and that this effect is mainly mediated by degradation of the anchorage of Na+/K+-ATPase to the membrane cytoskeleton.
Circulation Research 10/2005; 97(5):465-73. · 9.49 Impact Factor
