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Reports on the effect of obesity on the myocardial tolerance to ischemia are contradictory. We have described that obesity induced by high-fat diet (HFD) reduces infarct size in B6D2F1 mice submitted to transient coronary occlusion. In this study, we analysed the mechanism by which dietary obesity modifies the susceptibility to myocardial ischemia and the robustness of this effect. B6D2F1 (BDF), C57BL6/J (6J), C57BL6/N (6N) male mice and BDF female mice were fed with a HFD or control diet for 16 weeks. In all three strains, HFD induced obesity with hyperinsulinemia and hypercholesterolemia and without hyperglycemia, hypertension, ventricular remodelling or cardiac dysfunction. In obese mice from all three strains PDK4 was overexpressed and HSQC NMR spectroscopy showed reduced 13 C-glutamate and increased 13 C-lactate and 13 C-alanine, indicating uncoupling of glycolysis from glucose oxidation. In addition, HFD induced mild respiratory uncoupling in mitochondria from BDF and 6N mice in correlation with UCP3 overexpression. In studies performed in isolated perfused hearts submitted to transient ischemia these changes were associated with reduced ATP content and myocardial PCr/ATP ratio at baseline, and delayed pHi recovery ( 31 PNMR) and attenuated hypercontracture at the onset of reperfusion. Finally, in mice subjected to 45 min of coronary occlusion and 24 h of reperfusion, HFD significantly reduced infarct size respect to their respective control diet groups in male BDF (39.4 ± 6.1% vs. 19.9 ± 3.2%, P = 0.018) and 6N mice (38.0 ± 4.1 vs. 24.5 ± 2.6%, P = 0.017), and in female BDF mice (35.3 ± 4.4% vs. 22.3 ± 2.5%, P = 0.029), but not in male 6J mice (40.2 ± 3.4% vs. 34.1 ± 3.8%, P = 0.175). Our results indicate that the protective effect of HFD-induced obesity against myocardial ischemia/reperfusion injury is influenced by genetic background and appears to critically depend on inhibition of glucose oxidation and mild respiratory mitochondrial uncoupling resulting in prolongation of acidosis at the onset of reperfusion., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mitochondria play a central role in the protection conferred by ischemic preconditioning (IP) by not fully elucidated mechanisms. We investigated whether IP protects mitochondria against ischemia-reperfusion (IR) injury through mechanisms independent of cytosolic signaling. In isolated rat hearts, sublethal IR increased superoxide production and reduced complex-I- and II-mediated respiration in subsarcolemmal (SS), but not interfibrillar (IF) mitochondria. This effect of IR on mitochondrial respiration was significantly attenuated by IP. Similar results were obtained in isolated cardiac mitochondria subjected to in vitro IR. The reduction in SS mitochondrial respiration in the heart and in vitro model was paralleled by an increase in oxidized cysteine residues, which was also prevented by IP. IP was also protective in mitochondria submitted to lethal IR. The protective effect of IP against respiratory failure was unaffected by inhibition of mitochondrial KATP channels or mitochondrial permeability transition. However, IP protection was lost in mitochondria from genetically-modified animals in which connexin-43, a protein present in SS but not IF mitochondria, was replaced by connexin-32. Our results demonstrate the existence of a protective mitochondrial mechanism or "mitochondrial preconditioning" independent of cytosol that confers protection against IR-induced respiratory failure and oxidative damage, and requires connexin-43., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Myocardial edema occurs in a large number of myocardial pathologies particularly during ischemia-reperfusion, and may contribute to cell dysfunction and death occurring in these conditions. Cardiomyocyte cell volume is tightly regulated by modifications in cytosolic osmolality. Changes in membrane water permeability through aquaporin and connexin hemichannels also contribute to cell volume changes while caveolae may be important in sensing cell volume changes sensing and associated signaling. Ischemia-reperfusion alters these mechanisms and increases microvascular permeability by endothelial hypercontracture-induced gap formation, endothelial cell death and basal membrane disruption. Detection of myocardial edema by MRI has many useful diagnostic applications in acute myocardial infarction and other conditions. However, discrimination between intra and extracellular myocardial edema is presently difficult at the bench and impossible at the bedside. Developing methods to differentiate intra from extracellular myocardial water should allow a better understanding of the mechanisms and consequences of myocardial edema and, as a consequence lead to new diagnostic and therapeutic applications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Preserved mitochondrial function (respiration, calcium handling) and integrity (cytochrome c release) is central for cell survival following ischemia/reperfusion. Mitochondrial function also requires import of proteins from the cytosol via the translocase of the outer and inner membrane (TOM and TIM complexes). Since mitochondrial function following ischemia/reperfusion is better preserved by ischemic preconditioning (IP), we now investigated whether expression of parts of the import machinery is affected by ischemia/reperfusion without or with IP in vivo. We analyzed the mitochondrial content of the presequence receptor Tom20, the pore forming unit Tom40 and Tim23. Goettinger minipigs were subjected to 90 min of low-flow ischemia without or with preconditioning by 10 min ischemia and 15 min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall of the left ventricle and from the control posterior wall. Infarct size was significantly reduced by IP (20.1 +/- 1.6% of area at risk (non-preconditioned) vs. 6.5 +/- 2.5% of area at risk (IP)). Using Western blot analysis, the ratio of Tom20 (normalized to Ponceau S) between mitochondria isolated from the anterior ischemic and posterior control wall was reduced (0.72 +/- 0.11, a.u., n = 8), whereas the mitochondrial Tom20 content was preserved by IP (1.17 +/- 0.16 a.u., n = 7, P < 0.05). The mitochondrial Tom40, Tim23 and adenine nucleotide transporter (ANT) contents were not significantly different between non-preconditioned and preconditioned myocardium. The preservation of the mitochondrial Tom20 protein level may contribute to the improved mitochondrial function after IP.

Thrombin exerts multiple actions on cardiomyocytes leading to increased intracellular Na+ and Ca2+ concentrations, and to activation of a Ca2+-independent PLA2, and has been proposed to favor the genesis of arrhythmias and ischemic injury in acute coronary syndromes. However, the influence of thrombin on cardiomyocyte cell death during ischemia-reperfusion has not been studied. A beneficial influence of low thrombin concentrations has been described in other cell types. HL-1 cardiomyocytes were subjected to simulated ischemia (SI) and reperfusion (SR) and cell death was assessed by means of LDH release to the incubation media. Thrombin dose-dependently increased cell death in normoxic cells, in cells subjected to SI, and in cells subjected to SR (by 20+/-8%, 95+/-32% and 35+/-9%, respectively, at 100 U/ml). The effects of thrombin were associated to increased cytosolic Ca2+ overload, mimicked by 100 microM PAR-1 agonist peptide SFLLRNPNDKYEPF, and reversed by the direct thrombin inhibitor lepirudin (IC50=1.3+/-0.2 microg/ml). The presence of thrombin during simulated ischemia-reperfusion increases cardiomyocyte cell death by a mechanism that involves activation of PAR-1 receptors and can be prevented by the direct thrombin inhibitor lepirudin.

To investigate the role of Na(+)-H+ exchange occurring during hypoxia in the genesis of reoxygenation-induced myocardial oedema, isolated perfused rat hearts were submitted to 40 min of hypoxia and 90 min of reoxygenation. The influence of three factors on myocardial water content was analysed according to a 2 x 2 x 2 factorial design; the hearts were perfused at either pH = 7.4 or pH = 7.0, with either HCO3- buffer or HCO3(-)-free HEPES buffer, and in half of the experiments the hypoxic buffer contained HOE642 6.7 micromol/l. In an additional group, 160 min of normoxia resulted in no lactate dehydrogenase (LDH) release and in a 35.8% increase in myocardial water, independently of pH and of the presence of HCO3- in the buffer. In hearts perfused at pH = 7.4, reoxygenation induced LDH release which was reduced (P<0.05) by HOE642 by 20.1%, by HCO3(-)-free perfusion by 57.5%, and by the combination of both by 91.2%. Reoxygenation also induced severe myocardial oedema (26.3% increase (P<0.05) respect to normoxia). HOE642 reduced (P<0.05) reoxygenation oedema by 15.7%, HCO3(-)-free perfusion by 8.9%, and the combination of both by 24.6%. The effects of HCO3(-)-free perfusion could be mimicked in HCO3(-)-perfused hearts by blocking Na(+)-HCO3- cotransport with 4-4'-dibenzanidostilbene-2,2'-disulphonic acid (DIDS). The beneficial and additive effects of HOE642 and of HCO3(-)-free perfusion on oedema were not a mere consequence of their protective effects against the oxygen paradox, since they were observed in groups perfused at pH= 7.0, a condition which virtually prevented LDH release without preventing oedema (19.0% increase in myocardial water). Thus, reoxygenation-induced myocardial oedema may occur in the absence of necrosis, and is largely determined by Na+ gain during hypoxia via Na(+)-H+ exchange and Na(+)-HCO3- cotransport.