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Aims Previous studies demonstrated that pre-treatment with malonate, a reversible inhibitor of succinate dehydrogenase, given before ischaemia, reduces infarct size. However, it is unknown whether administration of malonate may reduce reperfusion injury. Methods and results Isolated mice hearts were treated, under normoxic conditions, with increasing concentrations of disodium malonate (0.03–30 mmol/L, n = 4). Malonate induced a concentration-dependent decrease in left ventricular developed pressure (LVdevP) (EC50 = 8.05 ± 2.11 mmol/L). In isolated hearts submitted to global ischaemia (35 min) followed by reperfusion (60 min), malonate 3 mmol/L given only during the first 15 min of reperfusion reduced lactate dehydrogenase release (125.41 ± 16.82 vs. 189.20 ± 13.74 U/g dry tissue/15 min in controls, P = 0.015) and infarct size (24.57 ± 2.32 vs. 39.84 ± 2.78%, P = 0.001, n = 7–8 per group) and improved recovery of LVdevP (20.06 ± 3.82 vs 7.76 ± 2.53% of baseline LVdevP, P = 0.017). 1H NMR spectroscopy demonstrated marked changes in the metabolic profile of malonate-treated hearts, including increased accumulation of succinate. Furthermore, malonate reduced reactive oxygen species (ROS) production, as measured by MitoSOX staining in myocardial samples obtained after 5 min of reperfusion and in mitochondrial preparations from these samples, preserved mitochondrial respiration, and reduced mitochondrial permeabilization, assessed by calcein retention. Treatment with malonate did not result in activation of RISK or SAFE signalling pathways in tissue extracts obtained 5 min after reperfusion. Conclusion Succinate dehydrogenase inhibition with malonate at the onset of reperfusion reduces infarct size in isolated mice hearts through reduction in ROS production and mitochondrial permeability transition pore opening.

Aims Remote ischaemic conditioning (RIC) has been shown to reduce myocardial infarct size in patients. Our objective was to investigate whether the combination of RIC with either exenatide or glucose–insulin–potassium (GIK) is more effective than RIC alone. Methods and results Pigs were submitted to 40 min of coronary occlusion followed by reperfusion, and received (i) no treatment, (ii) one of the following treatments: RIC (5 min ischemia/5 min reperfusion × 4), GIK, or exenatide (at doses reducing infarct size in clinical trials), or (iii) a combination of two of these treatments (RIC + GIK or RIC + exenatide). After 5 min of reperfusion ( n = 4/group), prominent phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) was observed, both in control and reperfused myocardium, in animals receiving GIK, and mitochondria from these hearts showed reduced ADP-stimulated respiration. 1H NMR-based metabonomics disclosed a shift towards increased glycolysis in GIK and exenatide groups. In contrast, oxidative stress (myocardial nitrotyrosine levels) and eNOS uncoupling were significantly reduced only by RIC. In additional experiments ( n = 7–10/group), ANOVA demonstrated a significant effect of the number of treatments after 2 h of reperfusion on infarct size (triphenyltetrazolium, % of the area at risk; 59.21 ± 3.34, 36.64 ± 3.03, and 21.04 ± 2.38% for none, one, and two treatments, respectively), and significant differences between one and two treatments ( P = 0.004) but not among individual treatments or between RIC + GIK and RIC + exenatide. Conclusions GIK and exenatide activate cardioprotective pathways different from those of RIC, and have additive effects with RIC on infarct size reduction in pigs.

New Findings What is the central question of this study? The microtubule network is disrupted during myocardial ischaemia–reperfusion injury. It was suggested that prevention of microtubule disruption with paclitaxel might reduce cardiac infarct size; however, the effects on infarction have not been studied. What is the main finding and its importance? Paclitaxel caused a reduction in microtubule disruption and cardiomyocyte hypercontracture during ischaemia–reperfusion. However, it induced a greater increase in cytosolic calcium, which may explain the lack of effect against infarction that we have seen in isolated rat hearts. The large increase in perfusion pressure induced by paclitaxel in this model may have clinical implications, because detrimental effects of the drug were reported after its clinical application. Microtubules play a major role in the transmission of mechanical forces within the myocardium and in maintenance of organelle function. However, this intracellular network is disrupted during myocardial ischaemia–reperfusion. We assessed the effects of prevention of microtubule disruption with paclitaxel on ischaemia–reperfusion injury in isolated rat cardiomyocytes and hearts. Isolated rat cardiomyocytes were submitted to normoxia (1 h) or 45 min of simulated ischaemia (pH 6.4, 0% O2, 37°C) and reoxygenation, without or with treatment with the microtubule stabilizer, paclitaxel (10−5 m), or the inhibitor of microtubule polymerization, colchicine (5 × 10−6 m). Simulated ischaemia leads to microtubule disruption before the onset of ischaemic contracture. Paclitaxel attenuated both microtubule disruption and the incidence of hypercontracture, whereas treatment with colchicine mimicked the effects of simulated ischaemia and reoxygenation. In isolated normoxic rat hearts, treatment with paclitaxel induced concentration-dependent decreases in heart rate and left ventricular developed pressure and increases in perfusion pressure. Despite protection against hypercontracture, paclitaxel pretreatment did not modify infarct size (60.37 ± 2.27% in control hearts versus 58.75 ± 10.25, 55.44 ± 10.32 and 50.06 ± 10.14% after treatment with 10−6, 3 × 10−6 and 10−5 m of paclitaxel) after 60 min of global ischaemia and reperfusion in isolated rat hearts. Lack of protection was correlated with a higher increase in cytosolic calcium levels during simulated ischaemia in cardiomyocytes treated with paclitaxel (2.32 ± 0.15 versus 1.13 ± 0.16 a.u. in the presence or absence of 10−6 m paclitaxel, respectively, P

Aims Remote ischaemic conditioning (RIC) releases a humoural factor able to exert cross-species cardioprotection when plasma dialysate is applied to isolated hearts. However, the exact chemical nature of this factor is currently unknown. Methods and results RIC (4 × 5min femoral occlusion/5min reperfusion) was applied to 10 male pigs, and blood was taken before and after the manoeuvre. Discriminant analysis of 1H-NMR spectra ( n = 10–12) obtained from plasma dialysates (12–14 kDa cut-off) allowed to demonstrate a different metabolic profile between control and postRIC samples, with lactate (2.671 ± 0.294 vs. 3.666 ± 0.291 μmol/mL, P = 0.020), succinate (0.062 ± 0.005 vs. 0.082 ± 0.008 μmol/mL, P = 0.035) and glycine (0.055 ± 0.009 vs. 0.471 ± 0.151 μmol/mL, P = 0.015) being the main responsible for such differences. Plasma dialysates were then given to isolated mice hearts submitted to global ischaemia (35 min) and reperfusion (60 min), for 30 min before ischaemia or during the first 15 min of reflow. Infarct size was significantly reduced when postRIC dialysate was applied before ischaemia as compared with hearts pretreated with control dialysate (44.81 ± 3.22 vs. 55.55 ± 2.53%, P = 0.012, n = 12). Blockade of glycine receptors with strychnine 10 μM inhibited the protective effect caused by pretreatment with postRIC dialysate (52.76 ± 6.94 vs. 51.92 ± 5.78%, P -NS, n = 5), whereas pretreatment with glycine 3 mmol/L, but not succinate 100 μmol/L, mimicked RIC protection (41.90 ± 4.50% in glycine-treated vs. 61.51 ± 5.16 and 64.73 ± 4.47% in succinate-treated and control hearts, respectively, P

What is the central question of this study? The microtubule network is disrupted during myocardial ischaemia-reperfusion injury. It was suggested that prevention of microtubule disruption with paclitaxel might reduce cardiac infarct size; however, the effects on infarction have not been studied. What is the main finding and its importance? Paclitaxel caused a reduction in microtubule disruption and cardiomyocyte hypercontracture during ischaemia-reperfusion. However, it induced a greater increase in cytosolic calcium, which may explain the lack of effect against infarction that we have seen in isolated rat hearts. The large increase in perfusion pressure induced by paclitaxel in this model may have clinical implications, because detrimental effects of the drug were reported after its clinical application. Microtubules play a major role in the transmission of mechanical forces within the myocardium and in maintenance of organelle function. However, this intracellular network is disrupted during myocardial ischaemia-reperfusion. We assessed the effects of prevention of microtubule disruption with paclitaxel on ischaemia-reperfusion injury in isolated rat cardiomyocytes and hearts. Isolated rat cardiomyocytes were submitted to normoxia (1 h) or 45 min of simulated ischaemia (pH 6.4, 0% O2 , 37 °C) and reoxygenation, without or with treatment with the microtubule stabilizer, paclitaxel (10(-5) M), or the inhibitor of microtubule polymerization, colchicine (5 × 10(-6) M). Simulated ischaemia leads to microtubule disruption before the onset of ischaemic contracture. Paclitaxel attenuated both microtubule disruption and the incidence of hypercontracture, whereas treatment with colchicine mimicked the effects of simulated ischaemia and reoxygenation. In isolated normoxic rat hearts, treatment with paclitaxel induced concentration-dependent decreases in heart rate and left ventricular developed pressure and increases in perfusion pressure. Despite protection against hypercontracture, paclitaxel pretreatment did not modify infarct size (60.37 ± 2.27% in control hearts versus 58.75 ± 10.25, 55.44 ± 10.32 and 50.06 ± 10.14% after treatment with 10(-6) , 3 × 10(-6) and 10(-5) m of paclitaxel) after 60 min of global ischaemia and reperfusion in isolated rat hearts. Lack of protection was correlated with a higher increase in cytosolic calcium levels during simulated ischaemia in cardiomyocytes treated with paclitaxel (2.32 ± 0.15 versus 1.13 ± 0.16 a.u. in the presence or absence of 10(-6) m paclitaxel, respectively, P0.05), but not with changes in aortic reactivity. In conclusion, microtubule stabilization with paclitaxel reduces hypercontracture in isolated rat cardiomyocytes but does not protect against infarction in isolated rat hearts.

Connexin 43 (Cx43) deficiency increases myocardial tolerance to ischemia-reperfusion injury and abolishes preconditioning protection. It is not known whether modifications in baseline signaling through protective RISK or SAFE pathways or in response to preconditioning may contribute to these effects. To answer this question we used Cx43(Cre-ER(T)/fl) mice, in which Cx43 expression is abolished after 4-hydroxytamoxifen (4-OHT) administration. Isolated hearts from Cx43(Cre-ER(T)/fl) mice, or from Cx43(fl/fl) controls, treated with vehicle or 4-OHT, were submitted to global ischemia (40 min) and reperfusion. Cx43 deficiency was associated with reduced infarct size after ischemia-reperfusion (11.17 ± 3.25 % vs. 65.04 ± 3.79, 59.31 ± 5.36 and 65.40 ± 4.91, in Cx43(fl/fl) animals treated with vehicle, Cx43(fl/fl) mice treated with 4-OHT, and Cx43(Cre-ER(T)/fl) mice treated with vehicle, respectively, n = 8-9, p 0.001). However, the ratio phosphorylated/total protein expression for Akt, ERK-1/2, GSK3β and STAT3 was not increased in normoxic samples from animals lacking Cx43. Instead, a reduction in the phosphorylation state of GSK3β was observed in Cx43-deficient mice (ratio: 0.15 ± 0.02 vs. 0.56 ± 0.11, 0.77 ± 0.15, and 0.46 ± 0.14, respectively, n = 5-6, p 0.01). Furthermore, ischemic preconditioning (IPC, 4 cycles of 3.5 min of ischemia and 5 min of reperfusion) increased phosphorylation of ERK-1/2, GSK3β, and STAT3 in all hearts without differences between groups (n = 5-6, p 0.05), although Cx43 deficient mice were not protected by either IPC or pharmacological preconditioning with diazoxide. Our data demonstrate that modification of RISK and SAFE signaling does not contribute to the role of Cx43 in the increased tolerance to myocardial ischemia-reperfusion injury and in preconditioning protection.