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Aims Ischaemic preconditioning (IPC) protects the heart against prolonged lethal ischaemia through a signalling cascade involving Akt, glycogen synthase kinase-3β (GSK-3β), and mitochondrial ATP-sensitive potassium channels (mitoKATP). We previously demonstrated the involvement of the Wnt pathway in IPC in vivo via GSK-3β. A downstream target might be mammalian target of rapamycin (mTOR) since Wnt can impair tuberous sclerosis complex-2 (TSC2) phosphorylation by inhibiting GSK-3β. Here, we investigate whether the mTOR pathway is involved in cardioprotection. Methods and results Isolated-perfused mouse hearts were subjected to IPC via four cycles of ischaemia/reperfusion or pharmacological preconditioning (PPC) by diazoxide, a selective mitoKATP activator. IPC, like PPC, induced an inhibition/phosphorylation of GSK-3β through Akt activation. Preconditioning also induced phosphorylation of mTOR, p70S6K, and 4E-BP1 that correlated with a significant reduction in infarct size after 40-min ischaemia and 120-min reperfusion when compared with non-preconditioned controls. Preconditioning was impaired in GSK3 knock-in mice. In transgenic mice hearts overexpressing secreted frizzled protein 1 (sFRP1, a Wnt/Frz antagonist), GSK-3β phosphorylation, mTOR activation, and cardioprotection were impaired. Cardioprotection and its signalling were also inhibited by rapamycin (an mTOR inhibitor), 5-HD (a mitoKATP blocker), and N -(2-mercaptopropionyl) glycine (MPG) as a reactive oxygen species (ROS) scavenger. Conclusions We propose that the preconditioning signalling pathway involving an amplification loop results in a downregulation of GSK-3β and a constant opening of mitoKATP with ROS generation to activate the mTOR pathway and induce cardioprotection. The disruption of the Wnt/Frz pathway by sFRP1 modulates this loop, inducing GSK-3β activation. This study provides evidence that cardioprotection involves both a pro-survival mTOR pathway and a developmental Wnt pathway targeting GSK-3β.

This study analyzed the oxidant generation during ischemia-reperfusion protocols of Langendorff-perfused rat hearts, preconditioned with a mitochondrial ATP-sensitive potassium channel (mitoKATP) opener (i.e., diazoxide). The autofluorescence of mitochondrial flavoproteins, and that of the total NAD(P)H pool on the one hand and the fluorescence of dyes sensitive to H2O2or O2•−[i.e., the dihydrodichlorofluoroscein (H2DCF) and dihydroethidine (DHE), respectively] on the other, were noninvasively measured at the surface of the left ventricular wall by means of optic fibers. Isolated perfused rat hearts were subjected to an ischemia-reperfusion protocol. Opening mitoKATPwith diazoxide (100 μM) 1) improved the recovery of the rate-pressure product after reperfusion (72 ± 2 vs. 16.8 ± 2.5% of baseline value in control group, P < 0.01), and 2) attenuated the oxidant generation during both ischemic (−46 ± 5% H2DCF oxidation and −40 ± 3% DHE oxidation vs. control group, P < 0.01) and reperfusion (−26 ± 2% H2DCF oxidation and −23 ± 2% DHE oxidation vs. control group, P < 0.01) periods. All of these effects were abolished by coperfusion of 5-hydroxydecanoic acid (500 μM), a mitoKATPblocker. During the preconditioning phase, diazoxide induced a transient, reversible, and 5-hydroxydecanoic acid-sensitive flavoprotein and H2DCF (but not DHE) oxidation. In conclusion, the diazoxide-mediated cardioprotection is supported by a moderate H2O2production during the preconditioning phase and a strong decrease in oxidant generation during the subsequent ischemic and reperfusion phases.

We showed recently that mitochondrial ATP-dependent K+channel (mitoKATP) opening is required for the inotropic response to ouabain. Because mitoKATPopening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na+-K+-ATPase with the opening of mitoKATP. In Langendorff-perfused rat hearts, 10–80 μM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoKATPopening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoKATPinhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection.

We have studied the protective effect of dexrazoxane on the cardiac toxicity induced by the anthracyclines currently used in clinics, doxorubicin, epirubicin, daunorubicin and idarubicin, with special emphasis on determining the optimal dose of dexrazoxane. This was performed using the model of isolated perfused rat heart after 12-day combination treatment of anthracyclines used at equi-cardiotoxic doses, and dexrazoxane used at 10-fold or 20-fold the anthracycline dose. We have shown in this study that dexrazoxane by itself was not cardiotoxic, and was able to significantly reduce anthracycline cardiac toxicity without increasing the general toxicity induced by these drugs. Using dexrazoxane at 20 times the anthracycline dose provided a better cardioprotection than using it at 10 times the anthracycline dose; at the higher dexrazoxane dose, the functional cardiac parameters (developed pressure, contractility and relaxation) were not different from those recorded in control animals.

Background The ability to assess myocardial perfusion in small animals is important, especially to investigate models of myocardial ischemia. Myocardial perfusion is usually assessed by postmortem techniques, eliminating the possibility of follow-up. We sought to evaluate whether contrast echocardiography was able to quantify myocardial perfusion in rats. Methods Twenty-four rats divided in 3 groups (sham-operated, and 8 and 21 days after left anterior descending coronary artery stenosis) underwent myocardial contrast echocardiography using intermittent triggered imaging. Peak plateau intensity and slope of refilling were compared with myocardial blood flow achieved with fluorescent microspheres. Results High-quality images were easily obtained for each experiment. Close correlation was found between myocardial contrast echocardiography and myocardial blood flow, especially for measurements of peak plateau intensity × slope of refilling relative to the control area (y = 1.15 × −0.14, r = 0.86). Conclusion Quantification of myocardial perfusion in rats is feasible by myocardial contrast echocardiography using intermittent triggered imaging.

The aim of this study was to investigate the effects of HMR1098, a selective blocker of sarcolemmal ATP-sensitive potassium channel (sarcK(ATP)), in Langendorff-perfused rat hearts submitted to ischemia and reperfusion. The recovery of heart hemodynamic and mitochondrial function, studied on skinned fibers, was analyzed after 30-min global ischemia followed by 20-min reperfusion. Infarct size was quantified on a regional ischemia model after 2-h reperfusion. We report that the perfusion of 10 microM HMR1098 before ischemia, delays the onset of ischemic contracture, improves recovery of cardiac function upon reperfusion, preserves the mitochondrial architecture, and finally decreases infarct size. This HMR1098-induced cardioprotection is prevented by 1 mM 2-mercaptopropionylglycine, an antioxidant, and by 100 nM nifedipine, an L-type calcium channel blocker. Concomitantly, it is shown that HMR1098 perfusion induces (i) a transient and specific inhibition of the respiratory chain complex I and, (ii) an increase in the averaged intracellular calcium concentration probed by the in situ measurement of indo-1 fluorescence. Finally, all the beneficial effects of HMR1098 were strongly inhibited by 5-hydroxydecanoate and abolished by glibenclamide, two mitoK(ATP) blockers. This study demonstrates that the HMR1098-induced cardioprotection occurs indirectly through extracellular calcium influx, respiratory chain complex inhibition, reactive oxygen species production and mitoK(ATP) opening. Taken together, these data suggest that a functional interaction between sarcK(ATP) and mitoK(ATP) exists in isolated rat heart ischemia model, which is mediated by extracellular calcium influx.

This study investigates the role of the mitochondrial ATP-sensitive K+ channel (mitoKATP) in response to positive inotropic stress. In Langendorff-perfused rat hearts, inotropy was induced by increasing perfusate calcium to 4 mM, by adding 80 μM ouabain or 0.25 μM dobutamine. Each of these treatments resulted in a sustained increase in rate-pressure product (RPP) of ∼60%. Inhibition of mitoKATP by perfusion of 5-hydroxydecanoate (5-HD) or tetraphenylphosphonium before induction of inotropic stress resulted in a marked attenuation of RPP. Inhibition of mitoKATP after induction of stress caused the inability of the heart to maintain a high-work state. In human atrial fibers, the increase in contractility induced by dobutamine was inhibited 60% by 5-HD. In permeabilized fibers from the Langendorff-perfused rat hearts, inhibition of mitoKATP resulted, in all cases, in an alteration of adenine nucleotide compartmentation, as reflected by a 60% decrease in the half-saturation constant for ADP [ K1/2 (ADP)]. We conclude that opening of cardiac mitoKATP is essential for an appropriate response to positive inotropic stress and propose that its involvement proceeds through the prevention of stress-induced decrease in mitochondrial matrix volume. These results indicate a physiological role for mitoKATP in inotropy and, by extension, in heart failure.

It has been proposed that activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) is part of signaling pathways triggering the cardioprotection afforded by ischemic preconditioning of the heart. This work was to analyze the mitochondrial function profile of Langendorff-perfused rat hearts during the different phases of various ischemia-reperfusion protocols. Specifically, skinned fibers of ischemic preconditioned hearts exhibit a decline in the succinate-supported respiration and complex II activity during ischemia, followed by a recovery during reperfusion. Meanwhile, the apparent affinity of respiration for ADP (which reflects the matrix volume expansion) is increased during preconditioning stimulus and, to a larger extent, during prolonged ischemia. This evolution pattern is mimicked by diazoxide and abolished by 5-hydroxydecanoate. It is concluded that opening the mitoK(ATP) channel mediates the preservation of mitochondrial structure-function via a mitochondrial matrix shrinkage and a reversible inactivation of complex II during prolonged ischemic insult.

Diazoxide opening of the mitochondrial ATP-sensitive K+(mitoKATP) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoKATPchannel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoKATPchannel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoKATPchannel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.

International audience; The aim of this study was to investigate mitochondrial alterations in an animal model of chronic myocardial ischemia in rats obtained by surgical constriction of the left coronary artery. Resting coronary blood flow was measured using the fluorescent microsphere technique. Contractile function, defined by rate-pressure product, and myocardial oxygen consumption were measured in a Langendorff preparation. The mitochondrial function was evaluated on permeabilized skinned fibers. Three weeks after surgery, ischemic hearts showed a significant decrease in coronary blood flow compared with sham. Hemodynamic measurements showed a significant systolic and diastolic dysfunction. Alterations in mitochondrial function in ischemic hearts were mainly characterized by a significant decrease in the maximal velocity and apparent half-saturation constant for ADP, loss of the stimulatory effect of creatine, and a stimulatory effect of exogenous cytochrome c. These functional alterations were supported by structural alterations characterized by mitochondrial clustering and swelling associated with membrane rupture. We conclude that the alterations in systolic function after chronic ischemia are supported by severe modifications of mitochondrial structure and function.

1. In order to develop a predictive model for the preclinical evaluation of anthracycline cardiotoxicity and the means of preventing it, we have studied the functional parameters of perfused hearts isolated from rats receiving repeated doses of several anthracyclines. 2. The anthracyclines studied were doxorubicin, epirubicin, pirarubicin and daunorubicin, and we also studied a liposomal formulation of daunorubicin (DaunoXome) and the co-administration of dexrazoxane (ICRF-187) and doxorubicin. 3. Anthracyclines were administered i.p. at equimolar doses corresponding to 3 mg kg-1 per injection of doxorubicin, every other day for a total of six doses. Dexrazoxane was used at the dose of 30 mg kg-1 per injection and was administered either 30 min before or 30 min after doxorubicin. We evaluated any general toxicity towards the animals as well as alterations of left ventricular contractility and relaxation ex vivo. 4. Epirubicin and daunorubicin were significantly less cardiotoxic than doxorubicin, and neither pirarubicin nor DaunoXome caused significant alterations in cardiac function. There was a direct relationship between the decrease in cardiac contractility or relaxation and anthracycline accumulation in the heart, evaluated after the same treatment schedule. 5. Dexrazoxane induced a significant protection against doxorubicin-induced cardiac toxicity when administered 30 min before doxorubicin, whereas this protection was ineffective when administered 30 min after doxorubicin. Direct perfusion of DaunoXome in isolated hearts of untreated animals resulted in a 12-fold reduction of the accumulation of daunorubicin in heart tissue as compared to the perfusion of free daunorubicin, and did not cause alterations in cardiac function at a dosage for which free daunorubicin induced major alterations. 6. The isolated perfused rat heart appears to be a valuable model for screening of new anthracyclines and of strategies for circumventing anthracycline cardiotoxicity.

We have compared the cardiotoxicity of 3 anthracyclines in a model of isolated perfused rat heart using the Langendorff technique. The contractile state and ventricular compliance were studied. Doxorubicin, epirubicin and pirarubicin were perfused at concentrations of 10(-6) and 10(-5) M during 70 min. The cardiac accumulation of the drugs was studied by HPLC. No significant alteration of cardiac functional parameters was observed at 10(-6) M. At 10(-5) M, epirubicin produced a significantly greater alteration of cardiac contractility than doxorubicin, whereas pirarubicin exerted first an inotropic effect followed by a recovery to initial values at the 60th min. Anthracycline accumulation in the heart was dose-dependent; epirubicin accumulated to a 30% greater extent than doxorubicin and pirarubicin heart concentrations were 4-5 times higher than those of doxorubicin at the end of the perfusion. These results suggest that doxorubicin and epirubicin have the same intrinsic cardiac toxicity, and that their distinct clinical cardiotoxicity must be explained by pharmacokinetic differences, whereas pirarubicin is much less cardiotoxic than the other anthracyclines because of different pharmacodynamic properties.

The aim of this study was to investigate the mechanisms of the cardiodepressive action of ionic and non-ionic contrast media currently used in coronary arteriography. Experiments carried out on isolated preparations of cat papillary muscle, treated with increasing concentrations of Telebrix 39, Radioselectan, Hexabrix and Iopamidol 370 and 300, on the one hand and, on the other hand, a pre-determined dose of Telebrix and Iopamidol 300 during hypoxia and subsequent reoxygenation, showed that, at constant Ca2+ concentrations: (1) The cardiodepressive effects of contrast media are correlated with the hyperosmolality that they induce. When osmolality was higher than 400 mOsm, all the products caused a reduction of the peak force (PF: 40.49 +/- 5.15%), the maximum velocity of contraction (Vmax: 39.85 +/- 3.66%) and of the peak velocity of relaxation (Vrelax: 23.30 +/- 2.20%) (P less than 0.01). The time to peak force (TPF), on the other hand, remained constant, whereas the half-relaxation time (THR) was increased. No significant differences were observed between these effects and those induced by control iso-osmolar solutions when the same osmolality was induced. In practice, however, the critical hyperosmolality value of 400 mOsm is never reached when using non-ionic contrast media such as Hexabrix and Iopamidol 300. This could explain the excellent tolerance to these substances. (2) During hypoxia and reoxygenation, the effect of hyperosmolality is more marked. Thus, the non-ionic contrast medium, Iopamidol 300 (340 mOsm), reduces the hypoxic contractility depression (PF: 53.10 +/- 2.60% compared to the control values of 47.60 +/- 5.00%, P less than 0.01), whereas, at the same dose, the ionic medium Telebrix is hyperosmolar (440 mOsm) and induces a more pronounced hypoxic depression of contractility (P less than 0.01). The critical hyperosmolality is never reached during ventriculography (320 mOsm), whatever the medium used, but it can be observed during coronary arteriography. It is, therefore, important to use non-ionic contrast media in the investigation of unstable angina and of acute myocardium infarction.

Wahrend der letzten Jahre befaste sich eine Anzahl von Studien mit den hamodynamischen Wirkungen der Nitrate [1,11]. Darin war insbesondere die Rede davon, das Nitrate unmittelbar fur das Gleichgewicht zwischen einem verminderten Sauerstoffbedarf und einem groseren Sauerstoffangebot verantwortlich sind, wodurch die myokardiale Kontraktilitat verbessert wird. Diese Befunde wiesen darauf hin, das Nitrate moglicherweise eine direkte Wirkung auf die Kontraktilitat ausuben.

Angiocardiographists are daily confronted with the problem of toxicity of the contrast media used. Most often it results in ECG changes and blood pressure decrease or fall. Fortunately, few accidents occur. They must be feared all the more as cardiac function is altered: myocardial failure, variant angina. Many workers have tried to determine precisely the advantages and the disadvantages of the various media used.

The purpose of the present study was to investigate the effects of low concentrations of nifedipine on the cat myocardium under normal conditions, and during hypoxia with subsequent reoxygenation, and, thus, to find out whether the protective effect on hypoxic myocardium described by different investigators existed at these levels. Experiments carried out on five series of each 10 papillary muscles showed that: (1) Nifedipine (0.02 μg/ml) exerted a negative ino tropic effect on maximum unloaded velocity of shortening and total force (Vmax: 92.91 ± 2.92%; PF: 92.10 ± 2.60%), whereas depression of peak velocity of relaxation became marked only at 0.2 μg/ml (Vrelax: 88.87 ± 3.91%). This dose-dependent effect is also Ca$ $-dependent. (2) Nifedipine (0.02 μ/ml) protected against the depression of Vmax, PF and Vrelax during hypoxia (PO2 = 25 mm Hg) Vmax: 82.60 ± 3.45% v. 78.45 ± 1.35% control; PF: 54.65 ± 4.41% v. 48.74 ±3.51% and Vrelax: 92.55 ± 4.50% v. 68.47 ± 1.30%). (3) The association of nifedipine (0.02 μ/ml) and nitroglycerin (0.3 μ/ml) accelerated the recovery of the depression of peak velocity of relaxation during reoxygenation (Vrelax: 99.90 ± 5.63% v. 89.96 ± 5.15% nifedipine alone).