Javier Inserte

Gap Junction Uncoupler Heptanol Prevents Cell-to-Cell Progression of Hypercontracture and Limits Necrosis During Myocardial Reperfusion

BACKGROUND The objective of this study was to test the hypothesis that chemical interaction through gap junctions may result in cell-to-cell progression of hypercontracture and that this phenomenon contributes to the final extent of reperfused infarcts. METHODS AND RESULTS Cell-to-cell transmission of hypercontracture was studied in pairs of freshly isolated adult rat cardiomyocytes. Hypercontracture induced by microinjection of a solution containing 1 mmol/L Ca2+ and 2% lucifer yellow (LY) was transmitted to the adjacent cell (11 of 11 pairs), and the gap junction uncoupler heptanol (2 mmol/L) prevented transmission in 6 of 8 pairs (P=.003), with a perfect association between passage of the LY and transmission of hypercontracture. In the isolated, perfused rat heart submitted to 30 minutes of hypoxia, addition of heptanol to the perfusion media during the first 15 minutes of reoxygenation had a dose-related protective effect against the oxygen paradox, as demonstrated by a reduction of diastolic pressure and marked recovery of developed pressure (P<.001), as well as less lactate dehydrogenase release during reoxygenation (P<.001) and less contraction band necrosis (P<.001) than controls. In the in situ pig heart submitted to 48 minutes of coronary occlusion, the intracoronary infusion of heptanol during the first 15 minutes of reperfusion at a final concentration of 1 mmol/L limited myocardial shrinkage, reflecting hypercontracture (P<.05), reduced infarct size after 5 hours of reperfusion by 54% (P=.04), and modified infarct geometry with a characteristic fragmentation of the area of necrosis. Heptanol at 1 mmol/L had no significant effect on contractility of nonischemic myocardium. CONCLUSIONS These results demonstrate that hypercontracture may be transmitted to adjacent myocytes through gap junctions and that heptanol may interfere with this transmission and reduce the final extent of myocardial necrosis during reoxygenation or reperfusion. These findings are consistent with the hypothesis tested and open a new approach to limitation of infarct size by pharmacological control of gap junction conductance.

Role of the reverse mode of the Na+/Ca2+ exchanger in reoxygenation-induced cardiomyocyte injury.

OBJECTIVE We have recently shown that spontaneous Ca2+ oscillations elicit irreversible hypercontracture of cardiomyocytes during reoxygenation. The aim of this study was to investigate whether influx of exterior Ca2+ through the reverse mode of the Na+/Ca2+ exchanger (NCE) contributes to the development of these oscillations and, therefore, to reoxygenation-induced hypercontracture. METHODS Isolated cardiomyocytes and hearts from rats were used as models. Cardiomyocytes were exposed to 60 min simulated ischemia (pH(o) 6.4) and 10 min reoxygenation (pH(o) 7.4). During reoxygenation cardiomyocytes were superfused with medium containing 1 mmol/l Ca2+ (control), with nominally Ca2+-free medium or with medium containing 10 micromol/l KB-R 7943 (KB), a selective inhibitor of the reverse mode of the NCE. RESULTS In reoxygenated cardiomyocytes rapid Ca2+ oscillations occurred which were reduced under Ca2+-free conditions or in presence of KB. Hypercontracture was also significantly reduced under Ca2+-free conditions or in presence of KB. After 30 min of normoxic perfusion isolated rat hearts were subjected to 60 min global ischemia and reperfusion. KB (10 micromol/l) was present during the first 10 min of reperfusion. LVEDP, LVdevP and lactate dehydrogenase (LDH) release were measured. Presence of KB reduced post-ischemic LVEDP and improved left ventricular function (LVdevP). In KB treated hearts the reperfusion induced release of LDH was markedly reduced from 81.1 +/- 9.9 (control) to 49.3 +/- 8.8 U/60 min/g dry weight. CONCLUSION Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury.

Effect of inhibition of Na+/Ca2+ exchanger at the time of myocardial reperfusion on hypercontracture and cell death

OBJECTIVE There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca(2+)](i) during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. METHODS The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca(2+)](i) and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. RESULTS In myocytes, KB-R7943 at concentrations up to 15 microM reduced [Ca(2+)](i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion. CONCLUSIONS These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.

Calcium-mediated cell death during myocardial reperfusion

Reperfusion may induce additional cell death in patients with acute myocardial infarction receiving primary angioplasty or thrombolysis. Altered intracellular Ca(2+) handling was initially considered an essential mechanism of reperfusion-induced cardiomyocyte death. However, more recent studies have demonstrated the importance of Ca(2+)-independent mechanisms that converge on mitochondrial permeability transition (MPT) and are shared by cardiomyocytes and other cell types. This article analyses the importance of Ca(2+)-dependent cell death in light of these new observations. Altered Ca(2+) handling includes increased cytosolic Ca(2+) levels, leading to activation of calpain-mediated proteolysis and sarcoplasmic reticulum-driven oscillations; this can induce hypercontracture, but also MPT due to the privileged Ca(2+) transfer between sarcoplasmic reticulum and mitochondria through cytosolic Ca(2+) microdomains. In the opposite direction, permeability transition can worsen altered Ca(2+) handling and favour hypercontracture. Ca(2+) appears to play an important role in cell death during the initial minutes of reperfusion, particularly after brief periods of ischaemia. Developing effective and safe treatments to prevent Ca(2+)-mediated cardiomyocyte death in patients with transient ischaemia, by targeting Ca(2+) influx, intracellular Ca(2+) handling, or Ca(2+)-induced cell death effectors, is an unmet challenge with important therapeutic implications and large potential clinical impact.

Calpain-Mediated Impairment of Na+/K+–ATPase Activity During Early Reperfusion Contributes to Cell Death After Myocardial Ischemia

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 α-fodrin and ankyrin-B and detachment of α1 and α2 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.

Delayed recovery of intracellular acidosis during reperfusion prevents calpain activation and determines protection in postconditioned myocardium

AIMS Indirect data suggest that delayed recovery of intracellular pH (pHi) during reperfusion is involved in postconditioning protection, and calpain activity has been shown to be pH-dependent. We sought to characterize the effect of ischaemic postconditioning on pHi recovery during reperfusion and on calpain-dependent proteolysis, an important mechanism of myocardial reperfusion injury. METHODS AND RESULTS Isolated Sprague-Dawley rat hearts were submitted to 40 min of ischaemia and different reperfusion protocols of postconditioning and acidosis. pHi was monitored by (31)P-NMR spectroscopy. Myocardial cell death was determined by lactate dehydrogenase (LDH) and triphenyltetrazolium staining, and calpain activity by western blot measurement of alpha-fodrin degradation. In control hearts, pHi recovered within 1.5 +/- 0.24 min of reperfusion. Postconditioning with 6 cycles of 10 s ischaemia-reperfusion delayed pHi recovery slightly to 2.5 +/- 0.2 min and failed to prevent calpain-mediated alpha-fodrin degradation or to elicit protection. Lowering perfusion flow to 50% during reperfusion cycles or shortening the cycles (12 cycles of 5 s ischemia-reperfusion) resulted in a further delay in pHi recovery (4.1 +/- 0.2 and 3.5 +/- 0.3 min, respectively), attenuated alpha-fodrin proteolysis, improved functional recovery, and reduced LDH release (47 and 38%, respectively, P < 0.001) and infarct size (36 and 32%, respectively, P < 0.001). This cardioprotection was identical to that produced by lowering the pH of the perfusion buffer to 6.4 during the first 2 min of reperfusion or by calpain inhibition with MDL-28170. CONCLUSION These results provide direct evidence that postconditioning protection depends on prolongation of intracellular acidosis during reperfusion and indicate that inhibited calpain activity could contribute to this protection.

Role of the parasympathetic nervous system in cardioprotection by remote hindlimb ischaemic preconditioning

•  What is the central question of this study? Ischaemia–reperfusion of peripheral tissues protects the heart from subsequent myocardial ischaemia–reperfusion‐induced injury and cardiac dysfunction, a phenomenon referred to as ‘remote ischaemic preconditioning’ (rIPC). This study addressed whether activation of sensory afferent nerves in the ischaemic hindlimb and vagal efferent nerves innervating the heart mediate rIPC. •  What is the main finding and its importance? Spinal cord section, bilateral vagotomy or blockade of muscarinic cholinergic receptors in vivo abolished rIPC and cardioprotection measured in vitro. Electrical stimulation of the vagus nerve induced cardioprotection, thus mimicking rIPC. The finding that sensory and parasympathetic neural mechanisms mediate rIPC confirms and extends previous results, with implications for translational studies in patients with coronary artery disease.

Contribution of calpains to myocardial ischaemia/reperfusion injury

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.

Intravenous administration of the natriuretic peptide urodilatin at low doses during coronary reperfusion limits infarct size in anesthetized pigs

OBJECTIVE It has been shown that cGMP content is reduced in post-ischemic myocardium, and that stimulation of cGMP synthesis prevents cardiomyocyte hypercontracture and cell death in vitro. This study was aimed at determining whether administration of the natriuretic peptide urodilatin (URO) at the time of reperfusion could limit myocardial cell death secondary to transient coronary occlusion. METHODS The relation between cGMP content in reperfused myocardium and the extent of cell death was investigated in isolated rat hearts (n=62) receiving different URO concentrations during initial reperfusion. The dose of intravenous URO necessary to obtain the targeted increase in cGMP in reperfused myocardium was investigated in ten pigs submitted to transient coronary occlusion (CO), and the effect of two selected doses of URO on infarct size was investigated in 22 pigs. RESULTS cGMP was severely reduced in post-ischemic rat hearts. Addition of 0.01 microM URO during the first 15 min of reperfusion had no effect on myocardial cGMP content, functional recovery or LDH release in hearts submitted to 40 or 60 min of ischemia. At 0.05 microM, URO increased myocardial cGMP to 111% of values in normoxic hearts, improved functional recovery (P=0.01) and reduced peak LDH released by 40% (P=0.02). The beneficial effect of urodilatin was abolished by ANP receptor inhibition. At 1 microM, URO increased cGMP in reperfused myocardium to 363% of normoxic controls and had no beneficial effect. In pigs allocated to 47 min of CO and 5 min of reperfusion, cGMP was markedly reduced in reperfused myocardium. Intravenous URO at 10 ng/kg per min during the first 25 min of reperfusion normalized myocardial cGMP after 5 min of reflow (95% of control myocardium), and reduced infarct size by 40% (P=0.04). At 50 ng/kg per min, urodilatin increased myocardial cGMP in reperfused myocardium to 335% of control myocardium and failed to significantly reduce infarct size (46 vs. 66%, P=0.125). None of these doses had detectable hemodynamic effects. CONCLUSIONS Intravenous low-dose URO at the time of reperfusion normalizes myocardial cGMP and limits necrosis. Large doses of URO increasing myocardial cGMP well over normal values may lack this beneficial effect.

Ischaemic conditioning and targeting reperfusion injury: a 30 year voyage of discovery

To commemorate the auspicious occasion of the 30th anniversary of IPC, leading pioneers in the field of cardioprotection gathered in Barcelona in May 2016 to review and discuss the history of IPC, its evolution to IPost and RIC, myocardial reperfusion injury as a therapeutic target, and future targets and strategies for cardioprotection. This article provides an overview of the major topics discussed at this special meeting and underscores the huge importance and impact, the discovery of IPC has made in the field of cardiovascular research.