Cardioprotective Properties of Mannitol—Involvement of Mitochondrial Potassium Channels

Cardiac preconditioning (PC) and postconditioning (PoC) are powerful measures against the consequences of myocardial ischemia and reperfusion (I/R) injury. Mannitol—a hyperosmolar solution—is clinically used for treatment of intracranial and intraocular pressure or promotion of diuresis in renal failure. Next to these clinical indications, different organ-protective properties—e.g., perioperative neuroprotection—are described. However, whether Mannitol also confers cardioprotection via a pre- and/or postconditioning stimulus, possibly reducing consequences of I/R injury, remains to be seen. Therefore, in the present study we investigated whether (1) Mannitol-induced pre- and/or postconditioning induces myocardial infarct size reduction and (2) activation of mitochondrial ATP-sensitive potassium (mKATP) channels is involved in cardioprotection by Mannitol. Experiments were performed on isolated hearts of male Wistar rats via a pressure controlled Langendorff system, randomized into 7 groups. Each heart underwent 33 min of global ischemia and 60 min of reperfusion. Control hearts (Con) received Krebs–Henseleit buffer as vehicle only. Pre- and postconditioning was achieved by administration of 11 mmol/L Mannitol for 10 min before ischemia (Man-PC) or immediately at the onset of reperfusion (Man-PoC), respectively. In further groups, the mKATP channel blocker 5HD, was applied with and without Mannitol, to determine the potential underlying cardioprotective mechanisms. Primary endpoint was infarct size, determined by triphenyltetrazolium chloride staining. Mannitol significantly reduced infarct size both as a pre- (Man-PC) and postconditioning (Man-PoC) stimulus compared to control hearts (Man-PC: 31 ± 4%; Man-PoC: 35 ± 6%, each p < 0.05 vs. Con: 57 ± 9%). The mKATP channel inhibitor completely abrogated the cardioprotective effect of Mannitol-induced pre- (5HD-PC-Man-PC: 59 ± 8%, p < 0.05 vs. Man-PC) and postconditioning (5HD-PoC-Man-PoC: 59 ± 10% vs. p < 0.05 Man-PoC). Infarct size was not influenced by 5HD itself (5HD-PC: 60 ± 14%; 5HD-PoC: 54 ± 14%, each ns vs. Con). This study demonstrates that Mannitol (1) induces myocardial pre- and postconditioning and (2) confers cardioprotection via activation of mKATP channels.

Vascular endothelial growth factor regulation of endothelial nitric oxide synthase phosphorylation is involved in isoflurane cardiac preconditioning

Aims Previous studies indicate that nitric oxide derived from endothelial nitric oxide synthase (eNOS) serves as both trigger and mediator in anaesthetic cardiac preconditioning. The mechanisms underlying regulation of eNOS by volatile anaesthetics have not been fully understood. Therefore, this study examined the role of vascular endothelial growth factor (VEGF) in isoflurane cardiac preconditioning. Methods and results Wistar rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Isoflurane given prior to ischaemia/reperfusion significantly decreased myocardial infarct size from 60 ± 1% in control to 40 ± 3% (n = 8 rats/group, P < 0.05). The beneficial effects of isoflurane were blocked by neutralizing antibody against VEGF (nVEGF). Coronary arterial endothelial cells (ECs) alone or together with cardiomyocytes (CMs) were subjected to hypoxia/reoxygenation injury. The expression of VEGF and eNOS was analysed by western blot, and nitric oxide was measured by ozone-based chemiluminescence. In co-cultured CMs and ECs, isoflurane administered before hypoxia/reoxygenation attenuated lactate dehydrogenase activity and increased the ratio of phosphorylated eNOS/eNOS and nitric oxide production. The protective effect of isoflurane on CMs was compromised by nVEGF and after VEGF in ECs was inhibited with hypoxia inducible factor-1α short hairpin RNA (shRNA). The negative effect of hypoxia inducible factor-1α shRNA was restored by recombinant VEGF. Conclusion Isoflurane cardiac preconditioning is associated with VEGF regulation of phosphorylation of eNOS and nitric oxide production.

Abstract 119: Tempol Pre-treatment Restores Isoflurane Preconditioning in the Aged Cardiomyocytes: Involvement of Pink1/Parkin-mediated Mitophagy

Background: Aging is associated with chronic oxidative stress and impaired cardiac preconditioning. Four-week pre-treatment with TEMPOL, an antioxidant, improves mitochondrial functions and restores isoflurane (ISO) preconditioning in the aging rat heart. Because mitophagy is implicated in cardiac preconditioning and declines with age, this study was designed to investigate how age influence mitophagy in response to preconditioning and whether TEMPOL pre-treatment improves it. Methods: Male Fischer 344 old (22-24 months) rats treated with or without 4-week TEMPOL (2 mM in drinking water) and young (4-6 months) untreated rats were used. Cardiomyocytes isolated from these rats were preconditioned with ISO and then collected for Western blot analyses of mitochondrial PINK1, Parkin and ubiquitination, or fixed for IF staining of co-localization of PINK1/Parkin with mitochondria. Autophagic flux was determined by IF staining of LC3 puncta, membrane-associated LC3-II and p62 in the presence and absences of bafilomycin A1, an autophagy inhibitor. ISO preconditioning was assessed by LDH release in a simulated hypoxia/reoxygenation model. Results: ISO protected young but not old cardiomyocytes. Four-week pre-treatment with TEMPOL in vivo restored ISO preconditioning in the old cardiomyocytes. In addition, ISO increased mitochondrial PINK1 and Parkin levels and promoted mitochondrial protein ubiquitination in the young, but not the old cardiomyocytes. TEMPOL pre-treatment improved these responses in the old cardiomyocytes. Aging impaired both baseline and ISO-induced autophagic flux, which was restored by TEMPOL pre-treatment. Inhibition of autophagy by bafilomycin abolished ISO preconditioning in the cardiomyocytes from the young and TEMPOL-pre-treated old rats. Conclusion: Aging reduces PINK1/Parkin-mediated mitophagy in response to ISO and impairs baseline as well as preconditioning-induced autophagic flux. A 4-week pre-treatment of TEMPOL reverses these changes and restores ISO preconditioning in the old cardiomyocytes. Our results may explain why cardiac preconditioning has failed in some clinical trials, because most patients were with pathologies, such as diabetes and advanced age, in which mitophagy is disrupted.

Preconditioning boosts regenerative programmes in the adult zebrafish heart

During preconditioning, exposure to a non-lethal harmful stimulus triggers a body-wide increase of survival and pro-regenerative programmes that enable the organism to better withstand the deleterious effects of subsequent injuries. This phenomenon has first been described in the mammalian heart, where it leads to a reduction of infarct size and limits the dysfunction of the injured organ. Despite its important clinical outcome, the actual mechanisms underlying preconditioning-induced cardioprotection remain unclear. Here, we describe two independent models of cardiac preconditioning in the adult zebrafish. As noxious stimuli, we used either a thoracotomy procedure or an induction of sterile inflammation by intraperitoneal injection of immunogenic particles. Similar to mammalian preconditioning, the zebrafish heart displayed increased expression of cardioprotective genes in response to these stimuli. As zebrafish cardiomyocytes have an endogenous proliferative capacity, preconditioning further elevated the re-entry into the cell cycle in the intact heart. This enhanced cycling activity led to a long-term modification of the myocardium architecture. Importantly, the protected phenotype brought beneficial effects for heart regeneration within one week after cryoinjury, such as a more effective cell-cycle reentry, enhanced reactivation of embryonic gene expression at the injury border, and improved cell survival shortly after injury. This study reveals that exposure to antecedent stimuli induces adaptive responses that render the fish more efficient in the activation of the regenerative programmes following heart damage. Our results open a new field of research by providing the adult zebrafish as a model system to study remote cardiac preconditioning.