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Major clinical trials with sodium glucose co-transporter-2 inhibitors (SGLT-2i) exhibit protective effects against heart failure events, whereas inconsistencies regarding the cardiovascular death outcomes are observed. Therefore, we aimed to compare the selective SGLT-2i empagliflozin (EMPA), dapagliflozin (DAPA) and ertugliflozin (ERTU) in terms of infarct size (IS) reduction and to reveal the cardioprotective mechanism in healthy non-diabetic mice. C57BL/6 mice randomly received vehicle, EMPA (10 mg/kg/day) and DAPA or ERTU orally at the stoichiometrically equivalent dose (SED) for 7 days. 24 h-glucose urinary excretion was determined to verify SGLT-2 inhibition. IS of the region at risk was measured after 30 min ischemia (I), and 120 min reperfusion (R). In a second series, the ischemic myocardium was collected (10th min of R) for shotgun proteomics and evaluation of the cardioprotective signaling. In a third series, we evaluated the oxidative phosphorylation capacity (OXPHOS) and the mitochondrial fatty acid oxidation capacity by measuring the respiratory rates. Finally, Stattic, the STAT-3 inhibitor and wortmannin were administered in both EMPA and DAPA groups to establish causal relationships in the mechanism of protection. EMPA, DAPA and ERTU at the SED led to similar SGLT-2 inhibition as inferred by the significant increase in glucose excretion. EMPA and DAPA but not ERTU reduced IS. EMPA preserved mitochondrial functionality in complex I&II linked oxidative phosphorylation. EMPA and DAPA treatment led to NF-kB, RISK, STAT-3 activation and the downstream apoptosis reduction coinciding with IS reduction. Stattic and wortmannin attenuated the cardioprotection afforded by EMPA and DAPA. Among several upstream mediators, fibroblast growth factor-2 (FGF-2) and caveolin-3 were increased by EMPA and DAPA treatment. ERTU reduced IS only when given at the double dose of the SED (20 mg/kg/day). Short-term EMPA and DAPA, but not ERTU administration at the SED reduce IS in healthy non-diabetic mice. Cardioprotection is not correlated to SGLT-2 inhibition, is STAT-3 and PI3K dependent and associated with increased FGF-2 and Cav-3 expression., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Aims: Remote ischemic conditioning (RIC) alleviates ischemia-reperfusion injury via several pathways, including micro-RNAs (miRs) expression and oxidative stress modulation. We investigated the effects of RIC on endothelial glycocalyx, arterial stiffness, LV remodelling, and the underlying mediators within the vasculature as a target for protection., Methods and Results: We block-randomised 270 patients within 48 h of STEMI post-PCI to either one or two cycles of bilateral brachial cuff inflation, and a control group without RIC. We measured: (a) the perfusion boundary region (PBR) of the sublingual arterial microvessels to assess glycocalyx integrity; (b) the carotid-femoral pulse wave velocity (PWV); (c) miR-144,-150,-21,-208, nitrate-nitrite (NOx) and malondialdehyde (MDA) plasma levels at baseline (T0) and 40 min after RIC onset (T3); and (d) LV volumes at baseline and after one year. Compared to baseline, there was a greater PBR and PWV decrease, miR-144 and NOx levels increase (p < 0.05) at T3 following single- than double-cycle inflation (PBR:ΔT0-T3 = 0.249 ± 0.033 vs 0.126 ± 0.034 μm, p = 0.03 and PWV:0.4 ± 0.21 vs -1.02 ± 0.24 m/s, p = 0.03). Increased miR-150,-21,-208 (p < 0.05) and reduced MDA was observed after both protocols. Increased miR-144 was related to PWV reduction (r = 0.763, p < 0.001) after the first-cycle inflation in both protocols. After one year, single-cycle RIC was associated with LV end-systolic volume reduction (LVESV) > 15% (odds-ratio of 3.75, p = 0.029). MiR-144 and PWV changes post-RIC were interrelated and associated with LVESV reduction at follow-up (r = 0.40 and 0.37, p < 0.05), in the single-cycle RIC., Conclusion: RIC evokes "vascular conditioning" likely by upregulation of cardio-protective microRNAs, NOx production, and oxidative stress reduction, facilitating reverse LV remodelling., Clinical Trial Registration: http://www.clinicaltrials.gov . Unique identifier: NCT03984123.

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., Competing Interests: HEB is shareholder of CellAegis Inc. PF is a founder and CEO of Pharmahungary, a group of R&D companies. GH served as a consultant to Servier. MO was a consultant for Neurovive Pharmaceuticals. DGD served as consultant to Neurovive Pharmaceuticals. All other authors have no relevant disclosures.

Postconditioning (POC) reduces lethal reperfusion injury under normal conditions, but its effectiveness under certain pathological states is in dispute. In the present study, we sought to determine the effect of chronic simvastatin treatment in hyperlipidemic animals with or without POC. Anesthetized rabbits were randomized into eight groups, as follows, and were subjected to 30-min myocardial ischemia followed by 3-h reperfusion. Normally fed animals: a Control group with no additional intervention, a Sim group treated with simvastatin for 3 weeks at a dose of 3 mg kg(-1), a POC group subjected to POC with eight cycles of 30-s ischemia/reperfusion, a Sim-POC group treated with simvastatin, and POC. Cholesterol fed (6 weeks) animals: a Chol group with no additional interventions, a Chol-Sim group treated with simvastatin for 3 weeks, a Chol-POC group subjected to POC, and a Chol-Sim-POC group treated with simvastatin and POC. Infarct size and plasma levels of malondialdehyde (MDA), nitrotyrosine (NT), NOx, total cholesterol, and LDL were evaluated. In a second series of experiments, heart tissue samples were taken for MDA, NT, and NOx assessment. Infarct size, circulating MDA, NT, NOx and cardiac MDA, NT, and NOx levels declined in POC and all Sim groups compared with Control, Chol, and Chol-POC (p < 0.05). Simvastatin also reduced total cholesterol and LDL plasma levels. In conclusion, a 3-week simvastatin treatment limits the infarct size and attenuates the oxidative and nitrosative stress both in normo- and in hyper-cholesterolemic rabbits subjected to ischemia-reperfusion irrespective of the presence of POC, while POC is effective only in normocholesterolemic animals.

Ischemic postconditioning, a simple mechanical maneuver at the onset of reperfusion, reduces infarct size after ischemia/reperfusion. After its first description in 2003 by Zhao et al. numerous experimental studies have investigated this protective phenomenon. Whereas the underlying mechanisms and signal transduction are not yet understood in detail, infarct size reduction by ischemic postconditioning was confirmed in all species tested so far, including man. We have now reviewed the literature with focus on experimental models and protocols to better understand the determinants of protection by ischemic postconditioning or lack of it. Only studies with infarct size as unequivocal endpoint were considered. In all species and models, the duration of index ischemia and the protective protocol algorithm impact on the outcome of ischemic postconditioning, and gender, age, and myocardial temperature contribute.

Redox signaling prior to a lethal ischemic insult is an important step in triggering the protected state in ischemic preconditioning. When the preconditioned heart is reperfused a second sequence of signal transduction events, the mediator pathway, occurs which is believed to inhibit mitochondrial permeability transition pore formation that normally destroys mitochondria in much of the reperfused tissue. Prominent among the mediator pathway's events is activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase. Recently it was found that both activation of PKC and generation of reactive oxygen species (ROS) at the time of reperfusion are required for protection in preconditioned hearts. To establish their relative order we tested whether ROS formation at reperfusion is required in hearts protected by direct activation of PKC at reperfusion. Isolated rabbit hearts were exposed to 30 min of regional ischemia and 2 h of reperfusion. Preconditioned hearts received 5 min of global ischemia and 10 min of reperfusion prior to the index ischemia. Another group of preconditioned hearts was exposed to 300 microM of the ROS scavenger N-(2-mercaptopropionyl) glycine (MPG) for 20 min starting 5 min prior to reperfusion. Infarct size was measured by triphenyltetrazolium staining. Preconditioning reduced infarct size from 36% +/- 2% of the ischemic zone in control hearts to only 18 +/- 2%. MPG during early reperfusion completely blocked preconditioning's protection (33 +/- 3% infarction). MPG given in the same dose and schedule to non-preconditioned hearts had no effect on infarct size. In the last group phorbol 12-myristate 13-acetate (PMA) (0.05 nM) was given to non-preconditioned hearts from 1 min before to 5 min after reperfusion in addition to MPG administered as in the other groups. MPG did not block protection from an infusion of PMA as infarct size was only 9 +/- 2% of the risk zone. We conclude that while redox signaling during the first few minutes of reperfusion is an essential component of preconditioning's protective mechanism, this step occurs upstream of PKC activation.

Post-conditioning in the early reperfusion period confers protection to the heart after a potentially lethal episode of prolonged ischemia. Protection from this novel intervention has been documented in rat, rabbit and canine hearts, but one group has reported that it is ineffective in pigs, a large-animal species that should be most relevant to humans. We hypothesized that this negative result was related to an inappropriate post-conditioning protocol rather than the species. The present study, therefore, tested whether an effective post-conditioning protocol could be identified that limits infarct size in anesthetized pigs. Domestic Landrace pigs weighing 25-29 kg were anesthetized, and after a mid-sternal thoracotomy and pericardiotomy the left anterior descending coronary artery was ligated for 60 min followed by 3 h of reperfusion. Three groups were studied: control group (n = 5) with no other intervention, 4-30 PostC group (n = 5) with 4 cycles of 30-s reperfusion/30-s ischemia, and 8-30 PostC group (n = 6) with 8 cycles of 30-s reperfusion/30-s ischemia. The two post-conditioning protocols started immediately after termination of the 60-min coronary occlusion. Region at risk and infarct size were delineated with the aid of pre-mortem monastral blue injection and postmortem staining with triphenyltetrazolium chloride, respectively. In control hearts 33.5 +/- 7.6% of the risk zone infarcted and 36.7 +/- 3.7% in the 4-30 PostC group (P = NS). But there was only 10.5 +/- 0.5% infarction in the 8-30 PostC group (P < 0.01 vs. the other two groups). Post-conditioning confers protection in pigs but requires more than 4 ischemia/reperfusion cycles. Post-conditioning may protect by inhibiting mitochondrial permeability transition pore formation by keeping the heart acidotic as it is reoxygenated. If true, then it would be difficult to employ too many occlusion cycles.

Previous studies have shown that the cardioprotective effect of ischemic preconditioning (IPC) can be mimicked pharmacologically with clinically relevant agents, including nitric oxide (NO) donors. However, whether pharmacological preconditioning shares the same molecular mechanism with IPC is not fully elucidated. The present study aimed to determine the activation of mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK and p46/p54 JNKs) during ischemia and at reperfusion in nitroglycerin-induced preconditioning as compared to IPC and to correlate this with the conferred cardioprotection in anesthetized rabbits. Sixty minutes of intravenous administration of nitroglycerin was capable of inducing both early and late phase preconditioning in anesthetized rabbits, as it was expressed by the reduction of infarct size. Despite the cardioprotective effect conferred by both ischemic and nitroglycerin-induced preconditioning, there was a differential phosphorylation of MAPKs between the studied groups. p38 MAPK was activated early in ischemia in both ischemic and the early nitroglycerin-induced preconditioning while JNKs were markedly increased only after IPC. Furthermore, in these groups, ERK1/2 were activated during reperfusion. A different profile was observed in the late preconditioning induced by nitroglycerin with increased p38 MAPK and ERK1/2 phosphorylation during late ischemia. No activation of JNKs was observed at any time point in this group. It seems that activation of individual MAPK subfamilies depends on the nature of preconditioning stimulus.

The aim of this study was to investigate if levels of circulating cyclic guanosine monophosphate (c-GMP) alter in preconditioning. Twenty-eight rabbits were divided into four groups. In vivo hearts were preconditioned, either with 5 min (group A, n = 8) or with 1 min (group B, n = 8) ischemia, followed by 10 min reperfusion, while groups C (n = 7) and D (n = 5) had no interventions. Protection was determined by subjecting groups A, B and C (but not D) to 30 min regional ischemia which was followed (including group D) by 2 h reperfusion. Seven blood samples were collected for the assessment of circulating c-GMP at different points of time. All results were expressed in pmol/ml using radio-immunoassay and the infarcted to risk area in percent using fluorescent particles and tetrazolium chloride (TTC). Circulating c-GMP increased during long ischemia only in group A (baseline value 47 +/- 4, long ischemic values 60.5 +/- 4 and 60.4 +/- 4, p < 0.05). Circulating c-GMP in group A was significantly higher in the middle of the long ischemia in comparison to the groups B, C and D (60.5 +/- 4 vs 43.9 +/- 4, 45.8 +/- 5 and 43.6 +/- 4, p < 0.05). Infarcted to risk ratio was lower in group A than in groups B and C (12.2 +/- 4 vs 29.6 +/- 6 and 34.2 +/- 6 respectively, p < 0.05). Circulating c-GMP is increased in classically preconditioned in comparison to ineffectively preconditioned hearts or to control groups. This elevation may be related to the protective effect of this phenomenon.