Is Intrinsic Cardioprotection a Laboratory Phenomenon or a Clinically Relevant Tool to Salvage the Failing Heart?

Cardiovascular diseases, especially ischemic heart disease, as a leading cause of heart failure (HF) and mortality, will not reduce over the coming decades despite the progress in pharmacotherapy, interventional cardiology, and surgery. Although patients surviving acute myocardial infarction live longer, alteration of heart function will later lead to HF. Its rising incidence represents a danger, especially among the elderly, with data showing more unfavorable results among females than among males. Experiments revealed an infarct-sparing effect of ischemic "preconditioning" (IPC) as the most robust form of innate cardioprotection based on the heart's adaptation to moderate stress, increasing its resistance to severe insults. However, translation to clinical practice is limited by technical requirements and limited time. Novel forms of adaptive interventions, such as "remote" IPC, have already been applied in patients, albeit with different effectiveness. Cardiac ischemic tolerance can also be increased by other noninvasive approaches, such as adaptation to hypoxia- or exercise-induced preconditioning. Although their molecular mechanisms are not yet fully understood, some noninvasive modalities appear to be promising novel strategies for fighting HF through targeting its numerous mechanisms. In this review, we will discuss the molecular mechanisms of heart injury and repair, as well as interventions that have potential to be used in the treatment of patients.

p55γ degrades RIP3 via MG53 to suppress ischaemia-induced myocardial necroptosis and mediates cardioprotection of preconditioning.

AIMS: Regulated necrosis (necroptosis) and apoptosis are important biological features of myocardial infarction, ischaemia-reperfusion (I/R) injury, and heart failure. However, the molecular mechanisms underlying myocardial necroptosis remain elusive. Ischaemic preconditioning (IPC) is the most powerful intrinsic cardioprotection against myocardial I/R injury. In this study, we aimed to determine whether IPC suppresses I/R-induced necroptosis and the underlying molecular mechanisms. METHODS AND RESULTS: We generated p55γ transgenic and knockout mice and used ligation of left anterior descending coronary artery to produce an in vivo I/R model. The effects of p55γ and its downstream molecules were subsequently identified using mass spectroscopy and co-immunoprecipitation and pulldown assays. We found that p55γ expression was down-regulated in failing human myocardium caused by coronary heart disease as well as in I/R mouse hearts. Cardiac-specific p55γ overexpression ameliorated the I/R-induced necroptosis. In striking contrast, p55γ deficiency (p55γ-/-) and cardiac-specific deletion of p55γ (p55γc-KO) worsened I/R-induced injury. IPC up-regulated p55γ expression in vitro and in vivo. Using reporter and chromatin immunoprecipitation assays, we found that Hif1α transcriptionally regulated p55γ expression and mediated the cardioprotection of IPC. IPC-mediated suppression of necroptosis was attenuated in p55γ-/- and p55γc-KO hearts. Mechanistically, p55γ overexpression decreased the protein levels of RIP3 rather than the mRNA levels, while p55γ deficiency increased the protein abundance of RIP3. IPC attenuated the I/R-induced up-regulation of RIP3, which was abolished in p55γ-deficient mice. Up-regulation of RIP3 attenuated the p55γ- or IPC-induced inhibition of necroptosis in vivo. Importantly, p55γ directly bound and degraded RIP3 in a ubiquitin-dependent manner. We identified MG53 as the E3 ligase that mediated the p55γ-induced degradation of RIP3. In addition, we also found that p55γ activated the RISK pathway during IPC. CONCLUSIONS: Our findings reveal that activation of the MG53-RIP3 signal pathway by p55γ protects the heart against I/R-induced necroptosis and underlies IPC-induced cardioprotection.

Endless Journey of Adenosine Signaling in Cardioprotective Mechanism of Conditioning Techniques: Clinical Evidence.

Myocardial ischemic injury is a primary cause of death among various cardiovascular disorders. The condition occurs due to an interrupted supply of blood and vital nutrients (necessary for normal cellular activities and viability) to the myocardium, eventually leading to damage. Restoration of blood supply to ischemic tissue is noted to cause even more lethal reperfusion injury. Various strategies, including some conditioning techniques, like preconditioning and postconditioning, have been developed to check the detrimental effects of reperfusion injury. Many endogenous substances have been proposed to act as initiators, mediators, and end effectors of these conditioning techniques. Substances, like adenosine, bradykinin, acetylcholine, angiotensin, norepinephrine, opioids, etc., have been reported to mediate cardioprotective activity. Among these agents, adenosine has been widely studied and suggested to have the most pronounced cardioprotective effects. The current review article highlights the role of adenosine signaling in the cardioprotective mechanism of conditioning techniques. The article also provides an insight into various clinical studies that substantiate the applicability of adenosine as a cardioprotective agent in myocardial reperfusion injury.

Effects of remote ischaemic preconditioning on myocardial injury after major abdominal surgery in patients at high risk for cardiovascular adverse events in China (RIPC-MAS): protocol for a randomised, sham-controlled, observer-blinded trial.

INTRODUCTION: Myocardial injury after non-cardiac surgery (MINS) caused by an ischaemic mechanism is common and is associated with adverse short-term and long-term prognoses. However, MINS is a recent concept, and few studies have prospectively used it as a primary outcome. Remote ischaemic preconditioning (RIPC) is a non-invasive procedure that induces innate cardioprotection and may reduce MINS. METHODS AND ANALYSIS: This is a multicentre, randomised, sham-controlled, observer-blinded trial. Patients with a high clinical risk of cardiovascular events who are scheduled to undergo major abdominal surgery will be enrolled. A total of 766 participants will be randomised (1:1 ratio) to receive RIPC or control treatment before anaesthesia. RIPC will comprise four cycles of cuff inflation for 5 min to 200 mm Hg and deflation for 5 min. In the controls, an identical-looking cuff will be placed around the arm but will not be actually inflated. The primary outcome will be MINS, defined as at least one postoperative cardiac troponin (cTn) concentration above the 99th percentile upper reference limit of the cTn assay as a result of a presumed ischaemic mechanism. This trial will test the concentration of high-sensitivity cardiac troponin T (hs-cTnT). The secondary outcomes will be hs-cTnT levels reaching/above the prognostically important thresholds, peak hs-cTnT and total hs-cTnT release during the initial 3 days after surgery, length of hospital stay after surgery, length of stay in the intensive care unit, myocardial infarction, major adverse cardiovascular events, cardiac-related death, all-cause death within 30 days, 6 months, 1 year and 2 years after surgery, and postoperative complications and adverse events within 30 days after surgery. ETHICS AND DISSEMINATION: This study protocol (version 5.0 on 7 April 2023) was approved by the Ethics Committee of Sixth Affiliated Hospital of Sun Yat-sen University. The findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05733208.

Attenuation of ST-segment elevation by ischemic preconditioning: Reflection of cardioprotection in Göttingen but not in Ossabaw minipigs.

BACKGROUND: Ischemic preconditioning (IPC; brief cycles of coronary occlusion/ reperfusion) reduces myocardial infarct size. The ST-segment elevation during coronary occlusion is progressively attenuated with increasing number of IPC cycles. Progressive attenuation of ST-segment elevation is considered a result of sarcolemmal KATP channel activation and has been considered to reflect and predict IPC's cardioprotection. We have recently demonstrated that IPC failed to reduce infarct size in minipigs of a particular strain (Ossabaw), which had a genetic predisposition to develop, but not yet established a metabolic syndrome. To determine whether or not Ossabaw minipigs nevertheless had attenuated ST-segment elevation over repetitive IPC cycles, we compared Göttingen vs. Ossabaw minipigs in which IPC reduces infarct size. METHODS AND RESULTS: We analyzed surface chest electrocardiographic (ECG) recordings of anesthetized open-chest contemporary Göttingen (n = 43) and Ossabaw minipigs (n = 53). Both minipig strains were subjected to 60 min coronary occlusion and 180 min reperfusion without or with IPC (3 × 5 min/ 10 min coronary occlusion/ reperfusion). ST-segment elevations during the repetitive coronary occlusions were analyzed. In both minipig strains, IPC attenuated ST-segment elevation with increasing number of coronary occlusions. IPC reduced infarct size in Göttingen minipigs (45 ± 10% without vs. 25 ± 13% of area at risk with IPC), whereas such cardioprotection was absent in Ossabaw minipigs (54 ± 11% vs. 50 ± 11%). CONCLUSION: Apparently, the block of signal transduction of IPC in Ossabaw minipigs occurs distal to the sarcolemma, where KATP channel activation still attenuates ST-segment elevation as it does in Göttingen minipigs.

Hydrogen peroxide preconditioning is of dual role in cardiac ischemia/reperfusion.

Moderate reactive oxygen species (ROS) at reperfusion would trigger cardioprotection and various antioxidants for pharmacological preconditioning failed to achieve cardioprotection. The causes for different roles of preischemic ROS during cardiac ischemia/reperfusion (I/R) require reevaluation. We investigated the precise role of ROS and its working model in this study. Different doses of hydrogen peroxide (H2O2, the most stable form of ROS) were added 5 min before ischemia using isolated perfused rat hearts, only moderate-dose H2O2 preconditioning (H2O2PC) achieved contractile recovery, whereas the low dose and high dose led to injury. Similar results were observed in isolated rat cardiomyocytes on cytosolic free Ca2+ concentration ([Ca2+]c) overload, ROS production, the recovery of Ca2+ transient, and cell shortening. Based on the data mentioned above, we set up a mathematics model to describe the effects of H2O2PC with the fitting curve by the percentage of recovery of heart function and Ca2+ transient in I/R. Besides, we used the two models to define the initial thresholds of H2O2PC achieving cardioprotection. We also detected the expression of redox enzymes and Ca2+ signaling toolkits to explain the mathematics models of H2O2PC in a biological way. The expression of tyrosine 705 phosphorylation of STAT3, Nuclear factor E2-related factor 2, manganese superoxide dismutase, phospholamban, catalase, ryanodine receptors, and sarcoendoplasmic reticulum calcium ATPase 2 were similar with the control I/R and low-dose H2O2PC but were increased in the moderate H2O2PC and decreased in the high-dose H2O2PC. Thus, we concluded that preischemic ROS are of dual role in cardiac I/R.

Exosomes as perioperative therapeutics to limit organ injury.

Perioperative organ injury is a frequent and major complication for the ∼240 million people undergoing surgery worldwide annually. Ischaemic preconditioning is a powerful technique that reduces organ injury in experimental models of heart, lung, gut, brain, and kidney ischaemia-reperfusion injury. However, ischaemic preconditioning has been a challenge to translate into clinical practice. We describe how utilising isolated pre-conditioned exosomes (secreted vesicles containing many cell-survival mediators), some of the translational hurdles of ischaemic preconditioning can be overcome. Delivery of exosomes in the perioperative period could become a promising new therapeutic strategy to prevent perioperative organ injury.

Remote Ischaemic Conditioning in STEMI Patients in Sub-Saharan AFRICA: Rationale and Study Design for the RIC-AFRICA Trial.

PURPOSE: Despite evidence of myocardial infarct size reduction in animal studies, remote ischaemic conditioning (RIC) failed to improve clinical outcomes in the large CONDI-2/ERIC-PPCI trial. Potential reasons include that the predominantly low-risk study participants all received timely optimal reperfusion therapy by primary percutaneous coronary intervention (PPCI). Whether RIC can improve clinical outcomes in higher-risk STEMI patients in environments with poor access to early reperfusion or PPCI will be investigated in the RIC-AFRICA trial. METHODS: The RIC-AFRICA study is a sub-Saharan African multi-centre, randomized, double-blind, sham-controlled clinical trial designed to test the impact of RIC on the composite endpoint of 30-day mortality and heart failure in 1200 adult STEMI patients without access to PPCI. Randomized participants will be stratified by whether or not they receive thrombolytic therapy within 12 h or arrive outside the thrombolytic window (12-24 h). Participants will receive either RIC (four 5-min cycles of inflation [20 mmHg above systolic blood pressure] and deflation of an automated blood pressure cuff placed on the upper arm) or sham control (similar protocol but with low-pressure inflation of 20 mmHg and deflation) within 1 h of thrombolysis and applied daily for the next 2 days. STEMI patients arriving greater than 24 h after chest pain but within 72 h will be recruited to participate in a concurrently running independent observational arm. CONCLUSION: The RIC-AFRICA trial will determine whether RIC can reduce rates of death and heart failure in higher-risk sub-optimally reperfused STEMI patients, thereby providing a low-cost, non-invasive therapy for improving health outcomes.

Plasma exosomes generated by ischaemic preconditioning are cardioprotective in a rat heart failure model.

BACKGROUND: Exosomes released into the plasma after brief cardiac ischaemia mediate subsequent cardioprotection. Whether donor exosomes can provide cardioprotection to recipients with chronic heart failure, which confers the highest perioperative risk, is unknown. We examined whether ischaemic preconditioning (IPC)-induced plasma exosomes exerted cardioprotection after their transfer from normal donors to post-infarcted failing hearts. METHODS: Plasma exosomes were obtained from adult rats after IPC or sham. An exosome inhibitor GW4869 was administrated before IPC in an in vivo model of ischaemia/reperfusion (I/R) injury in normal rats. The IPC exosomes or control exosomes from normal donor rats were perfused to the normal or post-infarcted failing rat hearts before ischaemia in Langendorff perfusion experiments. Infarct size, cardiac enzymes, cardiac function, and pro-survival kinases were quantified. RESULTS: The IPC stimulus increased the release of exosomes, whereas GW4869 inhibited the rise of plasma exosomes. Pre-treatment with GW4869 reversed IPC-mediated cardioprotection against in vivo I/R injury. In the Langendorff perfusion experiments, IPC exosomes from normal donor rats reduced mean infarct size from 41.05 (1.87)% to 31.43 (1.81)% and decreased lactate dehydrogenase activity in the post-infarcted failing rat hearts. IPC exosomes but not control exosomes activated pro-survival kinases in the heart tissues. CONCLUSIONS: Ischaemic preconditioning-induced exosomes from normal rats can restore cardioprotection in heart failure after myocardial infarction, which is associated with activation of pro-survival protein kinases. These results suggest a potential perioperative therapeutic role for ischaemic preconditioning-induced exosomes.

Exercise preconditioning improves electrocardiographic signs of myocardial ischemic/hypoxic injury and malignant arrhythmias occurring after exhaustive exercise in rats.

Exercise preconditioning (EP) has a good myocardial protective effect. This study explored whether EP improves electrocardiographic (ECG) signs of myocardial ischemic/hypoxic injury and the occurrence of malignant arrhythmia after exhaustive exercise. A total of 120 male SD rats were randomly divided into the control group (group C), early exercise preconditioning group (group EEP), late exercise preconditioning group (group LEP), exhaustive exercise group (group EE), early exercise preconditioning + exhaustive exercise group (group EEP + EE) and late exercise preconditioning + exhaustive exercise group (group LEP + EE). Changes in heart rate (HR), ST segment, T wave and QT corrected (QTc) intervals on ECG; hematoxylin-basic fuchsin-picric acid (HBFP) staining; and cTnI levels were used to study myocardial injury and the protective effect of EP. Compared with those in group C, the levels of plasma markers of myocardial injury, HBFP staining and ECG in group EE were significantly increased (P < 0.05). Compared with those in group EE, the levels of plasma markers of myocardial injury, HBFP staining and ECG in group EEP + EE and group LEP + EE were significantly decreased (P < 0.05). The results suggested that EP improved ECG signs of myocardial ischemic/hypoxic injury and malignant arrhythmias that occur after exhaustive exercise. The ST segment and T wave could also serve as indexes for evaluating exhaustive exercise-induced myocardial ischemia/hypoxia.

Creatine phosphate administration in cardiac ischemia-reperfusion injury in rats: focus on differences between preconditioning, perconditioning, and postconditioning protocols.

The aim of this study was to examine and compare the influence of preconditioning, perconditioning, and postconditioning with creatine phosphate (PCr) on functional recovery and production of prooxidants in isolated rat hearts subjected to ex vivo ischemic-reperfusion (I-R) injury on a Langendorff apparatus. Wistar albino rats (male, n = 40) were divided into four groups: control and groups in which PCr (0.5 mmol/L, 5 min) was perfused before (Pre group), after (Post group), or during (Per group) ex vivo induced ischemia. PCr application was associated with the great benefits of preserving cardiac contractility (in Pre group 100.96% for +(dP/dt max) and 97.61% for -(dP/dt max), in Per group 96.72% for +(dP/dt max) and 95.60% for -(dP/dt max), and in Post group 143.84% for +(dP/dt max) and 104.36% for -(dP/dt max) in relation to the stabilization). In addition, PCr application prevented the increase in prooxidative markers during I-R injury in all therapeutic modalities. The most intensive benefits in the current investigation were observed when PCr was applied during the period of ischemia because the lowest fluctuations in the parameters of cardiac function and oxidative stress were observed. Overall, the results of this study highlight PCr-induced cardioprotection with promising prospects for future clinical use.

Network-Assisted Systems Biology Analysis of the Mitochondrial Proteome in a Pre-Clinical Model of Ischemia, Revascularization and Post-Conditioning.

Infarct size is the major risk predictor for developing heart failure after an acute myocardial infarction (AMI). The discovery of the conditioning phenomena (i.e., repetitive brief cycles of ischemia applied either before or after a prolonged ischemic insult) has highlighted the existence of endogenous protective mechanisms of the heart potentially limiting infarct size after revascularization. However, most cardioprotective strategies, aiming at infarct size reduction, have failed in clinical studies. Thus, cardioprotection is an unmet clinical need. In the present study, we took a network-assisted systems biology approach to explore the mitochondrial proteomic signature of the myocardium after ischemia, ischemia with direct revascularization, and ischemia with re-establishment of blood flow by post-conditioning in a swine model of AMI. Furthermore, network extension with the ENCODE project human regulatory data allowed the prediction of potential transcription factors at play in the response to post-conditioning of the myocardium. Collectively, our results identify cardiac metabolism as a driver of cardioprotection, highlighting a dual role for post-conditioning promoting metabolic reprogramming of the myocardium, and a protective response mediated by VDAC2 and DJ-1 in the mitochondria.

Remote ischemic preconditioning can extend the tolerance to extended drug-coated balloon inflation time by reducing myocardial damage during percutaneous coronary intervention.

BACKGROUND: Remote ischemic preconditioning (RIPC) alleviates myocardial ischemia-reperfusion injury (IRI) that occurs during percutaneous coronary intervention (PCI) and increases the myocardial tolerance to ischemia and hypoxia. Prolonged inflation time of drug-coated balloons (DCBs) can improve the treatment effects of PCI and the long-term prognosis of patients. This study investigated whether preoperative RIPC improves the tolerance to extended DCB inflation time. METHODS AND RESULTS: Overall, 345 patients with coronary artery disease (CAD) were enrolled; 90, 96, 83, and 76 of these were randomized into the upper limb RIPC, lower limb RIPC, upper limb control, and lower limb control groups, respectively. Their baseline data were collected. Data on cardiac markers were analyzed. The DCB inflation time was recorded. The baseline data and cardiac marker levels before operation did not differ between RIPC and control groups. The post-PCI high-sensitivity troponin-T levels were lower in the RIPC groups (35.81 ± 14.02 and 34.65 ± 14.86 pg/mL) than in the control groups (41.63 ± 18.31 and 42.24 ± 14.38 pg/mL) (P = 0.001). The DCB inflation tolerance time was higher in the lower limb RIPC group (120 s [120,120]) than in the upper limb RIPC group (120 s [110,120]), and was the lowest in the upper limb control (100 s [90, 120]) and the lower limb control (100 s [90, 115]) groups (P < 0.001). CONCLUSIONS: RIPC reduces the level of myocardial damage that occurs during PCI and prolongs tolerance to increased DCB inflation time. The larger the ischemic area in RIPC, the better the improvement in the tolerance to extended DCB inflation time.

Synergetic protective effect of remote ischemic preconditioning and prolyl 4­hydroxylase inhibition in ischemic cardiac injury.

It has been reported that hypoxia­inducible factor 1α (HIF­1α) serves a key role in the protective effect of remote ischemic preconditioning (RIP) in ischemia/reperfusion (I/R)­induced cardiac injury. Moreover, inhibition of prolyl 4­hydroxylase (PHD), an enzyme responsible for HIF­1α degradation, prevents I/R­induced cardiac injury. However, whether their protective effects are synergetic remains to be elucidated. The present study aimed to investigate the protective effect of RIP, PHD inhibition using dimethyloxalylglycine (DMOG) and their combination on I/R­induced cardiac injury. Rabbits were randomly divided into seven groups: i) Sham; ii) I/R; iii) lung RIP + I/R; iv) thigh RIP + I/R; v) DMOG + I/R; vi) DMOG + lung RIP + I/R; and vii) DMOG + thigh RIP + I/R. I/R models were established via 30 min left coronary artery occlusion and 3 h reperfusion. For lung/thigh RIP, rabbits received left pulmonary artery (or left limb) ischemia for 25 min and followed by release for 5 min. Some rabbits were administered 20 mg/kg DMOG. The results demonstrated that both lung/thigh RIP and DMOG significantly decreased myocardial infarct size, creatine kinase activity and myocardial apoptosis in I/R rabbits. Furthermore, the combination of RIP and PHD inhibition exerted synergetic protective effects on these aforementioned changes. The mechanistic study indicated that both treatments increased mRNA and protein expression levels of HIF­1α and its downstream regulators, including vascular endothelial growth factor (VEGF), AKT and endothelial nitric oxide synthase (eNOS). In conclusion, the present study demonstrated that RIP and PHD inhibition exerted synergetic protective effects on cardiac injury via activation of HIF­1α and the downstream VEGF/AKT­eNOS signaling pathway.

Can Clinical and Functional Outcomes Be Improved with an Intelligent "Internet Plus"-Based Full Disease Cycle Remote Ischemic Conditioning Program in Acute ST-elevation Myocardial Infarction Patients Undergoing Percutaneous Coronary Intervention? Rationale and Design of the i-RIC Trial.

BACKGROUND: Acute ST-elevation myocardial infarction (STEMI) is associated with a high incidence of complications as well as a considerable hospitalization rate and economic burden. Preliminary evidence suggests that remote ischemic conditioning (RIC) is a promising non-invasive intervention that may effectively and safely reduce myocardial infarct size, subsequent cardiac events and complications, and mortality. However, RIC's cardio-protective effect remains under debate, especially for single timepoint RIC programs. Adequately powered large-scale randomized controlled trials investigating clinical outcomes are thus needed to clarify the role of full disease cycle RIC programs. METHODS: The intelligent "Internet Plus"-based full disease cycle remote ischemic conditioning (i-RIC) trial is a pragmatic, multicenter, randomized controlled, parallel group, clinical trial. The term, intelligent "Internet Plus"-based full disease cycle, refers to smart devices aided automatic and real-time monitoring of remote ischemic pre-, per- or post-conditioning intervention for patients with STEMI undergoing percutaneous coronary intervention (PCI). Based on this perspective, 4700 STEMI patients from five hospitals in China will be randomized to a control and an intervention group. The control group will receive PCI and usual care, including pharmacotherapy, before and after PCI. The intervention group will receive pre-, per-, and post-operative RIC combined with long-term i-RIC over a one-month period in addition. A smartphone application, an automated cuff inflation/deflation device and "Internet Plus"-based administration will be used in the long-term phase. The primary outcome is the combined cardiac death or hospitalization for heart failure rate. Secondary outcomes include clinical and functional outcomes: major adverse cardiac and cerebrovascular events rate, all-cause mortality, myocardial reinfarction rate, readmission rate for heart failure and ischemic stroke rate, unplanned revascularization rate, plasma concentration of myocardial infarction-related key biomarkers, infarct size, cardiac function, cardiopulmonary endurance, health-related quality of life, total hospital length of stay, total medical cost, and compliance with treatment regime. DISCUSSION: The i-RIC trial is designed to test the hypothesis that clinical and functional outcomes can be improved with the i-RIC program in STEMI patients undergoing PCI. The concept of RIC is expected to be enhanced with this intelligent "Internet Plus"-based program focusing on the full disease cycle. If the i-RIC program results in superior improvement in primary and secondary outcomes, it will offer an innovative treatment option for STEMI patients and form the basis of future recommendations. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry ( http://www.chictr.org.cn ): ChiCTR2000031550, 04 April 2020.

Preconditioning the rat heart with 5-azacytidine attenuates myocardial ischemia/reperfusion injury via PI3K/GSK3ß and mitochondrial KATP signaling axis.

5-Azacytidine is well known for its clinical usage in cancer treatments. The present study investigates the role of 5-azacytidine as a cardioprotective agent to ameliorate ischemia/reperfusion (I/R) injury. The cardioprotective effect of 5-azacytidine was evaluated in three experimental models: in vitro, ex vivo, and in vivo. The cardioprotective effect was evaluated via cell viability, hemodynamic indices, infarct size measurement, and assessment of histopathology, oxidative stress, and mitochondrial function. The experiments were repeated in the presence of PI3K/GSK3ß and mitochondrial KATP (mtKATP ) cardioprotective signaling pathway inhibitors to understand the underlying mechanism. 5-Azacytidine improved the cell viability by 29% in I/R-challenged H9C2 cells. Both isolated rat heart and LAD ligation model confirmed the infarct sparing effect of 5-azacytidine against I/R. It also provided a beneficial effect by normalizing the altered hemodynamics, reducing the infarct size and cardiac injury markers, reversing the perturbation of mitochondria, reduced oxidative stress, and improved the pPI3K and pAKT protein expression from I/R. In addition, it also augmented the activation of PI3K/AKT and mtKATP signaling pathway, confirmed by using wortmannin (PI3K inhibitor), SB216763 (GSK3ß inhibitor), and glibenclamide (mtKATP channel closer). The effectiveness of 5-azacytidine as a cardioprotective agent is attributed to its activation of the PI3K/GSK3ß and mtKATP channel signaling axis, thereby preserving mitochondrial function and reducing oxidative stress.

Influence of Hyperglycemia and Diabetes on Cardioprotection by Humoral Factors Released after Remote Ischemic Preconditioning (RIPC).

Remote ischemic preconditioning (RIPC) protects hearts from ischemia-reperfusion (I/R) injury in experimental studies; however, clinical RIPC trials were unsatisfactory. This discrepancy could be caused by a loss of cardioprotection due to comorbidities in patients, including diabetes mellitus (DM) and hyperglycemia (HG). RIPC is discussed to confer protective properties by release of different humoral factors activating cardioprotective signaling cascades. Therefore, we investigated whether DM type 1 and/or HG (1) inhibit the release of humoral factors after RIPC and/or (2) block the cardioprotective effect directly at the myocardium. Experiments were performed on male Wistar rats. Animals in part 1 of the study were either healthy normoglycemic (NG), type 1 diabetic (DM1), or hyperglycemic (HG). RIPC was implemented by four cycles of 5 min bilateral hind-limb ischemia/reperfusion. Control (Con) animals were not treated. Blood plasma taken in vivo was further investigated in isolated rat hearts in vitro. Plasma from diseased animals (DM1 or HG) was administered onto healthy (NG) hearts for 10 min before 33 min of global ischemia and 60 min of reperfusion. Part 2 of the study was performed vice versa-plasma taken in vivo, with or without RIPC, from healthy rats was transferred to DM1 and HG hearts in vitro. Infarct size was determined by TTC staining. Part 1: RIPC plasma from NG (NG Con: 49 ± 8% vs. NG RIPC 29 ± 6%; p < 0.05) and DM1 animals (DM1 Con: 47 ± 7% vs. DM1 RIPC: 38 ± 7%; p < 0.05) reduced infarct size. Interestingly, transfer of HG plasma showed comparable infarct sizes independent of prior treatment (HG Con: 34 ± 9% vs. HG RIPC 35 ± 9%; ns). Part 2: No infarct size reduction was detectable when transferring RIPC plasma from healthy rats to DM1 (DM1 Con: 54 ± 13% vs. DM1 RIPC 53 ± 10%; ns) or HG hearts (HG Con: 60 ± 16% vs. HG RIPC 53 ± 14%; ns). These results suggest that: (1) RIPC under NG and DM1 induces the release of humoral factors with cardioprotective impact, (2) HG plasma might own cardioprotective properties, and (3) RIPC does not confer cardioprotection in DM1 and HG myocardium.

Remote Ischemic Perconditioning Ameliorates Myocardial Ischemia and Reperfusion-Induced Coronary Endothelial Dysfunction and Aortic Stiffness in Rats.

BACKGROUND: Vascular stiffness and endothelial dysfunction are accelerated by acute myocardial infarction (AMI) and subsequently increase the risk for recurrent coronary events. AIM: To explore whether remote ischemic perconditioning (RIPerc) protects against coronary and aorta endothelial dysfunction as well as aortic stiffness following AMI. METHODS: Male OFA-1 rats were subjected to 30 min of occlusion of the left anterior descending artery (LAD) followed by reperfusion either 3 or 28 days with or without RIPerc. Three groups: (1) sham operated (Sham, without LAD occlusion); (2) myocardial ischemia and reperfusion (MIR) and (3) MIR + RIPerc group with 3 cycles of 5 minutes of IR on hindlimb performed during myocardial ischemia were used. Assessment of vascular reactivity in isolated septal coronary arteries (non-occluded) and aortic rings as well as aortic stiffness was assessed by wire myography either 3 or 28 days after AMI, respectively. Markers of pro-inflammatory cytokines, adhesion molecules were assessed by RT-qPCR and ELISA. RESULTS: MIR promotes impaired endothelial-dependent relaxation in septal coronary artery segments, increased aortic stiffness and adverse left ventricular remodeling. These changes were markedly attenuated in rats treated with RIPerc and associated with a significant decline in P-selectin, IL-6 and TNF-α expression either in infarcted or non-infarcted myocardial tissue samples. CONCLUSIONS: Our study for the first time demonstrated that RIPerc alleviates MIR-induced coronary artery endothelial dysfunction in non-occluded artery segments and attenuates aortic stiffness in rats. The vascular protective effects of RIPerc are associated with ameliorated inflammation and might therefore be caused by reduced inflammatory signaling.

Inhibition of PI3K/mTOR/KATP channel blunts sodium thiosulphate preconditioning mediated cardioprotection against ischemia-reperfusion injury.

Recent studies have shown that pre and postconditioning the heart with sodium thiosulfate (STS) attenuate ischemia-reperfusion (IR) injury. However, the underlying mechanism involved in the cardioprotective signaling pathway is not fully explored. This study examined the existing link of STS mediated protection (as pre and post-conditioning agents) with PI3K, mTOR, and mPTP signaling pathways using its respective inhibitors. STS was administered to the isolated perfused rat heart through Kreb's Heinselit buffer before ischemia (precondition: SIPC) and reperfusion (postcondition: SPOC) in the presence and absence of the PI3K, mTOR, and mPTP signaling pathway inhibitors (wortmannin, rapamycin, and glibenclamide respectively). SIPC failed to improve the IR injury-induced altered cardiac hemodynamics, increased infarct size, and the release of cardiac injury markers in the presence of these inhibitors. On the other hand, the SPOC protocol effectively rendered the cardioprotection even in the PI3K/mTOR/KATP inhibitors presence. Interestingly, the SIPC's identified mode of action viz reduction in oxidative stress and the preservation of mitochondrial function were lost in the inhibitors' presence. Based on the above results, we conclude that the underlying mechanism of SIPC mediated cardioprotection works via the PI3K/mTOR/KATP signaling pathway axis activation.

Remote and Anesthetic-Induced Myocardial Preconditioning Is Preserved in Atherosclerotic LDL Receptor-/- Mice In Vivo.

INTRODUCTION: In the animal model, preconditioning is a powerful weapon against ischemic damage. The reason why the human heart cannot be protected from ischemic damage by preconditioning remains unclear. There are assumptions that the lack of preconditioning in humans is caused by concomitant diseases such as dyslipoproteinemia and arteriosclerosis. This study investigates whether dyslipoproteinemia and the resulting arteriosclerosis can be a cause of a reduced precondition effect of heart in mice. METHODS: LDL receptor-deficient mice were fed a long-term (14-16 weeks) high-fat atherogenic diet to induce arteriosclerosis. Arteriosclerosis was identified by histological examination and vessel contraction tests. LDLR-/- and wild-type mice were randomly assigned to anesthetic-induced, remote ischemic, or no preconditioning. All mice were subjected to 45 minutes of coronary artery occlusion and 180 minutes of reperfusion. The area at risk and infarct size were determined by Evans Blue and triphenyltetrazolium chloride staining. RESULTS: Histopathological examination showed atherosclerosis in high-fat atherogenic fed LDLR-/- mice, and the vessel relaxation capacity was significantly reduced compared to wild-type mice. In the wild type, as expected, infarct size was significantly reduced by preconditioning compared to the control. In LDLR-/- mice, infarct size was significantly reduced by preconditioning compared to the control. Surprisingly, the LDLR-/- control group also had a significantly reduced infarct size compared to the wild-type control group. CONCLUSION: We were able to demonstrate that a high-fat diet morphologically and functionally triggered atherosclerosis in LDLR-/- mice. Interestingly, LDLR-/- mice with an atherogenic diet had smaller infarct sizes compared to wild-type mice. Moreover, preconditioning additionally reduced myocardial infarct size in LDLR-/- mice. A long-term high-fat atherogenic diet and preconditioning seem to result in additive cardioprotection in LDLR-/- mice.