EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection.

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Circadian Influences on Myocardial Ischemia-Reperfusion Injury and Heart Failure.

The impact of circadian rhythms on cardiovascular function and disease development is well established, with numerous studies in genetically modified animals emphasizing the circadian molecular clock's significance in the pathogenesis and pathophysiology of myocardial ischemia and heart failure progression. However, translational preclinical studies targeting the heart's circadian biology are just now emerging and are leading to the development of a novel field of medicine termed circadian medicine. In this review, we explore circadian molecular mechanisms and novel therapies, including (1) intense light, (2) small molecules modulating the circadian mechanism, and (3) chronotherapies such as cardiovascular drugs and meal timings. These promise significant clinical translation in circadian medicine for cardiovascular disease. (4) Additionally, we address the differential functioning of the circadian mechanism in males versus females, emphasizing the consideration of biological sex, gender, and aging in circadian therapies for cardiovascular disease.

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

Interaction of Cardiovascular Nonmodifiable Risk Factors, Comorbidities and Comedications With Ischemia/Reperfusion Injury and Cardioprotection by Pharmacological Treatments and Ischemic Conditioning.

Preconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities. SIGNIFICANCE STATEMENT: Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.

Cardioprotective properties of OMT-28, a synthetic analog of omega-3 epoxyeicosanoids.

OMT-28 is a metabolically robust small molecule developed to mimic the structure and function of omega-3 epoxyeicosanoids. However, it remained unknown to what extent OMT-28 also shares the cardioprotective and anti-inflammatory properties of its natural counterparts. To address this question, we analyzed the ability of OMT-28 to ameliorate hypoxia/reoxygenation (HR)-injury and lipopolysaccharide (LPS)-induced endotoxemia in cultured cardiomyocytes. Moreover, we investigated the potential of OMT-28 to limit functional damage and inflammasome activation in isolated perfused mouse hearts subjected to ischemia/reperfusion (IR) injury. In the HR model, OMT-28 (1 µM) treatment largely preserved cell viability (about 75 versus 40% with the vehicle) and mitochondrial function as indicated by the maintenance of NAD+/NADH-, ADP/ATP-, and respiratory control ratios. Moreover, OMT-28 blocked the HR-induced production of mitochondrial reactive oxygen species. Pharmacological inhibition experiments suggested that Gαi, PI3K, PPARα, and Sirt1 are essential components of the OMT-28-mediated pro-survival pathway. Counteracting inflammatory injury of cardiomyocytes, OMT-28 (1 µM) reduced LPS-induced increases in TNFα protein (by about 85% versus vehicle) and NF-κB DNA binding (by about 70% versus vehicle). In the ex vivo model, OMT-28 improved post-IR myocardial function recovery to reach about 40% of the baseline value compared to less than 20% with the vehicle. Furthermore, OMT-28 (1 µM) limited IR-induced NLRP3 inflammasome activation similarly to a direct NLRP3 inhibitor (MCC950). Overall, this study demonstrates that OMT-28 possesses potent cardio-protective and anti-inflammatory properties supporting the hypothesis that extending the bioavailability of omega-3 epoxyeicosanoids may improve their prospects as therapeutic agents.

MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Dynamin-related protein 1 is a critical regulator of mitochondrial calcium homeostasis during myocardial ischemia/reperfusion injury.

Dynamin-related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing reactive oxygen species (ROS) in cardiomyocytes. Drp1 has shown promise as a therapeutic target for reducing cardiac ischemia/reperfusion (IR) injury; however, the lack of specificity of some small molecule Drp1 inhibitors and the reliance on the use of Drp1 haploinsufficient hearts from older mice have left the role of Drp1 in IR in question. Here, we address these concerns using two approaches, using: (a) short-term (3 weeks), conditional, cardiomyocyte-specific, Drp1 knockout (KO) and (b) a novel, highly specific Drp1 GTPase inhibitor, Drpitor1a. Short-term Drp1 KO mice exhibited preserved exercise capacity and cardiac contractility, and their isolated cardiac mitochondria demonstrated increased mitochondrial complex 1 activity, respiratory coupling, and calcium retention capacity compared to controls. When exposed to IR injury in a Langendorff perfusion system, Drp1 KO hearts had preserved contractility, decreased reactive oxygen species (ROS), enhanced mitochondrial calcium capacity, and increased resistance to mitochondrial permeability transition pore (MPTP) opening. Pharmacological inhibition of Drp1 with Drpitor1a following ischemia, but before reperfusion, was as protective as Drp1 KO for cardiac function and mitochondrial calcium homeostasis. In contrast to the benefits of short-term Drp1 inhibition, prolonged Drp1 ablation (6 weeks) resulted in cardiomyopathy. Drp1 KO hearts were also associated with decreased ryanodine receptor 2 (RyR2) protein expression and pharmacological inhibition of the RyR2 receptor decreased ROS in post-IR hearts suggesting that changes in RyR2 may have a role in Drp1 KO mediated cardioprotection. We conclude that Drp1-mediated increases in myocardial ROS production and impairment of mitochondrial calcium handling are key mechanisms of IR injury. Short-term inhibition of Drp1 is a promising strategy to limit early myocardial IR injury which is relevant for the therapy of acute myocardial infarction, cardiac arrest, and heart transplantation.

Brown Adipose Tissue and BMP3b Decrease Injury in Cardiac Ischemia-Reperfusion.

BACKGROUND: Despite advances in treatment, myocardial infarction (MI) is a leading cause of heart failure and death worldwide, with both ischemia and reperfusion (I/R) causing cardiac injury. A previous study using a mouse model of nonreperfused MI showed activation of brown adipose tissue (BAT). Recent studies showed that molecules secreted by BAT target the heart. We investigated whether BAT attenuates cardiac injury in I/R and sought to identify potential cardioprotective proteins secreted by BAT. METHODS: Myocardial I/R surgery with or without BAT transplantation was performed in wild-type (WT) mice and in mice with impaired BAT function (uncoupling protein 1 [Ucp1]-deficient mice). To identify potential cardioprotective factors produced by BAT, RNA-seq (RNA sequencing) was performed in BAT from WT and Ucp1-/- mice. Subsequently, myocardial I/R surgery with or without BAT transplantation was performed in Bmp3b (bone morphogenetic protein 3b)-deficient mice, and WT mice subjected to myocardial I/R were treated using BMP3b. RESULTS: Dysfunction of BAT in mice was associated with larger MI size after I/R; conversely, augmenting BAT by transplantation decreased MI size. We identified Bmp3b as a protein secreted by BAT after I/R. Compared with WT mice, Bmp3b-deficient mice developed larger MIs. Increasing functional BAT by transplanting BAT from WT mice to Bmp3b-deficient mice reduced I/R injury whereas transplanting BAT from Bmp3b-deficient mice did not. Treatment of WT mice with BMP3b before reperfusion decreased MI size. The cardioprotective effect of BMP3b was mediated through SMAD1/5/8. In humans, the plasma level of BMP3b increased after MI and was positively correlated with the extent of cardiac injury. CONCLUSIONS: The results of this study suggest a cardioprotective role of BAT and BMP3b, a protein secreted by BAT, in a model of I/R injury. Interventions increasing BMP3b levels or targeting Smad 1/5 may represent novel therapeutic approaches to decrease myocardial damage in I/R injury.

Spatial Multiplexed Protein Profiling of Cardiac Ischemia-Reperfusion Injury.

BACKGROUND: Reperfusion therapy is critical to myocardial salvage in the event of a myocardial infarction but is complicated by ischemia-reperfusion injury (IRI). Limited understanding of the spatial organization of cardiac cells, which governs cellular interaction and function, has hindered the search for targeted interventions minimizing the deleterious effects of IRI. METHODS: We used imaging mass cytometry to characterize the spatial distribution and dynamics of cell phenotypes and communities in the mouse left ventricle following IRI. Heart sections were collected from 12 cardiac segments (basal, mid-cavity, apical, and apex of the anterior, lateral, and inferior wall) and 8 time points (before ischemia [I-0H], and postreperfusion [R-0H, R-2H, R-6H, R-12H, R-1D, R-3D, R-7D]), and stained with 29 metal-isotope-tagged antibodies. Cell community analysis was performed on reconstructed images, and the most disease-relevant cell type and target protein were selected for intervention of IRI. RESULTS: We obtained a total of 251 multiplexed images, and identified 197 063 single cells, which were grouped into 23 distinct cell communities based on the structure of cellular neighborhoods. The cellular architecture was heterogeneous throughout the ventricular wall and exhibited swift changes following IRI. Analysis of proteins with posttranslational modifications in single cells unveiled 13 posttranslational modification intensity clusters and highlighted increased H3K9me3 (tri-methylated lysine 9 of histone H3) as a key regulatory response in endothelial cells during the middle stage of IRI. Erasing H3K9 methylation, by silencing its methyltransferase Suv39h1 or overexpressing its demethylase Kdm4d in isolated endothelial cells, attenuated cardiac dysfunction and pathological remodeling following IRI. in vitro, H3K9me3 binding significantly increased at endothelial cell function-related genes upon hypoxia, suppressing tube formation, which was rescued by inhibiting H3K9me3. CONCLUSIONS: We mapped the spatiotemporal heterogeneity of cellular phenotypes in the adult heart upon IRI, and uncovered H3K9me3 in endothelial cells as a potential therapeutic target for alleviating pathological remodeling of the heart following myocardial IRI.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Upregulation of P2X7 Exacerbates Myocardial Ischemia-Reperfusion Injury through Enhancing Inflammation and Apoptosis in Diabetic Mice.

Diabetes-aggravated myocardial ischemia-reperfusion (MI/R) injury remains an urgent medical issue, and the molecular mechanisms involved with diabetes and MI/R injury remain largely unknown. Previous studies have shown that inflammation and P2X7 signaling participate in the pathogenesis of the heart under individual conditions. It remains to be explored if P2X7 signaling is exacerbated or alleviated under double insults. We established a high-fat diet and streptozotocin-induced diabetic mouse model, and we compared the differences in immune cell infiltration and P2X7 expression between diabetic and nondiabetic mice after 24 h of reperfusion. The antagonist and agonist of P2X7 were administered before and after MI/R. Our study showed that the MI/R injury of diabetic mice was characterized by increased infarct area, impaired ventricular contractility, more apoptosis, aggravated immune cell infiltration, and overactive P2X7 signaling compared with nondiabetic mice. The major trigger of increased P2X7 was the MI/R-induced recruitment of monocytes and macrophages, and diabetes can be a synergistic factor in this process. Administration of P2X7 agonist eliminated the differences in MI/R injury between nondiabetic mice and diabetic mice. Both 2 wk of brilliant blue G injection before MI/R and acutely administered A438079 at the time of MI/R injury attenuated the role of diabetes in exacerbating MI/R injury, as evidenced by decreased infarct size, improved cardiac function, and inhibition of apoptosis. Additionally, brilliant blue G blockade decreased the heart rate after MI/R, which was accompanied by downregulation of tyrosine hydroxylase expression and nerve growth factor transcription. In conclusion, targeting P2X7 may be a promising strategy for reducing the risk of MI/R injury in diabetes.

Glycyrrhizin attenuates myocardial ischemia reperfusion injury by suppressing Inflammation, oxidative stress, and ferroptosis via the HMGB1-TLR4-GPX4 pathway.

Ferroptosis, a form of regulated cell death process, play an important role in myocardial ischemia‒reperfusion (I/R) injury. Glycyrrhizin (GL), a natural glycoconjugate triterpene, has the property to improve growth rate, immune regulation, antioxidant, anti-inflammatory. However, whether GL can attenuate myocardial I/R injury by modulating ferroptosis or other mechanisms are still unclear. In this study, SD rats underwent in vivo myocardial ischemia/reperfusion (I/R) surgery, while H9C2 cells were subjected to the hypoxia/reoxygenation (H/R) model for in vitro experiments. In addition, TAK-242, a TLR4-specific antagonist, and GL were also used to evaluate the effect and mechanisms of GL on the cardiac function and expression of ferroptosis-related gene and protein in vivo and vitro. The results show that GL decreased not only the expression of the inflammation-related factors (HMGB1, TNF-α, IL-6, IL-18 and IL-1ß), but also reduced the number of TUNEL-positive cardiomyocytes, and mitigated pathological alterations in I/R injury. In addition, GL decreased the levels of MDA, promoted antioxidant capacity such as GSH, CAT, Cu/Zn-SOD, Mn-SOD, and SOD in vivo and vitro. More importantly, GL and TAK-242 regulate ferroptosis-related protein and gene expression in I/R and H/R model. Surprisingly, GL may ameliorate cardiomyocyte ferroptosis and ultimately improves cardiac function induced by H/R via the HMGB1-TLR4-GPX4 axis. Therefore, we have highlighted a novel mechanism by which GL regulates inflammation, oxidative stress, and ferroptosis via the HMGB1-TLR4-GPX4 pathway to prevent myocardial I/R injury. GL appears to be a potentially applicable drug for the treatment of myocardial I/R injury.

Myocardial reperfusion injury exacerbation due to ALDH2 deficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.

BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.

Classification of regulatory T cells and their role in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is closely related to the final infarct size in acute myocardial infarction (AMI). Therefore, reducing MIRI can effectively improve the prognosis of AMI patients. At the same time, the healing process after AMI is closely related to the local inflammatory microenvironment. Regulatory T cells (Tregs) can regulate various physiological and pathological immune inflammatory responses and play an important role in regulating the immune inflammatory response after AMI. However, different subtypes of Tregs have different effects on MIRI, and the same subtype of Tregs may also have different effects at different stages of MIRI. This article systematically reviews the classification and function of Tregs, as well as the role of various subtypes of Tregs in MIRI. A comprehensive understanding of the role of each subtype of Tregs can help design effective methods to control immune reactions, reduce MIRI, and provide new potential therapeutic options for AMI.

ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.

Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts.

Myocardial ischemia-reperfusion (IR) (stunning) injury triggers changes in the proteome and degradome of the heart. Here, we utilize quantitative proteomics and comprehensive degradomics to investigate the molecular mechanisms of IR injury in isolated rat hearts. The control group underwent aerobic perfusion, while the IR injury group underwent 20 min of ischemia and 30 min of reperfusion to induce a stunning injury. As MMP-2 activation has been shown to contribute to myocardial injury, hearts also underwent IR injury with ARP-100, an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2 to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy, we quantified 4468 proteins in ventricular extracts, whereby 447 proteins showed significant alterations among the three groups. We then used subtiligase-mediated N-terminomic labeling to identify more than a hundred specific cleavage sites. Among these protease substrates, 15 were identified following IR injury. We identified alterations in numerous proteins involved in mitochondrial function and metabolism following IR injury. Our findings provide valuable insights into the biochemical mechanisms of myocardial IR injury, suggesting alterations in reactive oxygen/nitrogen species handling and generation, fatty acid metabolism, mitochondrial function and metabolism, and cardiomyocyte contraction.

Alox15/15-HpETE Aggravates Myocardial Ischemia-Reperfusion Injury by Promoting Cardiomyocyte Ferroptosis.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) injury causes cardiac dysfunction to myocardial cell loss and fibrosis. Prevention of cell death is important to protect cardiac function after I/R injury. The process of reperfusion can lead to multiple types of cardiomyocyte death, including necrosis, apoptosis, autophagy, and ferroptosis. However, the time point at which the various modes of cell death occur after reperfusion injury and the mechanisms underlying ferroptosis regulation in cardiomyocytes are still unclear. METHODS: Using a left anterior descending coronary artery ligation mouse model, we sought to investigate the time point at which the various modes of cell death occur after reperfusion injury. To discover the key molecules involved in cardiomyocyte ferroptosis, we performed a metabolomics study. Loss/gain-of-function approaches were used to understand the role of 15-lipoxygenase (Alox15) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α) in myocardial I/R injury. RESULTS: We found that apoptosis and necrosis occurred in the early phase of I/R injury, and that ferroptosis was the predominant form of cell death during the prolonged reperfusion. Metabolomic profiling of eicosanoids revealed that Alox15 metabolites accumulated in ferroptotic cardiomyocytes. We demonstrated that Alox15 expression was specifically increased in the injured area of the left ventricle below the suture and colocalized with cardiomyocytes. Furthermore, myocardial-specific knockout of Alox15 in mice alleviated I/R injury and restored cardiac function. 15-Hydroperoxyeicosatetraenoic acid (15-HpETE), an intermediate metabolite derived from arachidonic acid by Alox15, was identified as a trigger for cardiomyocyte ferroptosis. We explored the mechanism underlying its effects and found that 15-HpETE promoted the binding of Pgc1α to the ubiquitin ligase ring finger protein 34, leading to its ubiquitin-dependent degradation. Consequently, attenuated mitochondrial biogenesis and abnormal mitochondrial morphology were observed. ML351, a specific inhibitor of Alox15, increased the protein level of Pgc1α, inhibited cardiomyocyte ferroptosis, protected the injured myocardium, and caused cardiac function recovery. CONCLUSIONS: Together, our results established that Alox15/15-HpETE-mediated cardiomyocyte ferroptosis plays an important role in prolonged I/R injury.

Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

BACKGROUND: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. METHODS: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. RESULTS: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-ß complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-ß) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-ß complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. CONCLUSIONS: Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

Nox4 regulates InsP3 receptor-dependent Ca2+ release into mitochondria to promote cell survival.

Cells subjected to environmental stresses undergo regulated cell death (RCD) when homeostatic programs fail to maintain viability. A major mechanism of RCD is the excessive calcium loading of mitochondria and consequent triggering of the mitochondrial permeability transition (mPT), which is especially important in post-mitotic cells such as cardiomyocytes and neurons. Here, we show that stress-induced upregulation of the ROS-generating protein Nox4 at the ER-mitochondria contact sites (MAMs) is a pro-survival mechanism that inhibits calcium transfer through InsP3 receptors (InsP3 R). Nox4 mediates redox signaling at the MAM of stressed cells to augment Akt-dependent phosphorylation of InsP3 R, thereby inhibiting calcium flux and mPT-dependent necrosis. In hearts subjected to ischemia-reperfusion, Nox4 limits infarct size through this mechanism. These results uncover a hitherto unrecognized stress pathway, whereby a ROS-generating protein mediates pro-survival effects through spatially confined signaling at the MAM to regulate ER to mitochondria calcium flux and triggering of the mPT.