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.

Inhibition of Hmbox1 Promotes Cardiomyocyte Survival and Glucose Metabolism Through Gck Activation in Ischemia/Reperfusion Injury.

BACKGROUND: Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. METHODS: We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. RESULTS: We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. CONCLUSIONS: Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury.

Transient Inhibition of Translation Improves Cardiac Function After Ischemia/Reperfusion by Attenuating the Inflammatory Response.

BACKGROUND: The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. METHODS: To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type-specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. RESULTS: Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)-4EBP1 (eIF4E-binding protein 1)-eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6Chi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. CONCLUSIONS: Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6Chi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

Lactate Contributes to Remote Ischemic Preconditioning-Mediated Protection Against Myocardial Ischemia Reperfusion Injury by Facilitating Autophagy via the AMP-Activated Protein Kinase-Mammalian Target of Rapamycin-Transcription Factor EB-Connexin 43 Axis.

Remote ischemic preconditioning (RIPC) exerts a protective role on myocardial ischemia/reperfusion (I/R) injury by the release of various humoral factors. Lactate is a common metabolite in ischemic tissues. Nevertheless, little is known about the role lactate plays in myocardial I/R injury and its underlying mechanism. This investigation revealed that RIPC elevated the level of lactate in blood and myocardium. Furthermore, AZD3965, a selective monocarboxylate transporter 1 inhibitor, and 2-deoxy-d-glucose, a glycolysis inhibitor, mitigated the effects of RIPC-induced elevated lactate in the myocardium and prevented RIPC against myocardial I/R injury. In an in vitro hypoxia/reoxygenation model, lactate markedly mitigated hypoxia/reoxygenation-induced cell damage in H9c2 cells. Further studies suggested that lactate contributed to RIPC, rescuing I/R-induced autophagy deficiency by promoting transcription factor EB (TFEB) translocation to the nucleus through activating the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway without influencing the phosphatidylinositol 3-kinase-Akt pathway, thus reducing cardiomyocyte damage. Interestingly, lactate up-regulated the mRNA and protein expression of connexin 43 (CX43) by facilitating the binding of TFEB to CX43 promoter in the myocardium. Functionally, silencing of TFEB attenuated the protective effect of lactate on cell damage, which was reversed by overexpression of CX43. Further mechanistic studies suggested that lactate facilitated CX43-regulated autophagy via the AMPK-mTOR-TFEB signaling pathway. Collectively, this research demonstrates that RIPC protects against myocardial I/R injury through lactate-mediated myocardial autophagy via the AMPK-mTOR-TFEB-CX43 axis.

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.

Platelets at the intersection of inflammation and coagulation in the APC-mediated response to myocardial ischemia/reperfusion injury.

Thromboinflammation is a complex pathology associated with inflammation and coagulation. In cases of cardiovascular disease, in particular ischemia-reperfusion injury, thromboinflammation is a common complication. Increased understanding of thromboinflammation depends on an improved concept of the mechanisms of cells and proteins at the axis of coagulation and inflammation. Among these elements are activated protein C and platelets. This review summarizes the complex interactions of activated protein C and platelets regulating thromboinflammation in cardiovascular disease. By unraveling the pathways of platelets and APC in the inflammatory and coagulation cascades, this review summarizes the role of these vital mediators in the development and perpetuation of heart disease and the thromboinflammation-driven complications of cardiovascular disease. Furthermore, this review emphasizes the significance of the counteracting effects of platelets and APC and their combined role in disease states.

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.

CITED4 gene therapy protects against maladaptive cardiac remodeling after ischemia/reperfusion injury in mice.

Cardiac signaling pathways functionally important in the heart's response to exercise often protect the heart against pathological stress, potentially providing novel therapeutic targets. However, it is important to determine which of these pathways can be feasibly targeted in vivo. Transgenic overexpression of exercise-induced CITED4 has been shown to protect against adverse remodeling after ischemia/reperfusion injury (IRI). Here we investigated whether somatic gene transfer of CITED4 in a clinically relevant time frame could promote recovery after IRI. Cardiac CITED4 gene delivery via intravenous AAV9 injections in wild type mice led to an approximately 3-fold increase in cardiac CITED4 expression. After 4 weeks, CITED4-treated animals developed physiological cardiac hypertrophy without adverse remodeling. In IRI, delivery of AAV9-CITED4 after reperfusion resulted in a 6-fold increase in CITED4 expression 1 week after surgery, as well as decreased apoptosis, fibrosis, and inflammatory markers, culminating in a smaller scar and improved cardiac function 8 weeks after IRI, compared with control mice receiving AAV9-GFP. Somatic gene transfer of CITED4 induced a phenotype suggestive of physiological cardiac growth and mitigated adverse remodeling after ischemic injury. These studies support the feasibility of CITED4 gene therapy delivered in a clinically relevant time frame to mitigate adverse ventricular remodeling after ischemic 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.

Vago-splenic signal transduction of cardioprotection in humans.

BACKGROUND AND AIMS: The spleen serves as an important relay organ that releases cardioprotective factor(s) upon vagal activation during remote ischaemic conditioning (RIC) in rats and pigs. The translation of these findings to humans was attempted. METHODS: Remote ischaemic conditioning or electrical auricular tragus stimulation (ATS) were performed in 10 healthy young volunteers, 10 volunteers with splenectomy, and 20 matched controls. Venous blood samples were taken before and after RIC/ATS or placebo, and a plasma dialysate was infused into isolated perfused rat hearts subjected to global ischaemia/reperfusion. RESULTS: Neither left nor right RIC or ATS altered heart rate and heart rate variability in the study cohorts. With the plasma dialysate prepared before RIC or ATS, respectively, infarct size (% ventricular mass) in the recipient rat heart was 36 ± 6% (left RIC), 34 ± 3% (right RIC) or 31 ± 5% (left ATS), 35 ± 5% (right ATS), and decreased with the plasma dialysate from healthy volunteers after RIC or ATS to 20 ± 4% (left RIC), 23 ± 6% (right RIC) or to 19 ± 4% (left ATS), 26 ± 9% (right ATS); infarct size was still reduced with plasma dialysate 4 days after ATS and 9 days after RIC. In a subgroup of six healthy volunteers, such infarct size reduction was abrogated by intravenous atropine. Infarct size reduction by RIC or ATS was also abrogated in 10 volunteers with splenectomy, but not in their 20 matched controls. CONCLUSIONS: In humans, vagal innervation and the spleen as a relay organ are decisive for the cardioprotective signal transduction of RIC and ATS.