Deubiquitinase USP5 regulates RIPK1 driven pyroptosis in response to myocardial ischemic reperfusion injury.

BACKGROUND: Gasdermin D (GSDMD) mediated pyroptosis plays a significant role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. However, the precise mechanisms regulating pyroptosis remain unclear. In the study, we aimed to investigate the underlying mechanism of pyroptosis in myocardial I/R injury. METHODS: In the present study, we analyzed the effects of USP5 on the RIPK1 kinase activity mediated pyroptosis in vitro after H/R (hypoxia/reoxygenation) and in vivo in a MI/R mouse model. TTC and Evan's blue dye, Thioflavin S and immunohistochemistry staining were performed in wild-type, RIPK1flox/flox Cdh5-Cre and USP5 deficiency mice. CMEC cells were transfected with si-USP5. HEK293T cells were transfected with USP5 and RIPK1 overexpression plasmid or its mutants. The levels of USP5, RIPK1, Caspase-8, FADD and GSDMD were determined by Western blot. Protein interactions were evaluated by immunoprecipitation. The protein colocalization in cells was monitored using a confocal microscope. RESULTS: In this study, our data demonstrate that RIPK1 is essential for limiting cardiac endothelial cell (CMEC) pyroptosis mediated by caspase-8 in response to myocardial I/R. Additionally, we investigate the role of ubiquitin-specific protease 5 (USP5) as a deubiquitinase for RIPK1. Mechanistically, USP5 interacts with RIPK1, leading to its deubiquitination and stabilization. CONCLUSIONS: These findings offer new insights into the role of USP5 in regulating RIPK1-induced pyroptosis.

The role and possible mechanism of the ferroptosis-related SLC7A11/GSH/GPX4 pathway in myocardial ischemia-reperfusion injury.

BACKGROUND: Myocardial ischemia-reperfusion injury (MI/RI) is an unavoidable risk event for acute myocardial infarction, with ferroptosis showing close involvement. We investigated the mechanism of MI/RI inducing myocardial injury by inhibiting the ferroptosis-related SLC7A11/glutathione (GSH)/glutathione peroxidase 4 (GPX4) pathway and activating mitophagy. METHODS: A rat MI/RI model was established, with myocardial infarction area and injury assessed by TTC and H&E staining. Rat cardiomyocytes H9C2 were cultured in vitro, followed by hypoxia/reoxygenation (H/R) modeling and the ferroptosis inhibitor lipoxstatin-1 (Lip-1) treatment, or 3-Methyladenine or rapamycin treatment and overexpression plasmid (oe-SLC7A11) transfection during modeling. Cell viability and death were evaluated by CCK-8 and LDH assays. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial membrane potential was detected by fluorescence dye JC-1. Levels of inflammatory factors, reactive oxygen species (ROS), Fe2+, malondialdehyde, lipid peroxidation, GPX4 enzyme activity, glutathione reductase, GSH and glutathione disulfide, and SLC7A11, GPX4, LC3II/I and p62 proteins were determined by ELISA kit, related indicator detection kits and Western blot. RESULTS: The ferroptosis-related SLC7A11/GSH/GPX4 pathway was repressed in MI/RI rat myocardial tissues, inducing myocardial injury. H/R affected GSH synthesis and inhibited GPX4 enzyme activity by down-regulating SLC7A11, thus promoting ferroptosis in cardiomyocytes, which was averted by Lip-1. SLC7A11 overexpression improved H/R-induced cardiomyocyte ferroptosis via the GSH/GPX4 pathway. H/R activated mitophagy in cardiomyocytes. Mitophagy inhibition reversed H/R-induced cellular ferroptosis. Mitophagy activation partially averted SLC7A11 overexpression-improved H/R-induced cardiomyocyte ferroptosis. H/R suppressed the ferroptosis-related SLC7A11/GSH/GPX4 pathway by inducing mitophagy, leading to cardiomyocyte injury. CONCLUSIONS: Increased ROS under H/R conditions triggered cardiomyocyte injury by inducing mitophagy to suppress the ferroptosis-related SLC7A11/GSH/GPX4 signaling pathway activation.

Depression exacerbates myocardial ischemia-reperfusion injury in mice via CNR2 gene and MIF-AMPK signaling pathway.

BACKGROUND: Myocardial ischemia-reperfusion(I/R)injury constitute the fundamental pathophysiology of acute myocardial infarction (AMI). Ischemic heart releases macrophage migration inhibitory factor (MIF), which activates MIF- AMPK signaling pathway. Depression is a significant risk factor for AMI. In a state of depression, peripheral expression of cannabinoid receptor 2 (CNR2) genes was downregulated. AIMS: We investigated the mechanism by which depression exacerbates myocardial I/R injury through the CNR2 and MIF-AMPK signaling pathways. METHODS: We established mouse models of depression and myocardial I/R. Left ventricular function was assessed using cardiac ultrasound and TTC staining. The protein levels of myocardial CNR2, MIF, AMPK, and ACC were determined by Western blot, while the expression level of CNR2 was measured using RT-qPCR. Additionally, MIF content in peripheral blood was quantified using ELISA. RESULTS: After I/R, the expression level of CNR2 was found to be lower in the depression group, leading to a deterioration in left heart function. Depressed mice exhibited lower secretion of MIF, accompanied by a decrease in the activation of the MIF-AMPK signaling pathway. However, injection of CNR2 agonist JWH133 prior to ischemia increased the activation of the MIF-AMPK signaling pathway, while CNR2 inhibitor AM630 decreased the activation. LIMITATIONS: Further research is needed to investigate the specific neuroendocrine mechanism affecting myocardial CNR2 expression in depression. And these experimental conclusions require further verification at the cellular level. CONCLUSIONS: The activation of CNR2 in myocardium following I/R is impeded by depression, thereby exacerbating myocardial I/R injury through attenuation of the MIF-AMPK signaling pathway activation.

Key subdomains of mesencephalic astrocyte-derived neurotrophic factor attenuate myocardial ischemia/reperfusion injury by JAK1/STAT1/NF-κB signaling pathway.

BACKGROUND: Myocardial ischemia/reperfusion (I/R) injury is a common pathological process in clinical practice. Developing effective therapeutic strategies to reduce or prevent this injury is crucial. The article aimed to investigate the role and mechanism of mesencephalic astrocyte-derived neurotrophic factor (MANF) and its key subdomains in modulating myocardial I/R-induced cardiomyocyte apoptosis. METHODS: MANF stable knockout cell line and MANF mutant overexpression plasmids were constructed. The effects of MANF and mutants on apoptosis and endoplasmic reticulum (ER) stress related proteins were evaluated in hypoxia/reoxygenation-induced HL-1 cardiomyocytes by western blot, immunofluorescence, Tunel and flow cytometry. Echocardiography, ELISA, TTC and Masson were used to observe the effects of recombinant MANF protein (rMANF) on cardiac function in myocardial I/R mice. RESULTS: This study observed increased expression of MANF in both myocardial infarction patients and I/R mice. MANF overexpression in cardiomyocytes decreased ER stress-induced apoptosis, while MANF knockout exacerbated it. rMANF improved cardiac function in I/R mice by reducing injury and inflammation. This study specifically demonstrates that mutations in the α-helix of MANF were more effective in reducing ER stress and cardiomyocyte apoptosis. Mechanistically, MANF and the α-helix mutant attenuated I/R injury by inhibiting the JAK1/STAT1/NF-κB signaling pathway in addition to reducing ER stress-induced apoptosis. CONCLUSION: These findings highlight MANF and its subdomains as critical regulators of myocardial I/R injury, offering promising therapeutic targets with significant clinical implications for I/R-related diseases.

TRAF3IP3 Blocks Mitophagy to Exacerbate Myocardial Injury Induced by Ischemia-Reperfusion.

To uncover the possible role of TRAF3IP3 in the progression of myocardial infarction (MI), clarify its role in mitophagy and mitochondrial function, and explore the underlying mechanism. GEO chip analysis, RT-qPCR, and LDH release assay were used to detect the expression of TRAF3IP3 in tissues and cells and its effects on cell damage. Immunostaining and ATP product assays were performed to examine the effects of TRAF3IP3 on mitochondrial function. Co-IP, CHX assays, Immunoblot and Immunostaining assays were conducted to determine the effects of TRAF3IP3 on mitophagy. TRAF3IP3 was highly expressed in IR rats and HR-induced H9C2 cells. TRAF3IP3 knockdown can alleviate H/R-induced H9C2 cell damage. In addition, TRAF3IP3 knockdown can induce mitophagy, thus enhancing mitochondrial function. We further revealed that TRAF3IP3 can promote the degradation of NEDD4 protein. Moreover, TRAF3IP3 knockdown suppressed myocardial injury in I/R rats. TRAF3IP3 blocks mitophagy to exacerbate myocardial injury induced by I/R via mediating NEDD4 expression.

Connexin43 overexpression promoted ferroptosis and increased myocardial vulnerability to ischemia-reperfusion injury in type 1 diabetic mice.

Enhancement of Connexin43 (Cx43) and ferroptosis are respectively associated with the exacerbation of myocardial ischemia-reperfusion injury (MIRI) in diabetes. Myocardial vulnerability to ischemic insult has been shown to vary during early and later phases of diabetes in experimental settings. Whether or not Connexin43 (Cx43) and ferroptosis interplay during MIRI in diabetes is unknown. We, thus, aimed to investigate whether or not the content of myocardial Cx43 may be attributable to myocardial vulnerability to MIRI at different stages of diabetes and also to explore the potential interplay between Cx43 and ferroptosis in this pathology. Age-matched control and subgroups of Streptozotocin-induced diabetic mice were subjected to MIRI induced by 30 minutes coronary artery occlusion and 2 hours reperfusion respectively at 1, 2 and 5 weeks of diabetes. Rat cardiac H9C2 cells were exposed to high glucose (HG) for 48h in the absence or presence of Cx43 gene knockdown followed by hypoxia/reoxygenation (HR) respectively for 6 and 12 hours. Post-ischemic myocardial infarct size was reduced in 1 and 2 weeks DM mice concomitant with enhanced GPX4 and reduced cardiac Cx43 and ferroptosis as compared to control. By contrast, cardiac GPX4 was significantly reduced while Cx43 increased at DM 5 weeks (D5w) which was correspondent to significant increases in ferroptosis and myocardial infarction. Post-ischemic cardiac function was improved in 1 and 2 weeks but worsened in 5w DM mice as compared with non-diabetic control. GAP19 (Cx43 inhibitor) significantly attenuated ferroptosis and reduced myocardial infarction in D5w mice. Erastin (ferroptosis activator) reversed the cardioprotective effect of GAP19. In vitro, HR significantly reduced cell viability accompanied with reduced GPX4 but elevated Cx43 expression, MDA production and ferroptosis. Cx43 gene knockdown in H9C2 resulted in a significant increase in GPX4, reduction in MDA and ferroptosis, and subsequently reduced post-hypoxic cell viability. The beneficial effects of Cx43 gene knock-down was minified or eliminated by Erastin. It is concluded that Cx43 overexpression exacerbates MIRI under diabetic conditions via promoting ferroptosis, while its down-regulation at early state of diabetes is attributable to enhanced myocardial tolerance to MIRI.

High­fat diet­induced LCN2 exacerbates myocardial ischemia­reperfusion injury by enhancing platelet activation.

Following acute myocardial infarction, the recovery of blood flow leads to myocardial ischemia­reperfusion (MI/R) injury, which is primarily characterized by the activation of inflammatory signals, microvascular obstruction, increased oxidative stress and excessive Ca2+ overload. It has also been demonstrated that platelets can exacerbate MI/R injury by releasing reactive oxygen species, inflammatory factors and chemokines, while also obstructing microvessels through thrombus formation. As a bioactive molecule with proinflammatory and chemotactic properties, lipocalin 2 (LCN2) exhibits a positive correlation with obesity, hyperglycemia, hypertriglyceridemia and insulin resistance index, which are all significant risk factors for ischemic cardiomyopathy. Notably, the potential role of LCN2 in promoting atherosclerosis may be related to its influence on the function of macrophages, smooth muscle cells and endothelial cells, but its effect on platelet function has not yet been reported. In the present study, the effect of a high­fat diet (HFD) on LCN2 expression was determined by detecting LCN2 expression levels in the liver and serum samples of mice through reverse transcription­quantitative PCR and enzyme linked immunosorbent assay, respectively. The effect of LCN2 on platelet function was evaluated by examining whether LCN2 affected platelet activation, aggregation, adhesion, clot retraction and P­selectin expression. To determine whether LCN2 aggravated MI/R injury in HFD­fed mice by affecting platelet and inflammatory cell recruitment, wild­type and LCN2 knockout mice fed a HFD were subjected to MI/R injury, then hearts were collected for hematoxylin and eosin staining and 2,3,5­triphenyltetrazolium chloride staining, and immunohistochemistry was employed to detect the expression of CD42b, Ly6G, CD3 and B220. Based on observing the upregulation of LCN2 expression in mice fed a HFD, the present study further confirmed that LCN2 could accelerate platelet activation, aggregation and adhesion. Moreover, in vivo studies validated that knockout of LCN2 not only mitigated MI/R injury, but also inhibited the recruitment of platelets and inflammatory cells in myocardial tissue following ischemia­reperfusion. In conclusion, the current findings suggested that the effect of HFD­induced LCN2 on aggravating MI/R injury may totally or partially dependent on its promotion of platelet function.

SIRT5 induces autophagy and alleviates myocardial infarction via desuccinylation of TOM1.

Myocardial infarction (MI) is a prevalent form of ischemic heart disease, significantly contributing to heart disease-related deaths worldwide. This condition is primarily caused by myocardial ischemic-reperfusion injury (MIRI). Sirtuin 5 (SIRT5) is a desuccinylase known for its ability to reduce protein succinylation. Recent studies have highlighted the potential role of SIRT5 in various human diseases, including MIRI. This study aims to investigate the specific role of SIRT5 in modulating autophagy and cardiomyocyte death in a MIRI model, as well as to identify the downstream protein targets of SIRT5. Initially, we established a hypoxia/reoxygenation (H/R)-induced MIRI cell model to measure SIRT5 expression and assess its functions. Our results indicated that H/R induction led to a downregulation of SIRT5 expression, decreased autophagy, and increased cell death. Notably, overexpression of SIRT5 effectively promoted autophagy and inhibited cell death in the MIRI cell model. Mechanistically, SIRT5 was found to directly interact with the target of myb1 membrane trafficking protein (TOM1) at the K48 site, inducing its desuccinylation and stabilization. Further rescue assays revealed that TOM1 knockdown reversed the changes in autophagy and apoptosis caused by SIRT5 overexpression in the MIRI cell model. In vivo experiments demonstrated that SIRT5 alleviated myocardial injury in MI models. In conclusion, this study uncovers the role of SIRT5-mediated desuccinylation of TOM1 in regulating autophagy-related cell death in MIRI, providing new insights into potential therapeutic strategies for MI.

Sevoflurane Affects Myocardial Autophagy Levels After Myocardial Ischemia Reperfusion Injury via the microRNA-542-3p/ADAM9 Axis.

This research focused on investigating the effects of sevoflurane (Sev) on myocardial autophagy levels after myocardial ischemia reperfusion (I/R) injury via the microRNA-542-3p (miR-542-3p)/ADAM9 axis. Mice underwent 30 min occlusion of the left anterior descending coronary (LAD) followed by 2 h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by echocardiography. Cardiac markers and oxidative stress factors were evaluated by ELISA. Autophagy-associated factors were detected by western blot. Relationship between miR-542-3p and ADAM9 was tested by dual-luciferase reporter gene assay, RT-qPCR, and western blot. Sev treatment ameliorated cardiac dysfunction, myocardial oxidative stress, and histopathological damages, decreased myocardial infarction size and myocardial apoptotic cells after myocardial I/R injury. Sev treatment elevated miR-542-3p expression and decreased ADAM9 expression in myocardial tissues after myocardial I/R injury. miR-542-3p overexpression could enhance the ameliorative effects of Sev on myocardial injury and myocardial autophagy in I/R mice. miR-542-3p targeted and negatively regulated ADAM9 expression. ADAM9 overexpression reversed the ameliorative effects of miR-542-3p up-regulation on myocardial injury and myocardial autophagy in Sev-treated I/R mice. Sev treatment could ameliorate myocardial injury and myocardial autophagy in I/R mice, mediated by mechanisms that include miR-542-3p up-regulation and ADAM9 down-regulation.

Kynurenic acid protects against ischemia/reperfusion injury by modulating apoptosis in cardiomyocytes.

Acute myocardial infarction, often associated with ischemia/reperfusion injury (I/R), is a leading cause of death worldwide. Although the endogenous tryptophan metabolite kynurenic acid (KYNA) has been shown to exert protection against I/R injury, its mechanism of action at the cellular and molecular level is not well understood yet. Therefore, we examined the potential involvement of antiapoptotic mechanisms, as well as N-methyl-D-aspartate (NMDA) receptor modulation in the protective effect of KYNA in cardiac cells exposed to simulated I/R (SI/R). KYNA was shown to attenuate cell death induced by SI/R dose-dependently in H9c2 cells or primary rat cardiomyocytes. Analysis of morphological and molecular markers of apoptosis (i.e., membrane blebbing, apoptotic nuclear morphology, DNA double-strand breaks, activation of caspases) revealed considerably increased apoptotic activity in cardiac cells undergoing SI/R. The investigated apoptotic markers were substantially improved by treatment with the cytoprotective dose of KYNA. Although cardiac cells were shown to express NMDA receptors, another NMDA antagonist structurally different from KYNA was unable to protect against SI/R-induced cell death. Our findings provide evidence that the protective effect of KYNA against SI/R-induced cardiac cell injury involves antiapoptotic mechanisms, that seem to evoke independently of NMDA receptor signaling.

Shexiang Tongxin dropping pill ameliorates microvascular obstruction via downregulating ALOX12 after myocardial ischemia-reperfusion.

BACKGROUND: Microvascular dysfunction (MVD) is common in patients with myocardial infarction receiving reperfusion therapy and is associated with adverse cardiac prognosis. Accumulating evidence suggests a protective role of Shexiang Tongxin dropping pill (STDP) in MVD. However, the specific effects and the underlying mechanisms of STDP in the context of MVD after myocardial ischemia-reperfusion (IR) remains unclear. AIMS: We aimed to elucidate the role of STDP in MVD induced by IR and the potential mechanisms involved. METHODS: Mice were orally administered with STDP or normal saline for 5 days before receiving myocardial IR. Cardiac function and microvascular obstruction was measured. Proteomics and single-cell RNA sequencing was performed on mouse hearts. In vitro hyoxia/reoxygenation model was established on mouse cardiac microvascular endothelial cells (MCMECs). RESULTS: STDP improved cardiac function and decreased microvascular obstruction (MVO) in mice after myocardial IR. Proteomics identified ALOX12 as an important target of STDP. Single-cell RNA sequencing further revealed that downregulation of ALOX12 by STDP mainly occurred in endothelial cells. The involvement of ALOX12 in the effect of STDP on MVO was validated by manipulating ALOX12 via endothelial-specific adeno-associated virus transfection in vivo and in vitro. In vivo, overexpression of ALOX12 increased whereas knockdown of ALOX12 decreased MVO and thrombus formation. STDP treatment alleviated the detrimental effects of overexpression of ALOX12. In vitro, overexpression of ALOX12 increased endothelial cell inflammation and platelet adhesion to endothelial cells, which was abolished by STDP treatment. CONCLUSION: Our findings suggest that STDP alleviates MVO after IR, with ALOX12 playing a crucial role.

ß3-Adrenergic receptor overexpression in cardiomyocytes preconditions mitochondria to withstand ischemia-reperfusion injury.

ß3-Adrenergic receptor (ß3AR) agonists have been shown to protect against ischemia-reperfusion injury (IRI). Since ß3ARs are present both in cardiomyocytes and in endothelial cells, the cellular compartment responsible for this protection has remained unknown. Using transgenic mice constitutively expressing the human ß3AR (hß3AR) in cardiomyocytes or in the endothelium on a genetic background of null endogenous ß3AR expression, we show that only cardiomyocyte expression protects against IRI (45 min ischemia followed by reperfusion over 24 h). Infarct size was also limited after ischemia-reperfusion in mice with cardiomyocyte hß3AR overexpression on top of endogenous ß3AR expression. hß3AR overexpression in these mice reduced IRI-induced cardiac fibrosis and improved long-term left ventricular systolic function. Cardiomyocyte-specific ß3AR overexpression resulted in a baseline remodeling of the mitochondrial network, characterized by upregulated mitochondrial biogenesis and a downregulation of mitochondrial quality control (mitophagy), resulting in elevated numbers of small mitochondria with a depressed capacity for the generation of reactive oxygen species but improved capacity for ATP generation. These processes precondition cardiomyocyte mitochondria to be more resistant to IRI. Upon reperfusion, hearts with hß3AR overexpression display a restoration in the mitochondrial quality control and a rapid activation of antioxidant responses. Strong protection against IRI was also observed in mice infected with an adeno-associated virus (AAV) encoding hß3AR under a cardiomyocyte-specific promoter. These results confirm the translational potential of increased cardiomyocyte ß3AR expression, achieved either naturally through exercise or artificially through gene therapy approaches, to precondition the cardiomyocyte mitochondrial network to withstand future insults.

Weighted gene co-expression network analysis and single-cell sequence analysis uncover immune landscape and reveal hub genes of necroptosis in macrophages in myocardial ischaemia-reperfusion injury.

Myocardial ischaemia-reperfusion injury (MIRI) caused by the treatment of acute myocardial infarction (AMI) is the primary cause of severe ventricular remodelling, heart failure (HF), and high mortality. In recent studies, research on the role of necroptosis in MIRI has focused on cardiomyocytes, but new biomarkers and immunocyte mechanisms of necroptosis are rarely studied. In the present study, weighted gene co-expression network analysis (WGCNA) algorithms were used to establish a weighted gene co-expression network, and Casp1, Hpse, Myd88, Ripk1, and Tpm3 were identified as biological markers of necroptosis using least absolute shrinkage, selection operator (LASSO) regression and support vector machine (SVM) feature selection algorithms. The role and discriminatory power of these five genes in MIRI had never been studied. Single-cell and cell-talk analyses showed that hub genes of necroptosis were focused on macrophages, which mediate the functions of monocytes, fibroblasts, haematopoietic stem cells, and cardiomyocytes, primarily through the TNF/TNFRSF1A interaction. The polarisation and functional activation of macrophages were affected by the MIF signalling network (MIF CD74/CXCR4 and MIF CD74/CD44) of other cells. The results of the immune infiltration assay showed that the five genes involved in necroptosis were significantly related to the infiltration and functional activity of M2 macrophages. TWS-119 is predicted to be a molecular drug that targets key MIRI genes. A mouse model was established to confirm the expression of five hub genes, and ventricular remodelling increased with time after ischaemia-reperfusion injury (IRI). Therefore, Casp1, Hpse, Myd88, Ripk1, and Tpm3 may be key genes regulating necroptosis and polarisation in macrophages, and causing ventricular remodelling.

Disruption of BACH1 Protects AC16 Cardiomyocytes Against Hypoxia/Reoxygenation-Evoked Injury by Diminishing CDKN3 Transcription.

Reperfusion after myocardial infarction (MI) can lead to myocardial ischemia/reperfusion (I/R) damage. The transcription factor (TF) broad-complex, tramtrack, and bric-a-brac (BTB) and cap'n'collar (CNC) homology 1 (BACH1) is implicated in the injury. However, the downstream mechanisms of BACH1 in affecting myocardial hypoxia/reoxygenation (H/R) damage are still fully understood. AC16 cells were stimulated with H/R conditions to model cardiomyocytes under H/R. mRNA analysis was performed by quantitative real-time PCR. Protein levels were gauged by immunoblot analysis. The effect of BACH1/cyclin-dependent kinase inhibitor 3 (CDKN3) on H/R-evoked injury was assessed by measuring cell viability via Cell Counting Kit-8 (CCK-8), apoptosis (flow cytometry and caspase 3 activity), ferroptosis via Fe2+, glutathione (GSH), reactive oxygen species (ROS) and malondialdehyde (MDA) markers and inflammation cytokines interleukin-1beta (IL-1ß) and tumor necrosis factor alpha (TNF-α). The BACH1/CDKN3 relationship was examined by chromatin immunoprecipitation (ChIP) experiment and luciferase assay. BACH1 was increased in MI serum and H/R-stimulated AC16 cardiomyocytes. Functionally, disruption of BACH1 mitigated H/R-evoked in vitro apoptosis, ferroptosis and inflammation of AC16 cardiomyocytes. Mechanistically, BACH1 activated CDKN3 transcription and enhanced CDKN3 protein expression in AC16 cardiomyocytes. Our rescue experiments validated that BACH1 disruption attenuated H/R-evoked AC16 cardiomyocyte apoptosis, ferroptosis and inflammation by downregulating CDKN3. Additionally, BACH1 disruption could activate the adenosine monophosphate-activated protein kinase (AMPK) signaling by downregulating CDKN3 in H/R-stimulated AC16 cardiomyocytes. Our study demonstrates that BACH1 activates CDKN3 transcription to induce H/R-evoked damage of AC16 cardiomyocytes partially via AMPK signaling.

Role of M6a Methylation in Myocardial Ischemia-Reperfusion Injury and Doxorubicin-Induced Cardiotoxicity.

Cardiovascular disease remains the leading cause of death worldwide, with acute myocardial infarction and anticancer drug-induced cardiotoxicity being the significant factors. The most effective treatment for acute myocardial infarction is rapid restoration of coronary blood flow by thrombolytic therapy or percutaneous coronary intervention. However, myocardial ischemia-reperfusion injury (MI/RI) after reperfusion therapy is common in acute myocardial infarction, thus affecting the prognosis of patients with acute myocardial infarction. There is no effective treatment for MI/RI. Anthracyclines such as Doxorubicin (DOX) have limited clinical use due to their cardiotoxicity, and the mechanism of DOX-induced cardiac injury is complex and not yet fully understood. N6-methyladenosine (m6A) plays a crucial role in many biological processes. Emerging evidence suggests that m6A methylation plays a critical regulatory role in MI/RI and DOX-induced cardiotoxicity (DIC), suggesting that m6A may serve as a novel biomarker and therapeutic target for MI/RI and DIC. M6A methylation may mediate the pathophysiological processes of MI/RI and DIC by regulating cellular autophagy, apoptosis, oxidative stress, and inflammatory response. In this paper, we first focus on the relationship between m6A methylation and MI/RI, then further elucidate that m6A methylation may mediate the pathophysiological process of MI/RI through the regulation of cellular autophagy, apoptosis, oxidative stress, and inflammatory response. Finally, briefly outline the roles played by m6A in DIC, which will provide a new methodology and direction for the research and treatment of MI/RI and DIC.

Mst1-mediated phosphorylation of FoxO1 and C/EBP-ß stimulates cell-protective mechanisms in cardiomyocytes.

The molecular mechanisms by which FoxO transcription factors mediate diametrically opposite cellular responses, namely death and survival, remain unknown. Here we show that Mst1 phosphorylates FoxO1 Ser209/Ser215/Ser218/Thr228/Ser232/Ser243, thereby inhibiting FoxO1-mediated transcription of proapoptotic genes. On the other hand, Mst1 increases FoxO1-C/EBP-ß interaction and activates C/EBP-ß by phosphorylating it at Thr299, thereby promoting transcription of prosurvival genes. Myocardial ischemia/reperfusion injury is larger in cardiac-specific FoxO1 knockout mice than in control mice. However, the concurrent presence of a C/EBP-ß T299E phospho-mimetic mutation reduces infarct size in cardiac-specific FoxO1 knockout mice. The C/EBP-ß phospho-mimetic mutant exhibits greater binding to the promoter of prosurvival genes than wild type C/EBP-ß. In conclusion, phosphorylation of FoxO1 by Mst1 inhibits binding of FoxO1 to pro-apoptotic gene promoters but enhances its binding to C/EBP-ß, phosphorylation of C/EBP-ß, and transcription of prosurvival genes, which stimulate protective mechanisms in the heart.

Valtrate Suppresses TNFSF14-Mediated Arrhythmia After Myocardial Ischemia-Reperfusion by Inducing N-linked Glycosylation of LTßR to Regulate MGA/MAX/c-Myc/Cx43.

ABSTRACT: Myocardial ischemia-reperfusion (MIR)-induced arrhythmia remains a major cause of death in patients with cardiovascular diseases. The reduction of Cx43 has been known as a major inducer of arrhythmias after MIR, but the reason for the reduction of Cx43 remains largely unknown. The aim of this study was to find the key mechanism underlying the reduction of Cx43 after MIR and to screen out an herbal extract to attenuate arrhythmia after MIR. The differentially expressed genes in the peripheral blood mononuclear cell (PBMCs) after MIR were analyzed using the data from several gene expression omnibus data sets, followed by the identification in PBMCs and the serum of patients with myocardial infarction. Tumor necrosis factor superfamily protein 14 (TNFSF14) was increased in PBMCs and the serum of patients, which might be associated with the injury after MIR. The toxic effects of TNFSF14 on cardiomyocytes were investigated in vitro . Valtrate was screened out from several herbal extracts. Its protection against TNFSF14-induced injury was evaluated in cardiomyocytes and animal models with MIR. Recombinant TNFSF14 protein not only suppressed the viability of cardiomyocytes but also decreased Cx43 by stimulating the receptor LTßR. LTßR induces the competitive binding of MAX to MGA rather than the transcriptional factor c-Myc, thereby suppressing c-Myc-mediated transcription of Cx43. Valtrate promoted the N-linked glycosylation modification of LTßR, which reversed TNFSF14-induced reduction of Cx43 and attenuated arrhythmia after MIR. In all, valtrate suppresses TNFSF14-induced reduction of Cx43, thereby attenuating arrhythmia after MIR.

Isoflurane preconditioning attenuates OGD/R-induced cardiomyocyte cytotoxicity by regulating the miR-210/BNIP3 axis.

Isoflurane, a commonly used inhaled anesthetic, has been found to have a cardioprotective effect. However, the precise mechanisms have not been fully elucidated. Here, we found that isoflurane preconditioning enhanced OGD/R-induced upregulation of miR-210, a hypoxia-responsive miRNA, in AC16 human myocardial cells. To further test the roles of miR-210 in regulating the effects of isoflurane preconditioning on OGD/R-induced cardiomyocyte injury, AC16 cells were transfected with anti-miR-210 or control anti-miRNA. Results showed that isoflurane preconditioning attenuated OGD/R-induced cardiomyocyte cytotoxicity (as assessed by cell viability, LDH and CK-MB levels), which could be reversed by anti-miR-210. Isoflurane preconditioning also prevented OGD/R-induced increase in apoptotic rate, caspase-3 and caspase-9 activities, and Bax level and decrease in Bcl-2 expression level, while anti-miR-210 blocked these effects. We also found that anti-miR-210 prevented the inhibitory effects of isoflurane preconditioning on OGD/R-induced decrease in adenosine triphosphate content; mitochondrial volume; citrate synthase activity; complex I, II, and IV activities; and p-DRP1 and MFN2 expression. Besides, the expression of BNIP3, a reported direct target of miR-210, was significantly decreased under hypoxia condition and could be regulated by isoflurane preconditioning. In addition, BNIP3 knockdown attenuated the effects of miR-210 silencing on the cytoprotection of isoflurane preconditioning. These findings suggested that isoflurane preconditioning exerted protective effects against OGD/R-induced cardiac cytotoxicity by regulating the miR-210/BNIP3 axis.

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.

Identification of biomarkers related to angiogenesis in myocardial ischemia-reperfusion injury and prediction of potential drugs.

Myocardial ischemia-reperfusion injury (MIRI) refers to the secondary damage to myocardial tissue that occurs when blood perfusion is rapidly restored following myocardial ischemia. This process often exacerbates the injury to myocardial fiber structure and function. The activation mechanism of angiogenesis is closely related to MIRI and plays a significant role in the occurrence and progression of ischemic injury. In this study, we utilized sequencing data from the GEO database and employed WGCNA, Mfuzz cluster analysis, and protein interaction network to identify Stat3, Rela, and Ubb as hub genes involved in MIRI-angiogenesis. Additionally, the GO and KEGG analysis of differentially expressed genes highlighted their broad participation in inflammatory responses and associated signaling pathways. Moreover, the analysis of sequencing data and hub genes revealed a notable increase in the infiltration ratio of monocytes and activated mast cells. By establishing key cell ROC curves, using independent datasets, and validating the expression of hub genes, we demonstrated their high diagnostic value. Moreover, by scrutinizing single-cell sequencing data alongside trajectory analysis, it has come to light that Stat3 and Rela exhibit predominant expression within Dendritic cells. In contrast, Ubb demonstrates expression across multiple cell types, with all three genes being expressed at distinct stages of cellular development. Lastly, leveraging the CMap database, we predicted potential small molecule compounds for the identified hub genes and validated their binding activity through molecular docking. Ultimately, our research provides valuable evidence and references for the early diagnosis and treatment of MIRI from the perspective of angiogenesis.