Coronary Artery Ligation Research Articles

Sodium-Glucose Cotransporter 2 inhibitor Empagliflozin Enhances Autophagy and Reverses Remodeling in Hearts with Large, Old Myocardial Infarctions.

Large clinical trials recently showed that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve the prognosis of heart failure patients with or without diabetes. Using a mouse model of large myocardial infarction, we investigated the therapeutic effects and underlying molecular mechanisms of the highly selective SGLT2 inhibitor empagliflozin in heart failure. Four weeks after myocardial infarction induced by left coronary artery ligation, the surviving mice were assigned to vehicle or empagliflozin groups and treated for 8 weeks. Empagliflozin did not alter body weight, blood pressure, glycohemoglobin, blood glucose or beta-hydroxybutyrate levels but significantly attenuated cardiac dysfunction and left ventricular dilatation (remodeling). Hearts from empagliflozin-treated mice showed less fibrosis, less cardiomyocyte hypertrophy, and lower myocardial ANP levels than those from vehicle-treated mice. Autophagy was augmented in cardiomyocytes from empagliflozin-treated mice, as indicated by increased myocardial microtubule-associated protein-1 LC3 (light chain 3)-II levels and LC-3-II/I ratio as well as increased levels of cathepsin D and ATP. Additionally, numerous autophagic vacuoles and lysosomes were observed, accompanied by increased AMP-activated protein kinase (AMPK) phosphorylation and suppression of mammalian target of rapamycin phosphorylation. Myocardial sodium-hydrogen antiporter (NHE)-1 expression was increased in infarcted mice, and that effect was unchanged by empagliflozin. In vitro, empagliflozin increased autophagic flux and induced an intracellular pH drop, AMPK activation and ATP production in cardiomyocytes. These effects were similar to those of the NHE-1 inhibitor cariporide, suggesting a possibility that they both act on the same pathway. Empagliflozin is a beneficial pharmacological tool that enhances autophagy to reverse remodeling in the postinfarction heart.

Left superior cervical ganglia lymph node mimicry and its role in rat ventricular arrhythmias following myocardial infarction.

Sympathetic overactivation may lead to severe ventricular arrhythmias (VAs) post-myocardial infarction (MI). The superior cervical ganglion (SCG) is an extracardiac sympathetic ganglion which regulates cardiac autonomic tone. We aimed to investigate the characteristics and functional significance of SCG on neuro-cardiac communication post-MI. Constructed MI rat model by left anterior descending coronary artery ligation, and electrophysiological, SCG sympathetic nerve activity testing, echocardiography and histology study were performed. The proteins and gene expression were detected using RNA-seq, spatial transcriptomics, quantitative PCR, and western blotting. The SCG neuronal remodeling was recognized by significant increase in adrenergic tyrosine hydroxylase (TH) (+) neurons and decrease in neuronal size. Top differentially expressed genes enriched in pro-inflammatory profile and nerve regulatory factor in left SCG (LSCG) post-MI. Interleukin (IL)-1β and IL-6 increased significantly at Day 3, ahead of nerve growth factor (NGF) which peaked at Day 7 post-MI. Spatial transcriptomics further identified the relativity of TH enrichment with macrophages and cytokines. Therapeutic LSCG-ectomy successfully triggered cardiac denervation and improved VA vulnerability. Eventually, cardiac denervation attenuated macrophage/mast cell infiltration at para-infarct regions, thus improved cardiac dysfunction. Mechanism study revealed that genetic knockdown of NGF receptor trkA in LSCG reversed sympathetic remodeling and cardiac inflammation, which may be partially mediated by substance P and calcitonin gene-related peptide (CGRP). Extracardiac sympathetic LSCG remodeling participated in arrhythmogenesis and cardiac inflammation/function post-MI. NGF bridged neuro-immune crosstalk between pro-inflammatory shifting and sympathetic overdrive. Targeting LSCG modification facilitated cardiac protection and prevented VAs post-MI.

Leptin drives glucose metabolism to promote cardiac protection via OPA1-mediated HDAC5 translocation and Glut4 transcription

Metabolic reprogramming, the shifting from fatty acid oxidation to glucose utilization, improves cardiac function as heart failure (HF) progresses. Leptin plays an essential role in regulating glucose metabolism. However, the crosstalk between leptin and metabolic reprogramming is poorly understood. We tested the hypothesis that leptin improves cardiac function after myocardial infarction via enhancing glucose metabolism. In the isoproterenol (ISO)-induced heart failure model in vitro, H9c2 cell apoptosis was assessed by the TUNEL and Annexin V/PI staining assay. Leptin-mediated mitochondrial fusion was performed via TEM, and glucose oxidation was explored, as well as the ECAR, OCR, and protein expression of the vital metabolic enzymes. By blocking OPA1 expression or HDAC5 inhibition, the mitochondrial dynamic and glucose metabolic were detected to evaluate the role of OPA1 and HDAC5 in leptin-stimulated glucose metabolism. In the mouse model of HF in vivo, intraperitoneal leptin administration appreciably increased glucose oxidation and preserved cardiac function 56 days after coronary artery ligation. In vitro, we identified the OPA1-dependent HDAC5 nucleus export as a crucial process in boosting glucose utilization by activating MEF2 to upregulate Glut4 expression using the RNA interference technique in H9c2 cells. In vivo, leptin promotes glucose utilization and confers heart functional and survival benefits in chronic ischemic HF. The current study provided a novel insight into the role of leptin in metabolic reprogramming and revealed potential therapeutic targets for chronic HF.

Dichloroacetate: A metabolic game-changer in alleviating macrophage inflammation and enhancing recovery after myocardial infarction.

Dichloroacetate (DCA) has shown potential in modulating cellular metabolism and inflammation, particularly in cardiac conditions. This study investigates DCA's protective effects in a mouse model of myocardial infarction (MI), focusing on its ability to enhance cardiac function, reduce inflammation, and shift macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. An acute MI model was created using left anterior descending coronary artery ligation. Mice were assigned to four groups: normal control, MI control, MI + 50 mM DCA, and MI + 100 mM DCA. Cardiac fibrosis and injury were assessed through H&E staining. Cardiac function was evaluated via echocardiography, and serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were measured. Inflammation and apoptosis were analyzed through immunohistochemistry, ELISA, western blotting, and flow cytometry in heart tissue and RAW264.7 cells. Additionally, macrophage polarization and relevant signaling pathways were examined. DCA significantly improved cardiac function in MI mice, evidenced by reduced myocardial injury and lower CK-MB and LDH levels. It also decreased inflammatory cytokines (TNF-α, IL-6 and IL-1β) and facilitated macrophage polarization from M1 to M2. Western blotting revealed that DCA inhibited iNOS and COX2 while enhancing Arg1 expression, alongside improved mitochondrial function and reduced apoptosis. Additionally, by injecting AAV-PDHK4 (pyruvate dehydrogenase kinase) into MI mice, we found that DCA effectively inhibited the progression of MI through the suppression of PDHK4. DCA protects against myocardial infarction by enhancing cardiac function, reducing inflammation, and promoting macrophage polarization, likely through inhibition of PDHK4 and NF-κB pathways, positioning it as a potential therapeutic strategy for cardiac repair post-MI.

Melatonin inhibits ferroptosis through the ATF3/GPX4 signaling pathway to relieve myocardial ischemia-reperfusion injury in rats.

Melatonin (MEL), functioning as a circulating hormone, is important for the regulation of ferroptosis in different health scenarios and acts as a crucial antioxidant in cardiovascular diseases. However, its specific function in ferroptosis related to myocardial ischemia-reperfusion injury (MIRI) remains to be fully elucidated. In our research, we utilized a rat model of MIRI induced by coronary artery ligation, along with a cell model subjected to hypoxia/reoxygenation (H/R). We evaluated relevant genes and proteins by real-time fluorescent quantitative PCR and Western blot analysis. To evaluate myocardial tissue damage and cell injury, we employed cell counting kit-8 assays, flow cytometry, hematoxylin-eosin staining, and 2,3,5-triphenyltetrazolium chloride staining techniques. Our results show that administering MEL notably reduces the concentrations of cTnT, CK-MB, and lactate dehydrogenase in the serum of MIRI rats, mitigates the extent of myocardial infarction, improves the recovery of pathological conditions in myocardial tissues, and reduces the concentrations of Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) in the myocardial tissue, while also promoting increased glutathione levels. Moreover, MEL can also restore the reduced viability of H9C2 cells caused by H/R or ferroptosis inducers (RSL3), reduce the cellular content of Fe2+, MDA, and ROS, and inhibit ferroptosis. Mechanistically, MEL promotes the expression of GPX4 by downregulating the expression of ATF3, thereby inhibiting ferroptosis in cardiomyocytes and ultimately alleviating the process of MIRI. Our study demonstrates that MEL ameliorates MIRI by inhibiting ferroptosis.

Extracellular vesicle-mediated VEGF-A mRNA delivery rescues ischaemic injury with low immunogenicity.

Lackluster results from recently completed gene therapy clinical trials of VEGF-A delivered by viral vectors have heightened the need to develop alternative delivery strategies. This study aims to demonstrate the pre-clinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischaemic vascular disease. After encapsulation of full-length VEGF-A mRNA into fibroblast-derived EVs via cellular nanoporation (CNP), collected VEGF-A EVs were delivered into mouse models of ischaemic injury. Target tissue delivery was verified by in situ analysis of protein and gene expression. Functional rescue was confirmed by in vivo imaging and histology. The safety of single and serial delivery was demonstrated using immune-based assays. VEGF-A EVs were generated with high mRNA content using a CNP methodology. VEGF-A EV administration demonstrated expression of exogenous VEGF-A mRNA by in situ RNA hybridization and elevated protein expression by western blot, microscopy, and enzyme-linked immunosorbent assay. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischaemic tissues with increased arterial perfusion and improvement in left ventricular function, respectively. Serial delivery of VEGF-EVs in injured skin showed improved wound healing with repeat administration. Importantly, as compared with adeno-associated viral and lipid nanoparticle VEGF-A gene therapy modalities, murine VEGF-A EV delivery did not trigger innate or adaptive immune responses at the injection site or systemically. This study demonstrated that VEGF-A EV therapy offers efficient, dose-dependent VEGF-A protein formation with low immunogenicity, resulting in new vessel formation in murine models of ischaemic vascular disease.

Inflammatory adhesion mediates myocardial segmental necroptosis induced by mixed lineage kinase domain-like protein in acute myocardial infarction

PurposeCardiomyocyte death is a major cytopathologic response in acute myocardial infarction (AMI) and involves complex inflammatory interactions. Although existing reports indicating that mixed lineage kinase domain-like protein (MLKL) is involved in macrophage necroptosis and inflammasome activation, the downstream mechanism of MLKL in necroptosis remain poorly characterized in AMI.MethodsMLKL knockout mice (MLKLKO), RIPK3 knockout mice (RIPK3KO), and macrophage-specific MLKL conditional knockout mice (MLKLM−KO) were established. AMI was induced by coronary artery ligation. The role of MLKL in regulating myocardial morphological necroptosis was evaluated using immunofluorescence staining, flow cytometry, qRT-PCR, Western blot, CCK-8 assay, and ELISA.ResultsOur findings revealed that myocardial segmental necroptosis (MSN), a unique morphological characteristics of cell death observed post-AMI, was promoted by intercellular inflammatory adhesion mediated by MLKL. The key features of MSN included localized cytomembrane perforation, segmental attenuation of myofilaments, MLKL-mediated filling, and macrophage inflammatory adhesion. In a mouse model of AMI, we observed MSN, which was absent in immunosuppressed mice. Pharmacological depletion of macrophages or genetic knockout of macrophage-specific MLKL (MLKLM−KO) reduced the occurrence of MSN. This reduction was reversed upon reinfusion of wild-type macrophages. Additionally, myocardial injury was significantly ameliorated in MLKLM−KO mice following AMI. In a macrophage-cardiomyocyte co-culture system, MLKLM−KO attenuated hypoxia-induced MSN and inhibited macrophage-mediated inflammatory adhesion. Furthermore, MLKL was found to trigger the formation of membrane pores and the polymerization of integrin αvβ1, thereby enhancing inflammatory adhesion in the co-culture system. Notably, MLKL-enhanced inflammatory adhesion was not entirely dependent on RIPK3.ConclusionOur study demonstrates that MLKL is directly involved in myocardial segmental necroptosis by interacting with macrophages through inflammatory adhesion, and possibly independently of RIPK3.

Mechanistic Insights into Melatonin's Antiarrhythmic Effects in Acute Ischemia-Reperfusion-Injured Rabbit Hearts Undergoing Therapeutic Hypothermia.

The electrophysiological mechanisms underlying melatonin's actions and the electrophysiological consequences of superimposed therapeutic hypothermia (TH) in preventing cardiac ischemia-reperfusion (IR) injury-induced arrhythmias remain largely unknown. This study aimed to unveil these issues using acute IR-injured hearts. Rabbits were divided into heart failure (HF), HF+melatonin, control, and control+melatonin groups. HF was induced by rapid right ventricular pacing. Melatonin was administered orally (10 mg/kg/day) for four weeks, and IR was created by 60-min coronary artery ligation and 30-min reperfusion. The hearts were then excised and Langendorff-perfused for optical mapping studies at normothermia, followed by TH. Melatonin significantly reduced ventricular fibrillation (VF) maintenance. In failing hearts, melatonin reduced the spatially discordant alternans (SDA) inducibility mainly by modulating intracellular Ca2+ dynamics via upregulation of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and calsequestrin 2 and attenuating the downregulation of phosphorylated phospholamban protein expression. In control hearts, melatonin improved conduction slowing and reduced dispersion of action potential duration (APDdispersion) by upregulating phosphorylated connexin 43, attenuating the downregulation of SERCA2a and phosphorylated phospholamban and attenuating the upregulation of phosphorylated Ca2+/calmodulin-dependent protein kinase II. TH significantly retarded intracellular Ca2+ decay slowed conduction, and increased APDdispersion, thereby facilitating SDA induction, which counteracted the beneficial effects of melatonin in reducing VF maintenance.

The Immune System: An Arrow to the Heart and Principles of Cardioimmunology as an Emerging Branch of Medicine.

Cardioimmunology is an emerging branch of medicine whose development has been facilitated by more sophisticated diagnostic procedures. Recent studies have mainly focused on the immune response during myocardial infarction (MI), and there is evidence that both resident and external immune cells participate in acute inflammatory disease, as well as tissue remodeling. Cardiac Innate Immune Cells: Following MI, macrophages, dendritic cells (DCs) and mast cells (MCs) are the main players in the heart. Under steady-state conditions, cardiac resident macrophages (CRMs) protect the heart against stress and infectious events, being involved in cell and matrix turnover, as well as phagocytosis of apoptotic cells. Moreover, CRMs contribute to the resolution of inflammation via release of interleukin (IL)-10, and efferocytosis of dying cells. Conversely, CCR2+ monocyte-derived macrophages promote inflammation in the acute phase of myocardial damage, with the release of pro-inflammatory cytokines. Conventional (c) DCs possess enhanced capacity to present antigens to T lymphocytes. In MI patient autopsies, massive infiltration of T helper (Th) cells and CDs has been detected in the myocardium. Cardiac MCs play a dual role during MI, with the production of cytokines for early inflammatory response, and the release of anti-inflammatory cytokines, IL10 and IL-13 during the resolution phase. Adaptive Immune Response: In experimental coronary artery ligation, the myocardium is infiltrated with Th1, Th2, Th17, and T regulatory (TREG) cells, which participate in the acute inflammation. In cardiac repair, T cell reparative response is mediated by TREG cells, with improved ventricular remodeling and function post-ischemia. In this review, emphasis will be placed on the innate and adaptive immune response during and post-MI. At the same time, immunotherapy-based cardiac failure following chimeric antigen receptor T-cell and immune checkpoint inhibitory therapy will be pointed out.

Arachidonic acid synergizes with aspirin preventing myocardial ischemia-reperfusion injury and mitigates bleeding risk.

The therapeutic efficacy of coronary revascularization is compromised by myocardial ischemia-reperfusion (MI/R) injury. Higher levels of circulating arachidonic acid (AA) are reportedly associated with lower risk of cardiovascular disease. The cyclooxygenase (COX) pathway metabolizes AA into prostaglandins (PGs) and the platelet-activating thromboxane A2 (TXA2), which is inhibited by aspirin. We aimed to explore whether AA or its combination with aspirin modulates MI/R injury and aspirin-caused gastric bleeding. Mice were subjected to 30min coronary artery ligation followed by reperfusion. AA reduced MI/R injury in mice, and its combination with aspirin provided further cardioprotection. Aspirin inhibited MI/R-triggered platelet activation and ameliorated microvascular obstruction immediately upon reperfusion, whereas AA improved microvascular perfusion at a later stage of reperfusion, coinciding with increased coronary vasodilatation. Co-administration of AA and aspirin markedly reduced cardiac neutrophil infiltration and vascular permeability and improved microcirculation. AA increased urinary metabolites of PGI2 and PGE2, not TXA2, and this selective augmentation was further enhanced by co-treatment with aspirin. Elevation in PGI2 and PGE2 correlated with reduced infarction and improved ventricular function, and inhibiting COX-2 attenuated the synergistic cadioprotection. Furthermore, oral administration of AA with aspirin after reperfusion provided a maximal cardioprotection and abolished aspirin-caused gastric bleeding. AA synergizes with aspirin in protecting against MI/R injury, while minimizing the related bleeding risk, a major concern for patients with acute myocardial infarction. This is attributable to the selective augmentation of PGI2 and PGE2 that is amplified by TXA2 suppression by aspirin, underscoring improved microcirculation and ameliorated inflammation.

Novel Selective Cardiac Myosin-Targeted Inhibitors Alleviate Myocardial Ischaemia–Reperfusion Injury

Abstract Purpose Reperfusion of the ischaemic heart is essential to limit myocardial infarction. However, reperfusion can cause cardiomyocyte hypercontracture. Recently, cardiac myosin-targeted inhibitors (CMIs), such as Mavacamten (MYK-461) and Aficamten (CK-274), have been developed to treat patients with cardiac hypercontractility. These CMIs are well tolerated and safe in clinical trials. We hypothesised that, by limiting hypercontraction, CMIs may reduce hypercontracture and protect hearts in the setting of ischaemia and reperfusion (IR). Methods We investigated the ability of MYK-461 and CK-274 to inhibit hypercontracture of adult rat cardiomyocytes (ARVC) in vitro following ATP depletion. A suitable dose of CMIs for subsequent in vivo IR studies was identified using cardiac echocardiography of healthy male Sprague Dawley rats. Rats were anaesthetized and subject to coronary artery ligation for 30 min followed by 2 h of reperfusion. Prior to reperfusion, CMI or vehicle was administered intraperitoneally. Ischaemic preconditioning (IPC) was used as a positive control group. Infarct size was assessed by tetrazolium chloride staining and extent of hypercontracture was assessed by histological staining. Results Treatment with CMIs inhibited ARVC hypercontracture in vitro. MYK-461 (2 mg/kg) and CK-274 (0.5 mg/kg to 2 mg/kg) significantly reduced infarct size vs. vehicle. IR caused extensive contraction band necrosis, which was reduced significantly by IPC but not by CMIs, likely due to assay limitations. GDC-0326, an inhibitor of PI3Kα, abrogated CK-274-mediated protection following IR injury. GDC-0326 reduced phosphorylation of AKT when administered together with CK-274. Conclusion This study identifies CMIs as novel cardioprotective agents in the setting of IR injury.

Pericarpium Trichosanthis Inhibits TGF-β1-Smad3 Pathway-Induced Cardiac Fibrosis in Heart Failure Rats via Upregulation of microRNA-29b.

Cardiac dysfunction and adverse consequences induced by cardiac fibrosis have been well documented. However, the cardiac fibrosis pathway in chronic heart failure (CHF) remains unclear, and it is therefore necessary to conduct further research for the sake of developing more effective therapeutic strategies for CHF. Some recent studies suggest that Pericarpium Trichosanthis (PT) may help improve the progression of fibrotic diseases. To validate this possibility, we conducted an experiment to evaluate the effect of PT on cardiac fibrosis and explore the hidden mechanism. In the experiment, we induced cardiac fibrosis in rats by left anterior descending (LAD) coronary artery ligation. The findings revealed that PT reduced myocardial fibrosis and increased cardiac activity in CHF rats receiving LAD ligation. In addition, the TGF-β1 level was decreased, and the miR-29b expression was increased in CHF rats after PT treatment. Our invitro experiment also demonstrated that PT treatment suppressed fibroblast activation and collagen synthesis in cardiac fibroblasts stimulated by TGF-β1， and at the same time decreased the TGF-β1 level and increased the miR-29b expression. We further verified that this action was correlated with the TGF-β/Smad3 signaling pathway. We also observe that miR-29b could suppress the TGF-β1 expression, and the suppression of miR-29b weakened the anti-fibrotic effect of PT. This suggests that PT could cure cardiac fibrosis and dysfunction both invitro and invivo via the TGF-β/Smad3 signaling pathway, while miR-29b may participate in this action.

REPOLARIZATION DISPERSION AS A PREDICTOR FOR LIFE-THREATENING VENTRICULAR ARRHYTHMIAS IN SHORT-TERM EXPERIMENTAL DIABETES MELLITUS

Diabetes mellitus (DM) is a well-known risk factor for cardiovascular diseases (CVDs). It can cause myocardial metabolic dysfunction, oxidative stress, inflammation, cardiomyocyte apoptosis, structural remodeling, ventricular dysfunction and lethal arrhythmias. The aim of this study was to find a model for diabetes mellitus increasing the frequency of ventricular arrhythmias induced by myocardial ischemia/reperfusion and to study the electrophysiological properties of the ventricular myocardium in the obtained models. Materials and Methods. The study was conducted on 32 rats with uncontrolled streptozotocin-induced diabetes mellitus lasting 4 (short-term) and 8 (long-term) weeks. The control group consisted of 23 rats. During epicardial mapping, depolarization and repolarization parameters, and ventricular myocardial repolarization dispersion were measured. Left ventricular myocardial ischemia and reperfusion were caused by coronary artery ligation to provoke ventricular tachycardia and/or ventricular fibrillation. Results. The repolarization duration was significantly higher in both groups of rats with diabetes mellitus compared to the control. However, the repolarization dispersion differed from the control only in the group with short-term diabetes mellitus (4 weeks). In this group, reperfusion ventricular arrhythmias developed significantly more often compared with the group with long-term diabetes mellitus (8 weeks). Conclusions: The incidence of reperfusion-induced ventricular tachycardia and/or ventricular fibrillation was higher in rats with short-term diabetes mellitus (4 weeks). It may be due to greater dispersion of left ventricular myocardial repolarization in this group.

Comparative efficacy study of experimental chronic heart failure

Introduction. Combination therapy of chronic heart failure (CHF) is an urgent and important treatment strategy, as it allows to achieve an improvement in the prognosis, optimal control of the symptoms of the disease and an increase in the quality of life of patients.Aim. Comparison of the effectiveness of the malonic acid derivative ethmaben, the sodium-glucose cotransporter inhibitor empagliflozin and their combinations with the variability of the starting drug in experimental post-infarction chronic heart failure in rats.Materials and methods. In a model of post-infarction chronic heart failure, reproduced by permanent ligation of the left coronary artery in rats, an echocardiographic study was used to compare etmaben and empagliflozin monotherapy with a combined regimen of these drugs. The concentration of etmaben in the myocardium was assessed by HPLC.Results and discussion. Antagonism between etmaben and empagliflozin was revealed, manifested by a decrease in the effect of combination therapy in relation to any of the components, a decrease in the concentration of malonic acid derivative in the myocardium under the action of a type 2 sodium-glucose cotransporter inhibitor was shown.Conclusion. Based on the data obtained, additional studies of pharmacokinetics and pharmacodynamics of etmaben were planned.

Activation of Imprinted Gene PW1 Promotes Cardiac Fibrosis After Ischemic Injury.

Cardiac fibrosis, characterized by excessive extracellular matrix (ECM) deposition in the myocardium, is an important target for heart disease treatments. Pw1 (paternally expressed gene 3) is an imprinted gene expressed from the paternal allele, and de novo purine biosynthesis (DNPB) is a crucial pathway for nucleotide synthesis. However, the roles of PW1 and DNPB in ECM production by cardiac fibroblasts during myocardial ischemia are not yet understood. To induce myocardial damage, we performed left anterior descending coronary artery ligation. We generated Pw1CreER-2A-eGFP and Pw12A-CreER knock-in mouse lines to evaluate the expression of the 2 Pw1 alleles in normal and injured hearts. Bisulfite sequencing was used to analyze the DNA methylation of the Pw1 imprinting control region. We identified the phosphoribosylformylglycinamidine synthase (Pfas) gene, encoding the DNPB enzyme PFAS, as a direct target of PW1 using chromatin immunoprecipitation sequencing and real-time quantitative polymerase chain reaction. The role of DNPB in ECM production and cardiac fibrosis after injury was examined in vitro using cultured cardiac fibroblasts and in vivo with Pfas-deficient mice. Our study demonstrates that myocardial infarction reduces DNA methylation at the imprinting control region of the maternally imprinted gene Pw1, triggering a switch from monoallelic imprinting to biallelic expression of Pw1 in cardiac fibroblasts. In activated cardiac fibroblasts, increased Pw1 expression promotes purine biosynthesis and induces ECM production by transcriptionally activating the DNPB factor Pfas. We identified that DNPB is essential for ECM production in activated fibroblasts and that loss of Pfas in fibroblasts limits cardiac fibrosis and improves heart function after injury. This study demonstrates that Pw1 imprinting is disrupted after injury and reveals a novel role for the downstream target PFAS in ECM production and cardiac fibrogenesis. Targeting the PW1/PFAS signaling pathway presents a promising therapeutic strategy for improving cardiac repair after injury.

The Metabolically Resistant Apelin-17 Analogue LIT01-196 Reduces Cardiac Dysfunction and Remodelling in Heart Failure After Myocardial Infarction.

To protect patients after myocardial infarction (MI) and preserve cardiac function, the development of new therapeutics remains an important issue. Apelin, a neuro-vasoactive peptide, increases aqueous diuresis and cardiac contractility while reducing vascular resistance. However, its invivo half-life is very short. We therefore developed a metabolically resistant apelin-17 analogue, LIT01-196 and investigated its effects on cardiac function and remodelling in a murine MI model. The selectivity of LIT01-196 toward the apelin receptor was checked invitro. Its invivo half-life was assessed in male Swiss mice using radioimmunoassay. After permanent coronary artery ligation to induce MI, mice received subcutaneous administration of LIT01-196 (MI+ LIT01-196, 9 mg/kg/d) or saline (MI+ vehicle) for 4 weeks. Left ventricular (LV) function was assessed using echocardiography and Millar (Houston, TX) catheter, vascular density using immunofluorescence, and cardiac fibrosis using Sirius red staining. Real-time quantitative PCR was used to measure mRNA expression of heart failure (HF) fibrosis biomarkers and sarco/endoplasmic reticulum Ca2+-ATPase-2. The invivo half-life of LIT01-196, a specific and selective apelin receptor agonist, was 2.5 hours. MI+ LIT01-196 showed significantly improved LV function, reduced HF biomarkers, and enhanced cardiac contractility and sarco/endoplasmic reticulum Ca2+-ATPase-2 expression compared with MI+ vehicle. LIT01-196 treatment almost doubled cardiac vascular density and maintained LV wall thickness post MI. It also significantly reduced cardiac fibrosis and fibrosis biomarkers, without decreasing arterial blood pressure. Chronic LIT01-196 treatment post MI improves LV function without decreasing blood pressure, increases cardiac vascular density, and reduces cardiac remodelling. This suggests that apelin receptor activation by LIT01-196 might constitute an original pharmacological approach for HF treatment after MI.

Ultrasound-Targeted β-Catenin Gene Therapy Improves the Cardiac Function in Mice After Myocardial Infarction

Gene therapy has received great attention as a therapeutic approach to improve cardiac function post-myocardial infarction (MI), but its limitation lies in the lack of targeting. This study explored the use of ultrasound-targeted microbubble destruction (UTMD) technique to deliver β-catenin gene to the myocardium, aiming to evaluate its efficacy in preventing cardiac dysfunction post-MI. A cationic microbubble solution containing β-catenin gene pcDNA3.1 plasmid was injected through the tail vein at a rate of 0.6 mL/h, and ultrasound beams were delivered to the heart using GE Vivid 7 Medical Ultrasound System M3s Transducer. Bioluminescence imaging was used to analyze the efficiency of UTMD gene transfection into the myocardium. β-catenin levels were detected by real-time polymerase chain reaction and western blot. Additionally, MI was induced in mice by surgical ligation of the left coronary artery, and cardiac function was evaluated using echocardiography at 14 and 28 days post-surgery. Masson’s trichrome staining was employed to determine infarct size. Blood vessel density was also measured. TUNEL assay was used to measure cardiomyocyte apoptosis. Furthermore, mouse cardiac stem cells were isolated using flow cytometry, and Giemsa stain was applied to evaluate the colony adhesion. UTMD delivered the gene to the heart with high efficiency and specificity in vivo. The β-catenin expression was significantly increased in the myocardium (P < 0.01). After MI, the β-catenin group exhibited a notable improvement in the gene therapy-induced neovascularization in the border zone (P < 0.01) and the number and function of cardiac stem cells (P < 0.01), and a significant decrease in cardiomyocyte apoptosis in the heart tissue (P < 0.01). β-catenin gene pre-treated with UTMD can reduce the impact of myocardial injury and promote cardiac self-repair after MI.

Synergistic combinations of Angelica sinensis for myocardial infarction treatment: network pharmacology and quadratic optimization approach

Background and aimAngelica sinensis (Oliv.) Diels (Danggui, DG), exhibits potential in myocardial infarction (MI) treatment. However, research on its synergistic combinations for cardioprotective effects has been limited owing to inadequate approaches.Experimental procedureWe identified certain phenolic acids and phthalein compounds in DG. Network pharmacology analysis and experimental validation revealed the components that protected H9c2 cells and reduced lactate dehydrogenase levels. Subsequently, a combination of computational experimental strategies and a secondary phenotypic optimization platform was employed to identify effective component combinations with synergistic interactions. The Chou-Talalay and Zero Interaction Potency (ZIP) models were utilized to quantify the synergistic relationships. The optimal combination identified, Z-Ligustide and Chlorogenic acid (Z-LIG/CGA), was evaluated for its protective effects on cardiac function and cardiomyocytes apoptosis induced by inflammatory in a mouse model of induced by left anterior descending coronary artery ligation. Flow cytometry was further utilized to detect the polarization ratio of M1/M2 macrophages and the expression of inflammatory cytokines in serum was measured, assessing the inhibition of inflammatory responses and pro-inflammatory signaling factors by Z-LIG/CGA.Key resultsQuadratic surface analysis revealed that the Z-LIG/CGA combination displayed synergistic cardioprotective effects (combination index value &amp;lt;1; ZIP value &amp;gt;10). In vivo, Z-LIG/CGA significantly improved cardiac function and reduced the fibrotic area in mice post-MI, surpassing the results in groups treated with Z-LIG or CGA alone. Compared to the MI group, the Z-LIG/CGA group exhibited decreased ratios of the myocardial cell apoptosis-related proteins BAX/Bcl-2 and Cleaved Caspase-3/Caspase-3 in mice. Further research revealed that Z-LIG/CGA treatment significantly increased IL-1R2 levels, significantly decreased IL-17RA levels, and inhibited the activation of p-STAT1, thereby alleviating cell apoptosis after MI. Additionally, the Z-LIG/CGA combination significantly inhibited the ratio of M1/M2 macrophages and suppressed the expression levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α in the serum.Conclusion and implicationsWe successfully identified a synergistic drug combination, Z-LIG/CGA, which improves MI outcomes by inhibiting cardiomyocyte apoptosis and inflammatory damage through modulating macrophage polarization and regulating the IL-1R2/IL-17RA/STAT1 signaling pathway. This study provides a charming paradigm to explore effective drug combinations in traditional Chinese medicine and a promising treatment for MI.

Cross-species single-cell RNA-seq analysis reveals disparate and conserved cardiac and extracardiac inflammatory responses upon heart injury

The immune system coordinates the response to cardiac injury and controls regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Adult mice and humans lack the ability to fully recover while adult zebrafish spontaneously regenerate after heart injury. Here we profile the inflammatory response to heart cryoinjury in zebrafish and coronary artery ligation in mouse using single cell transcriptomics. We interrogate the extracardiac reaction to cardiomyocyte necrosis to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages play a critical role in determining tissue homeostasis by healing versus scarring. We identify distinct transcriptional clusters of monocytes/macrophages (mono/Mϕ) in each species and find analogous pairs in zebrafish and mice. However, the reaction to myocardial injury is largely disparate between mice and zebrafish. The dichotomous response to heart damage between the murine and zebrafish mono/Mϕ and/or the presence of distinct zebrafish mono/Mϕ subtypes may underlie the impaired regenerative process in adult mammals and humans. Our study furnishes a direct cross-species comparison of immune responses between regenerative and profibrotic myocardial injury models, providing a useful resource to the fields of regenerative biology and cardiovascular research.

Mechanism of jianxin granules in the treatment of heart failure based on proteomics and metabolomics

BackgroundHeart failure (HF) is associated with high mortality and rehospitalization rates, highlighting the need for novel therapeutic approaches. Jianxin (JX) granules, a Traditional Chinese Medicine formulation, have been patented for the treatment of HF. However, the specific therapeutic effects and underlying mechanisms of JX granules have not been fully elucidated. This study aimed at investigating the effects and mechanism of JX granules in the treatment of HF based on proteomics and metabolomic profiling.MethodsHF model was established in rats by ligation of left coronary artery. The successfully modeled rats were randomly divided into three groups: the model group, the JX granules group, and Sacubitril/Valsartan (S/V) group. Four weeks after treatment, left ventricular (LV) function was evaluated via echocardiography. LV fibrosis and apoptosis were examined through histological analyses, while mitochondrial morphology was assessed using transmission electron microscopy. Quantitative assessment of oxidative stress was also conducted. Proteomics was used to identify the differentially expressed proteins and potential pathways. Metabolomics was utilized to elucidate the variations in metabolism. Then western blotting and in vitro analyses were performed.ResultsA rat model of HF was established, evidenced by a decrease in left ventricular ejection fraction (LVEF), stroke volume (SV), and left ventricular fractional shortening (LVFS), alongside diminished adenosine triphosphate (ATP) content, elevated oxidative stress, augmented apoptosis, and disrupted pyruvate metabolism. Treatment with JX granules ameliorated these effects, improving systolic function, reducing ventricular chamber size, and increasing LVEF, SV, and LVFS, as assessed by echocardiography. Additionally, JX granules attenuated cardiac fibrosis and improved mitochondrial structure, as evidenced by less vacuolation and clearer mitochondrial cristae, when compared to the model group. The treatment also regulated apoptosis-related protein expression, partially reversing the increase in cleaved Caspase-9, cleaved Caspase-3, and Bax and the suppression of Bcl-2 observed in the heart failure rats. All of these effects were similar to S/V. Proteomic and metabolomic analyses identified key differential genes, such as triosephosphate isomerase 1 (TPI1), lactate dehydrogenase B (LDHB), pyruvate kinase M (PKM), protein kinase B (Akt), Pyruvate Dehydrogenase Beta (PDHB) and lactate dehydrogenase A (LDHA), as well as vital pathways including carbon metabolism, the PI3K-Akt signaling pathway, pyruvate metabolism, and HIF-1α signaling pathway. Moreover, JX granules mitigated oxidative stress, inhibited apoptosis, and activated Akt in H9c2 cells exposed to angiotensin II, which could be reversed by the PI3K inhibitor LY294002.ConclusionJX granules improve HF in parallel to the efficacy of S/V, at least in part, through enhancing pyruvate metabolism, inhibiting oxidative stress and activating PI3K/Akt pathway.