Mouse Model Of Myocardial Infarction Research Articles

NAT10-mediated RNA ac4C acetylation contributes to the myocardial infarction-induced cardiac fibrosis.

Cardiac fibrosis is featured cardiac fibroblast activation and extracellular matrix accumulation. Ac4C acetylation is an important epigenetic regulation of RNAs that has been recently discovered, and it is solely carried out by NAT10, the exclusive enzyme used for the modification. However, the potential regulatory mechanisms of ac4C acetylation in myocardial fibrosis following myocardial infarction remain poorly understood. In our study, we activated fibroblasts invitro using TGF-β1 (20 ng/mL), followed by establishing a myocardial infarction mouse model to evaluate the impact of NAT10 on collagen synthesis and cardiac fibroblast proliferation. We utilized a NAT10 inhibitor, Remodelin, to attenuate the acetylation capacity of NAT10. In the cardiac fibrosis tissues of chronic myocardial infarction mice and cultured cardiac fibroblasts (CFs) in response to TGF-β1 treatment, there was an elevation in the levels of NAT10 expression. This increase facilitated proliferation, the accumulation of collagens, as well as fibroblast-to-myofibroblast transition. Through the administration of Remodelin, we effectively reduced cardiac fibrosis in myocardial infarction mice by inhibiting NAT10's ability to acetylate mRNA. Inhibition of NAT10 resulted in changes in collagen-related gene expression and ac4C acetylation levels. Mechanistically, we found that NAT10 upregulates the acetylation modification of BCL-XL mRNA and enhances the stability of BCL-XL mRNA, thereby upregulating its protein expression, inhibiting the activation of Caspase3 and blocking the apoptosis of CFs. Therefore, the crucial involvement of NAT10-mediated ac4C acetylation is significant in the cardiac fibrosis progression, affording promising molecular targets for the treatment of fibrosis and relevant cardiac diseases.

Hyperuricemia suppresses lumican, exacerbating adverse remodeling after myocardial infarction by promoting fibroblast phenotype transition

BackgroundHyperuricemia is independently associated with a poor prognosis in patients with myocardial infarction (MI). Furthermore, MI induces activation of the repair response in local fibroblasts, resulting in extracellular matrix accumulation that generates a stable fibrotic scar in the infarcted area. However, researchers have not determined whether hyperuricemia affects fibroblast activation and its involvement in postinfarction cardiac remodeling.ObjectivesWe aimed to trigger hyperuricemia by administering potassium oxonate in a mouse model of MI to evaluate the role of hyperuricemia in MI pathogenesis.MethodsMicroarray datasets and single-cell sequencing data from gout patients, heart failure patients, and model mice were used to identify the underlying mechanisms responsible for the effect of hyperuricemia on MI progression. A hyperuricemia-related MI mouse model was established. Cardiac function was assessed, followed by sample collection and a uric acid assay. We conducted an enzyme-linked immunosorbent assay, histological detection, immunofluorescence, sequencing data processing, single-cell RNA-seq, and functional enrichment analysis. We then isolated and cultured cardiac fibroblasts and performed Western blotting, quantitative real-time polymerase chain reaction, and shRNA-mediated lumican knockdown assays.ResultsHyperuricemia decreased cardiac function, increased mortality, and aggravated adverse fibrosis remodeling in mice after MI. These outcomes were closely related to reduced levels of fibroblast-derived lumican. This reduction activated the TGF-β/SMAD signaling pathway to induce aberrant myofibroblast activation and extracellular matrix deposition in the infarcted area. Furthermore, lumican supplementation or uric acid-lowering therapy with allopurinol alleviated hyperuricemia-mediated abnormal cardiac remodeling.ConclusionHyperuricemia aggravates postinfarction cardiac remodeling by reducing lumican expression and promoting fibroblast phenotype transition. We highlight the clinical importance of lowering uric acid levels in hyperuricemia-related MI to prevent adverse ventricular remodeling.

Cardiomyocyte-Specific Overexpression of Activated Yes-Associated Protein Modified-RNA Promotes Cardiomyocyte Proliferation and Myocardial Regeneration.

The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes-associated protein, 5SA [active] mutant), but persistent overexpression of proliferation-inducing genes could lead to hypertrophy and arrhythmia, whereas off-target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte-specific modified mRNA translation system (YAP5SACM-SMRTs or GFPCM-SMRTs, respectively). YAP5SA-cardiomyocyte specificity was confirmed via invitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced- pluripotent stem cells and in human umbilical-vein endothelial cells, and the regenerative potency of YAP5SACM-SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced-pluripotent stem cells-cardiomyocytes, YAP was abundantly expressed for 3 days after YAP5SACM-SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near-background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after GFPCM-SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced-pluripotent stem cells-cardiac fibroblasts and human umbilical-vein endothelial cells on day 1 after GFPCM-SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in YAP5SACM-SMRTs-treated mice than in vehicle-treated control animals, and YAP5SACM-SMRTs appeared to promote cardiomyocyte proliferation. The CM-SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

Protective role of Metrnl on macrophage recruitment during the acute inflammatory phase in a mouse model of myocardial infarction

Abstract Introduction Meteorin-like protein (Metrnl) is a cytokine involved in the attenuation of inflammation. Previously, a cardioprotective role of Metrnl against cardiac hypertrophy and ischemia/reperfusion has been described. After myocardial infarction (MI), macrophages recruited from the spleen, and their capacity to shift from inflammatory (M1) to reparative (M2) phases determines the fate of cardiac repair. Purpose The aim of this study was to analyse the function of Metrnl in a mouse model of permanent MI during the acute inflammatory phase. Methods Wild-type (WT), Metrnl Knock-out (Metrnl-/-) and Metrnl-reconstituted Metrnl-/- mice (AAV9-Metrnl, with myocardial-specific tropism) were subjected to MI by coronary artery ligation. After 4 days, cardiac function was assessed using echocardiography at baseline and 4 days post-MI. Monocyte populations were analysed in spleen and blood by flow cytometry, and macrophages infiltrating the infarcted myocardium by gene expression analyses. Results Echocardiographic analysis demonstrated that Metrnl deficiency led to cardiac dysfunction (FAC p=0.02) accompanied by myocardial dilation 4 days post-MI (EDV p=0.02; ESV p=0.01). On the contrary, the overexpression of Metrnl in the myocardium of AVV9-Metrnl mice restored cardiac function compared to Metrnl-/- group (FAC p=0.01, EDV p=0.04, ESV p=0.09). Furthermore, we observed a reduction in splenic monocytes (CD11b+F4/80+) in Metrnl-/- mice compared to the WT group at 4 days post-MI (p=0.06). In particular, a significant decrease in pro-inflammatory Ly6CHigh and CD86+ monocytes was noted in the spleens of Metrnl-/- mice compared to the WT group (p=0.03; p=0.04 respectively), suggesting their mobilization out of the spleen, but not in the AVV9-Metrnl group. Although the total number of Ly6CHigh and Ly6CLow monocytes in the bloodstream was similar in all groups, there was a trend to different expression in the CCR2 and CCR5 chemokine receptors (p=0.07; p=0.06 respectively). At the same time, M1 macrophage recruitment chemokines (CCL2) and inflammatory markers (TNFα) were significantly induced in the ischemic area of the infarcted myocardium compared to the remote zone in the WT and Metrnl-/- groups (CCL2 p=0.03; p=0.01 respectively; TNFα p=0.008;p=0.04 respectively). In contrast, TNFα and CCL2 genes were not induced in the ischemic area of AVV9-Metrnl mice. Regarding M2 macrophages, the Arg1 marker was significantly reduced in the ischemic zone of Metrnl-/- mice compared to the WT group (p= 0.01), while in the AVV9-Metrnl group was recovered (p= 0.04). Conclusions Our results demonstrate that the absence of Metrnl promotes the recruitment of inflammatory macrophages to the ischemic myocardium and is associated to cardiac dysfunction.

Direct in vivo cardiac reprogramming with circular RNA of single transcription factor delivered by fibroblast-targeting lipid nanoparticles

Abstract Background Adult mammalian cardiomyocytes (CMs) hold limited capacity for self-regeneration, thus leading to permanent loss of functional myocardium once suffering from injury, especially myocardial infarction (MI). Therefore, cardiac regeneration therapy has been regarded as a potential path to help restore damaged tissue. Direct cardiac reprogramming is one of the most promising ways to boost cardiac regeneration as it could convert resident cardiac fibroblasts into functional induced cardiomyocytes (iCMs). However, difficulty of delivery has been one of the biggest problems that hinder the further clinical translation of this technique such as concerns of mostly used retrovirus vectors and lack of fibroblast-specific targeting ability. In our previous research, we have found that after knocking down of Ybx1 gene, single transcriptional factor Tbx5 could induce cardiac reprogramming of fibroblasts without adding traditional Mef2c and Gata4. But whether this cocktail could achieve in vivo direct cardiac reprogramming still remains to be explored. Objective We planned to develop fibroblast-targeting lipid nanoparticles (LNP) to delivery circular RNA(circRNA) of Tbx5 and small interfering RNA(siRNA) of Ybx1 to realize in vivo cardiac reprogramming in a mouse model of myocardial infarction. Results We first screened different formulations of tdTomato mRNA delivered-LNP both in primary neonatal cardiac fibroblasts and in the myocardial infarction model and found that formulation XF4d could realize specific selectively uptake by cardiac fibroblasts instead of cardiomyocytes. Then we succeeded in synthesizing XF4d LNP delivering Tbx5 circRNA and Ybx1 siRNA (LNPXF4d@Tbx5+siYbx1) and validated its stability at 4°C. Next, after infecting LNPXF4d@Tbx5+siYbx1, primary neonatal CFs and mouse embryonic fibroblasts could be reprogrammed into functional iCMs. We further measured the protein expression of Tbx5 and Ybx1 in CFs after LNPXF4d@Tbx5+siYbx1 treatment. What’s more, we used fibroblast-specific lineage tracing mice (Fsp1Cre/tdTomato mice) to prove the successful conversion of resident CFs into iCMs in the mouse model of myocardial infarction by intramyocardial injection of LNPXF4d@Tbx5+siYbx1. At last, we found that LNPXF4d@Tbx5+siYbx1 could help improve cardiac function after injury and reduce fibrosis, as measured by echocardiography and Masson’s trichrome staining. Conclusion We suggest that our fibroblast specific LNPs delivering single transcriptional factors could a promising method to realize in vivo cardiac reprogramming and help treat myocardial injury, with high possibility of clinical translation and less biosafety concerns.Graphical Abstract

MiRNA-519e promotes cardiomyocyte cell division accompanied by pro-angiogenic and anti-apoptotic effects

Abstract Background MicroRNAs have the therapeutic potential for heart repair after myocardial infarction(MI). Using a functional high-throughput screening approach, we identified microRNA-519e(miR-519e) as an inducer of cell-cycling in human-derived iPSC-cardiomyocytes(hiPSC-­CM). Purpose This study examines miR-519e for cell division in hiPSC-­CM, additional beneficial functions in heart-related non-CMs, and direct target identification and validation in vitro and in vivo. Methods MiR-519e-mimics or miR-scrambled(miR-scr) were transfected into hiPSC-­CM, human aortic endothelial cells(HAEC) and human cardiac fibroblasts(cFB). Proliferative markers (EdU(5-Ethynyl-2'- deoxyuridine), H3P(Phospho-Histone H3) and AURKB(Aurora B-kinase)) and apoptotic response using MTT assay after exposure to doxorubicin were assessed in hiPSC­-CMs. Angiogenic capacity of HAEC was evaluated by tube formation assay and fibrotic response of cFBs using EdU and α-SMA(smooth muscle actin) staining. In vivo, a mouse MI model with ligation of the left anterior descending followed by intramyocardial injection of miR-519e or miR-scr was used. Direct target identification of miR-519e was assessed using in silico analysis combined with RNA-Seq of miR-519e-mimic treated hiPSC-CMs and validated using Ago2-RNA immunoprecipitation(RIP) followed by qPCR. Results After transfection of miR-519e-mimics in hiPSC-­CM, a significant proliferative response indicative of cell division was observed in immunofluorescence staining and on protein level as compared with miR-scr treated hiPSC-CMs (EdU, H3P and AURKB, p<0.01). Apoptosis was blunted in miR-519e-treated hiPSC-CMs (p<0.05). MiR-519e-treatment of HAEC resulted in more meshes (p<0.01) and longer tube length (p<0.05), indicating enhanced pro-angiogenic capacity. Importantly, EdU-uptake and α-SMA were similar in miR-519e-treated cFB as compared to miR-scr, suggesting neutral effects of miR-519e on myofibroblast transition. In mice undergoing MI, injection of miR-519e-mimics significantly downregulated CDKN1A/P21(Cyclin dependent kinase inhibitor 1A) (p<0.05 vs. miR-scr), a direct target of miR-519e and negative master regulator of cell-cycling. For further novel target identification of miR-519e, we performed in silico analysis combined with RNA-Seq data from miR-519e-treated hiPSC-CM and validation using Ago2-RIP followed by qPCR. Herein, we identified PTEN(Phosphatase and tensin homolog) and TGFBR2(Transforming growth factor β receptor 2) that are involved in cardiomyocyte proliferation and hypoxic response, as direct targets of miR-519e. Conclusion Collectively, miR-519e is a potent inducer of cell division in human cardiomyocytes, with anti-apoptotic and pro-angiogenic capacity in the absence of pro-fibrotic effects. We show that miR-519e treatment downregulates key negative cell-cycle regulators in vivo and we identified novel direct targets of miR-519e that are involved in regulatory pathways for heart regeneration.

Inhibiting chondroitin sulfate synthase 1 ameliorates hyperplastic arterial remodelling

Abstract Introduction Hyperplastic arterial remodelling is characterized by intima-media thickening, lumen narrowing, inflammation and increased extracellular matrix (ECM) deposition. Chondroitin sulfate (CS) glycosaminoglycans, the unbranched polysaccharide chains of proteoglycans, are important components of ECM. We previously found CS accumulated in adversely remodelled heart and artery, aggravating cardiac deterioration. Objective We aim to explore a therapeutic strategy to interfere with CS synthesis and investigate its potential in ameliorating hyperplastic arterial remodelling. Methods Through single-cell RNA sequencing of non-cardiomyocytes cardiac cells from mouse myocardial infarction model, chondroitin sulfate synthase 1 (CHSY1) was identified as the primary CS synthase and was increased during disease progression. A Chsy1 knockout (KO) mouse strain was generated. Fibrosis and arterial remodelling were induced using: (i) 4 weeks of angiotensin II/phenylephrine (AngII/PE) infusion through subcutaneous osmotic minipumps; and (ii) transverse aortic constriction surgery. To assess the therapeutic potential of CHSY1 inhibition in ameliorating hyperplastic arterial remodelling, Chsy1 was knockdown by dsiRNA post disease onset. Mechanism of action was investigated using primary vascular smooth muscle cells (VSMCs). Results Chsy1 KO reduced CS and its stereoisomer dermatan sulfate in the heart to 42 ± 22% and 36 ± 7% respectively, whereas other types of glycosaminoglycans remain intact. KO mice were protected against phenotypes of hyperplastic arterial remodelling induced by the two disease models in both the coronary and carotid arteries. For example, 90.1% of coronary arterial fibrosis and 79.7% of macrophage infiltration induced by AngII/PE was declined in KO mice compared to WT mice, while cardiac function as stroke volume was augmented by 41.3%. Concomitantly, carotid arterial wall thickening was lower by 36.5% and distensibility was higher by 36.7%, validating Chsy1 as a potent target. As a therapeutic approach, weekly intravenous injection of dsiRNA achieved Chsy1 reduced to 30.2 ± 5.5% in the carotid artery and 72.5 ± 4.7% in the heart. Post disease onset, Chsy1 KD therapy reduced fibrosis by 59.9% in the coronary artery (P = 0.006) and improved stroke volume by 25.6% (P = 0.036). The structural integrity and function of carotid arteries were well-reserved, manifesting as improved distensibility (29%, P = 0.016), increased elastin/perimeter ratio (15%, P = 0.002), and reduced macrophage infiltration (38%, P = 0.029). In vitro, CHSY1 inhibition suppressed migration and cytokine production of macrophages, fibroblast-to-myofibroblast transition, and VSMC hyperproliferation. NOTCH3 signalling was found to be positively correlated with CHSY1 modulation. Conclusions We demonstrated that inhibiting CHSY1 is necessary and sufficient to block CS synthesis. Chsy1 KD by dsiRNA is an effective therapy to ameliorate hyperplastic arterial remodelling.

Mechanical stimulation promotes the maturation of cardiomyocyte-like cells from P19 cells and the function in a mouse model of myocardial infarction.

In this study, we aimed to promote the maturation of cardiomyocytes-like cells by mechanical stimulation, and evaluate their therapeutic potential against myocardial infarction. The cyclic tensile strain was used to induce the maturation of cardsiomyocyte-like cells from P19 cells in vitro. Western blot and qPCR assays were performed to examine protein and gene expression, respectively. High-resolution respirometry was used to assay cell function. The induced cells were then evaluated for their therapeutic effect. In vitro, we observed cyclic tensile strain induced P19 cell differentiation into cardiomyocyte-like cells, as indicated by the increased expression of cardiomyocyte maturation-related genes such as Myh6, Myl2, and Gja1. Furthermore, cyclic tensile strain increased the antioxidant capacity of cardiomyocytes by upregulating the expression Sirt1, a gene important for P19 maturation into cardiomyocyte-like cells. High-resolution respirometry analysis of P19 cells following cyclic tensile strain showed enhanced metabolic function. In vivo, stimulated P19 cells enhanced cardiac function in a mouse model of myocardial infarction, and these mice showed decreased infarction-related biomarkers. The current study demonstrates a simple yet effective mean to induce the maturation of P19 cells into cardiomyocyte-like cells, with a promising therapeutic potential for the treatment of myocardial infarction.

MG53 Protein-Mediated biomimetic nanotherapeutics for the treatment of acute myocardial infarction by driving the impaired plasma membrane resealing

Myocardial infarction (MI) remains a leading global cause of mortality. The frequent disruptions of cardiomyocyte plasma membranes underscore the pivotal role played by MG53, a tissue repair protein secreted by skeletal muscle in response to physical exercise, in cardioprotection. However, the clinical application of recombinant human MG53 (rhMG53) is challenged by its short circulation half-life, necessitating frequent administration, and its low cell membrane permeability. Here we introduce an innovative MG53 protein-mediated biomimetic nanotherapeutic (Rm@mSiO2@MG53) to address the poor delivery. Rm@mSiO2@MG53 nanotherapeutics, attributed to the high specific surface area and adjustable pore size of mSiO2 and the expression of CD47 on erythrocyte membranes, enable sustained protein delivery, enhance their ability to evade the mononuclear phagocytic system (MPS), and extend the half-life of rhMG53 proteins in circulation. In a mouse MI model, Rm@mSiO2@MG53 demonstrated superior efficacy in protecting injured cardiomyocytes and preserving heart function post-MI. Our findings developed a novel protein biomimetic nanotherapeutics that mediates heart repair by driving cell membrane resealing, offering the potential to expedite the clinical deployment of rhMG53 as a viable therapeutic for treating MI and other tissue injuries.

TMT-based proteomics reveals methylprotodioscin alleviates oxidative stress and inflammation via COX6C in myocardial infraction

The effect of methylprotodioscin (MPD), a steroidal saponin obtained from medicinal plants, on myocardial infarction (MI) remains elusive. In this study, HL-1 and AC16 cells were subjected to injury induced by hypoxic environment, and a mouse model of MI was established by ligating the left anterior descending. MPD significantly increased viabilities and proliferations, improved the stability of MMP, reduced ROS and inflammatory factor levels in hypoxia cardiomyocytes. Moreover, MPD significantly improved cardiac functions, increased the ventricular ejection fraction and short axis shortening rate of mice with MI, reduced the infarction area, alleviated oxidative stress and increased ATPase activities. Then, differentially expressed proteins (DEPs) were discovered and evaluated using tandem mass tag (TMT)-based proteomics and bioinformatics approaches. Compared with sham group, there were 420 DEPs in the cardiac tissue of MI group, likewise, 163 DEPs in MPD group were identified compared to MI group. By validating, the expression of COX6C was elevated in MI group and declined in MPD groups, consistent with the TMT-based proteomics results. Correspondingly, p-NF-κB expression was downregulated, while Nrf2 and SOD expressions were upregulated by MPD. Moreover, si-COX6C transfection blocked the regulatory effects of MPD on COX6C-mediated inflammation and oxidative stress in MI. Our findings indicate that MPD, a naturally occurring active ingredient, could effectively improve cardiac function. Its ability may result from regulating COX6C to reduce oxidative stress and suppress inflammation, suggesting that MPD is very attractive for the treatment of MI.

Specific peptides targeting the myocardiocyte are prognostic markers for heart attack: Function of α-SMA protein

Myocardial infarction (MI) is a serious cardiovascular disease that often results in a significant decline in heart function and associated complications. α-SMA (α-smooth muscle cell actin) is an important biomarker in the process of cardiac remodeling and repair, and its expression level is closely related to myocardial remodeling and prognosis. Therefore, the purpose of this study was to investigate the potential of nanoparticles containing cardiomyocyte targeting peptides in predicting prognosis and α-SMA protein expression after myocardial infarction, with a view to providing new therapeutic strategies and clinical guidelines. In this study, a novel targeting nanoparticle was constructed, using cardiomyocyte specific peptides as targeting ligands, and characterized by loading different drugs. Subsequently, a mouse model of myocardial infarction was used to systematically evaluate the effect of nanoparticles on α-SMA protein expression and prognosis prediction ability after MI. The expression level of α-SMA was analyzed by Western blot and immunohistochemistry, and the prognosis was evaluated by cardiac function assessment. The study found that nanoparticles containing cardiomyocyte targeting peptides significantly increased α-SMA expression levels and improved heart function in animal models of myocardial infarction. Compared with the control group, the application of targeted nanoparticles was closely related to the level of myocardial cell repair and fibrosis, and could effectively predict the prognosis after myocardial infarction. Therefore, nanoparticles containing cardiomyocyte targeting peptides can not only effectively improve the expression of α-SMA, but also serve as an important prognostic tool after myocardial infarction.

Engineered model of heart tissue repair for exploring fibrotic processes and therapeutic interventions

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless, a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process, including hypoxic stress, fibrosis, and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions, leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology, demonstrating that the phased combination of TGFβ inhibitor SB431542, Rho kinase inhibitor Y27632, and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction, providing a crucial platform for discovering therapeutic targets for ischemic heart disease.

Legumain-Guided Ferulate-Peptide Self-Assembly Enhances Macrophage-Endotheliocyte Partnership to Promote Therapeutic Angiogenesis After Myocardial Infarction.

Promoting angiogenesis and modulating the inflammatory microenvironment are promising strategies for treating acute myocardial infarction (MI). Macrophages are crucial in regulating inflammation and influencing angiogenesis through interactions with endothelial cells. However, current therapies lack a comprehensive assessment of pathological and physiological subtleties, resulting in limited myocardial recovery. In this study, legumain-guided ferulate-peptide nanofibers (LFPN) are developed to facilitate the interaction between macrophages and endothelial cells in the MI lesion and modulate their functions. LFPN exhibits enhanced ferulic acid (FA) aggregation and release, promoting angiogenesis and alleviating inflammation. The multifunctional role of LFPN is validated in cells and an MI mouse model, where it modulated macrophage polarization, attenuated inflammatory responses, and induces endothelial cell neovascularization compare to FA alone. LFPN supports the preservation of border zone cardiomyocytes by regulating inflammatory infiltration in the ischemic core, leading to significant functional recovery of the left ventricle. These findings suggest that synergistic therapy exploiting multicellular interaction and enzyme guidance may enhance the clinical translation potential of smart-responsive drug delivery systems to treat MI. This work emphasizes macrophage-endothelial cell partnerships as a novel paradigm to enhance cell interactions, control inflammation, and promote therapeutic angiogenesis.

Sacubitril/valsartan alleviates myocardial infarction-induced inflammation in mice by promoting M2 macrophage polarisation via regulation of PI3K/Akt pathway

Background Macrophage polarisation-mediated inflammation plays a critical role in ventricular remodelling after myocardial infarction (MI). Sacubitril/Valsartan (Sac/Val) is an angiotensin receptor-neprilysin inhibitor that has shown beneficial effects on MI and heart failure. This study aims to further explore the mechanisms by which Sac/Val exerts its protective effects against MI. Methods A mouse MI model was induced by ligating the left anterior descending coronary artery, followed by Sac/Val administration. TTC staining and Masson trichrome staining were employed for estimating myocardial infarct size and fibrosis, respectively. The expression levels of proinflammatory factors were determined by ELISA and RT-qPCR. Flow cytometry and immunofluorescence staining were implemented to detect CD206-positive cell infiltration in mouse hearts. Western blotting was conducted to assess protein levels of Arg1, pro-fibrotic factors, and PI3K/Akt signalling-related markers. Results Sac/Val treatment reduced myocardial infarct size and fibrosis in mice after MI. Sac/Val administration decreased proinflammatory cytokine production and facilitated M2 macrophage polarisation in MI mouse cardiac tissues. Sac/Val activated PI3K/Akt signalling in MI mouse hearts. Blocking PI3K/Akt signalling counteracted Sac/Val-mediated protective effects in MI mice. Conclusion Sac/Val ameliorates MI-induced inflammation by facilitating M2 macrophage polarisation and activating PI3K/Akt signalling.

Abstract P396: A Selective Agonist of the Beta 2 Retinoic Acid Receptor Reduces Necrosis, Lipid Peroxidation, and Oxidative Stress in Doxorubicin-induced Cardiotoxicity

Objective: Doxorubicin (Doxo) is used as an antiproliferative agent to treat various cancers. However, a significant limitation of Doxo is cardiotoxicity, that can induce heart failure in patients. Mounting evidence indicates that an increased generation of reactive oxygen species (ROS), lipid peroxidation, and necrosis, are major causes of Doxo-induced heart failure. Recently, we reported that a highly selective and potent agonist of the b2 retinoic acid receptor (RARb2), improved cardiac dysfunction in mice model of myocardial infarction by inhibiting oxidative stress. In this study we investigated cardioprotective effects of RARb2 stimulation on Doxo-induced heart failure. Methods: We performed in vitro and in vivo experiments on an established murine model of Doxo-induced heart failure. Necrosis, oxidative stress, and lipid peroxidation were evaluated, assessing both the prevention and the rescue capacities of RARb2 stimulation. Results: An increased ROS production both at the mitochondrial and cellular level was observed in cardiomyocytes isolated from Doxo-treated mice. RARb2 stimulation significantly attenuated Doxo-induced ROS production. In addition, this treatment mitigated lipid peroxidation induced by Doxo in the left ventricle, marked by 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). RARb2 stimulation also mitigated the increased expression of High Mobility Group Box 1 (HMGB1), a necrosis marker, in the heart and in the serum of Doxo-treated mice. Conclusion: Our data demonstrate that RARb2 stimulation can attenuate necrosis, lipid peroxidation, and ROS production in a mouse model of Doxo cardiotoxicity.

Macrophage MKL1 contributes to cardiac fibrosis in a mouse model of myocardial infarction

AimsCardiac fibrosis is characterized by aberrant collagen deposition in the heart. Macrophage polarization or infiltration is the main reason to accelerate the collagen deposition. We attempted to investigate the involvement of MKL1 in macrophages during the development of cardiac fibrosis. Materials and methodsCardiac fibrosis is induced by myocardial infarction (MI). The MKL1f/f mice were crossed to the Lyz2-cre mice to generate macrophage conditional MKL1 knockout mice (MKL1ΔMφ). In addition, macrophage conditional MKL1 overexpression mice (MKL1Mϕ-OE) were constructed by crossing Lyz2-cre mice to MKL1ΔN200-Rosa26 mice. Key findingsMKL1 expression was significantly increased in macrophages of both ischemic cardiomyopathy (ICM) patients and mice induced to develop myocardial infarction. Deletion of MKL1 in macrophages improved the heart function after MI-induced cardiac fibrosis. Consistently, MKL1Mϕ-OE mice displayed more severe cardiac fibrosis and worsened heart function than the control mice after MI. Moreover, administration of a small-molecule MKL1 inhibitor CCG-1423 also decreased the collagen deposition after MI. SignificanceOur data demonstrate that MKL1 in macrophages contributes to cardiac fibrosis pathogenesis and reinforce the notion that targeting MKL1 may yield effective antifibrotic therapeutics in the heart.

Exosomes from Hypoxic Pretreatment ADSCs Ameliorate Cardiac Damage Post-MI via Activated circ-Stt3b/miR-15a-5p/GPX4 Signaling and Decreased Ferroptosis.

Accumulation studies confirmed that oxidative stress caused by ischemia after myocardial infarction (MI) is an important cause of ventricular remodeling. Exosome secretion through hypoxic pretreatment adipose-derived mesenchymal stem cells (ADSCs) ameliorates myocardial damaging post-MI. However, if ADSCs exosome can improve the microenvironment and ameliorate cardiac damage post-MI still unknown. Next-generation sequencing (NGS) was used to study abnormally expressed circRNAs in hypoxic pretreatment ADSC exosomes (HExos) and untreated ADSC exosomes (Exos). Bioinformatics and luciferase reporting were used to elucidate interaction correlation related to circRNA, mRNA, and miRNA. HL-1 cells were used to analyze the reactive oxygen species (ROS) and apoptosis under hypoxic conditions using immunofluorescence and flow cytometry. An MI mouse model was constructed and the therapeutic effect of Exos was determined using immunohistochemistry, immunofluorescence, and ELISA. The results showed that HExos had a more pronounced treatment effect than ADSC Exos on cardiac damage amelioration after MI. NGS showed that circ-Stt3b plays a role in HExo-mediated cardiac damage repair after MI. Overexpression of circ-Stt3b decreased apoptosis, ROS level, and inflammatory factor expression in HL-1 cells under hypoxic conditions. Bioinformatics and luciferase reporting data validated miR-15a-5p and GPX4 as downstream circ-Stt3b targets. GPX4 downregulation or miR-15a-5p overexpression reversed protective effect regarding circ-Stt3b upon HL-1 cells after exposure to a hypoxic microenvironment. Overexpression of circ-Stt3b increased the treatment effect of ASDSC Exos on cardiac damage amelioration after MI. Taken together, the study results demonstrated that Exos from hypoxic pretreatment ADSCs ameliorate cardiac damage post-MI through circ-Stt3b/miR-15a-5p/GPX4 signaling activation and decreased ferroptosis.

A novel theranostic strategy for myocardial infarction through neutralization of endogenous SO2 using an endoplasmic reticulum-targeted fluorescent probe

Myocardial infarction (MI), one of the leading causes of death worldwide, urgently needs further understanding of the pathological process and effective therapies. SO2 in endoplasmic reticulum in several cardiovascular diseases has been reported to be particularly important. However, the role of endogenous SO2 in endoplasmic reticulum in treating myocardial infarction is still ambiguous and needs to be elucidated. Herein, we developed TPA-HI-SO2 as the first endoplasmic reticulum-targeting fluorescent agent for specific imaging and detection of sulfur dioxide derivatives both in vitro and in vivo. TPA-HI-SO2 shows a highly sensitive and selective response to SO2 derivatives over other anions in aqueous solution with a satisfactory response time and detection limit. Furthermore, TPA-HI-SO2 decreased the SO2 concentration in H9C2 cells treated with H2O2 and in an MI mouse model. Most importantly, TPA-HI-SO2 protects H9C2 cells from H2O2-induced apoptosis and obviously protects against myocardial infarction in vivo through neutralization of endogenous SO2. Taken together, we developed the first ER-targeting ratiometric fluorescent probe for endogenous SO2 with excellent biocompatibility, high selectivity and sensitivity in this paper. More importantly, we demonstrated an obvious increase of the endogenous SO2 concentration in a myocardial infarction mouse model for the first time, which suggests that neutralization of endogenous SO2 in endoplasmic reticulum could be a promising therapeutic strategy for myocardial infarction.

Abstract Mo130: Epigenetic Mechanisms Regulate Sex Differences in Cardiac Reparative Functions of Bone Marrow Progenitor Cells

Historically, lower incidence of CVD and related deaths in women as compared with men of the same age has been attributed to female sex hormones, particularly estrogen and its receptors. Autologous Bone marrow stem cell (BMSC) clinical trials for cardiac cell therapy overwhelmingly included male patients; however, meta-analysis data on these trials suggest a better functional outcome in women as compared with men. A direct comparison of the mechanisms governing sex-specific cardiac reparative activity in bone marrow-derived mesenchymal stem cells (BMSCs), with and without the influence of sex hormones, remains unexplored. This study was designed to identify mechanisms that regulate superior reparative properties of female endothelial progenitor cells (EPCs) post-MI, particularly epigenetic mechanisms. Male (M), female (F), and ovariectomized female (OVX) mice-derived EPCs were subjected to a series of molecular and epigenetic analyses followed by in vivo functional assessments of cardiac repair. RNA sequencing and other quantitative assays showed a similar genetic profile between F-EPCs and OVX-EPCs, distinct from M-EPCs that displayed significant up-regulation of inflammation-related genes. F-EPCs and OVX EPCs secrete higher levels of proangiogenic factors, lower levels of proinflammatory cytokines and show better cardiac reparative activity after intra-cardiac injections in a male mouse model of myocardial infarction (MI). Epigenetic sequencing revealed a marked difference in the occupancy of the gene repressive H3K9me3 mark, particularly at transcription start sites of key angiogenic and proinflammatory genes in male EPCs compared with female and ovariectomized EPCs. Our study unveiled that functional sex differences in EPCs is, in part, mediated by differential epigenetic regulation of the proinflammatory and anti-angiogenic gene CCL3, orchestrated by the control of H3K9me3 by histone methyltransferase, G9a/Ehmt2. Our research highlights the importance of considering sex of donor cells for progenitor-based tissue repair.

Abstract Tu046: Mechanisms of Epicardium-Directed Cardiac Repair

Background: In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability. Methods: We used the Wt1 (Wilms Tumor 1) CreERT2 ; R26 tdTomato mouse line for tamoxifen-inducible epicardial-specific lineage tracing and Pdgfra nGFP to label cardiac fibroblasts ( Wt1 CreERT2 ; R26 tdT ; Pdgfra nGFP ). Tagged epicardial-derived cells (tdTomato + ) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed in vitro studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI. Results: We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with Wnt signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in Wt1 -expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition. Conclusions: Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.