Murine Model Of Myocardial Infarction Research Articles

Single cell multi-omics analysis unveils a distinct mechanism of exercise-mediated cardioprotection in ischemic cardiomyopathy

Abstract Background Exercise is pivotal in heart disease prevention, with proven cardioprotective effects in clinical trials. However, its precise molecular mechanisms remain elusive. Recent advancements in single-cell omics have revealed cellular heterogeneity within heart tissues, offering a deeper understanding of the pathophysiology of cardiac diseases. Purpose To investigate the exercise-induced physiological changes in heart tissue at the single-cell level and elucidate the molecular mechanisms underlying its cardioprotective effect on ischemic cardiomyopathy. Methods Murine myocardial infarction (MI) model was established by ligating the left ascending coronary artery, and a six-week voluntary wheel running exercise regimen was implemented to investigate the effect of aerobic exercise on ischemic cardiomyopathy. Integrated multi-omics analyses of single-cell DNA methylation profiling, single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) were conducted to clarify the molecular mechanism of exercise-mediated cardioprotection at single-cell resolution. Overexpression and short hairpin RNA-mediated knockdown of gene X were conducted for murine MI model using cardiomyocyte-specific Myo-AAV2a vector. snRNA-seq of heart tissues from heart failure patients was performed to validate the clinical significance of the expression of gene X in cardiomyocytes. Results Exercise attenuates pathological cardiac remodeling, inducing physiological hypertrophy in non-infarcted areas while suppressing pathological remodeling of cardiomyocytes in infarct border zones. Single-cell epigenetic analysis unveiled intricate molecular mechanisms underlying exercise-induced transcriptome changes, providing insights into the gene regulatory networks modulated by exercise in the heart. In addition, integrated with snRNA-seq, we identified exercise-specific cardiomyocyte clusters, highlighting the substantial role of gene X in the molecular mechanism of exercise. Functional studies confirmed the essential role of gene X in exercise-induced cardioprotection, as its overexpression suppressed the adverse remodeling after MI and knockdown abolished the beneficial effects of exercise. Furthermore, snRNA-seq of heart failure patients revealed that higher expression of gene X was associated with ventricular reverse remodeling and favorable clinical outcomes, underscoring its significance in the pathophysiology of patients with heart failure. Conclusion Our study elucidates exercise-specific gene expression regulation networks in ischemic cardiomyopathy by unraveling the cellular heterogeneity and molecular changes associated with exercise. We identified gene X as a central player in exercise-mediated cardioprotection and further confirmed its significance in clinical settings, providing a potential therapeutic target for heart failure.

A newly developed small-molecule ErbB4 agonist reduces reactive cardiac fibrosis and adverse ventricular remodelling after myocardial infarction in a sex-specific manner

Abstract Background The beneficial effects of the neuregulin/ErbB pathway on the diseased heart have been well-established. The anti-fibrotic and cardioprotective aspects of neuregulin are largely attributable to the activation of the ErbB4 receptor. We recently developed a small-molecule selective ErbB4 agonist, which reduced collagen production in cultured fibroblasts and prevented oxidative-stress induced cardiomyocyte death in vitro. Purpose In order to evaluate the in vivo effects of this small-molecule ErbB4 agonist (referred to as compound, Cpd), we assessed its effect on left ventricular remodelling, cardiac fibrosis and cardiac function in a murine model of myocardial infarction (MI). Since there is a potential interaction between ErbB4 and the oestrogen receptor, we also aimed to evaluate whether these effects were sex-dependent. We hypothesised that Cpd would attenuate left ventricular remodelling, reduce cardiac fibrosis and help retain cardiac function in a sex-specific manner. Methods Mice were divided by sex and then further divided into three experimental groups: a sham group, an MI group receiving Cpd (MI/Cpd), and a control MI group receiving vehicle only (MI/Veh). MI was induced by ligating the left descending coronary artery. Seven days after MI, mice started treatment with either Cpd (2mg/kg/d) or its vehicle through subcutaneous osmotic minipumps. Cardiac ultrasound was performed weekly until mice were sacrificed after four weeks of treatment. Hearts were excised and stained with Masson’s trichrome to assess reactive cardiac fibrosis in the myocardium of the left ventricle, located remote from the infarction scar. A WGA-isolectinB4-DAPI immunofluorescent stain was performed to assess cardiomyocyte cross-sectional area in order to evaluate cardiomyocyte hypertrophy. Results In females (n=25), Cpd significantly decreased left ventricular end-diastolic volume compared to vehicle (LVEDV, Fig.1A, 80±23 µl vs.108±29 µl, p=0.045) and induced a slightly increased left ventricular ejection fraction (LVEF, Fig.1B, 32±12% vs. 22±9%, p=0.086). In addition, Cpd significantly reduced reactive interstitial fibrosis myocardium (Fig.1C, fibrotic area, 1.4±1.0% vs. vehicle: 3.8±3.1%, p=0.0363). In males (n=30), Cpd had no significant effects on either of the parameters mentioned above, (Fig.1E-G). There was no significant treatment effect in either group on cardiomyocyte cross sectional area (Fig.1D, Fig.1H). Conclusion Treatment with the novel small-molecule ErbB4 agonist attenuates left ventricular dilation and reactive interstitial fibrosis after MI in female mice, but not in males. These data support the premise that small molecule-induced ErbB4 activation is a potential new therapy for heart failure, especially in female patients. The mechanisms underlying the sex-dependent effects warrant further exploration.Figure 1

CD14 blockade preserves left ventricular structure and systolic function in a murine model of ST-elevation myocardial infarction

Abstract Background Cluster of differentiation-14 (CD14) facilitates the excessive pro-inflammatory monocyte-macrophage response to myocardial injury. Both acute and chronic infiltration of pro-inflammatory macrophages are implicated in the immunological progression of ischemic heart failure. However, investigation of clinically practicable strategies of CD14 blockade are yet to be reported. Purpose This fully blinded and randomised preclinical study evaluated the efficacy of a neutralising anti-CD14 antibody given at reperfusion in a murine model of reperfused ST-elevation myocardial infarction (STEMI) with key clinical features. Methods Large anterior STEMI was induced in 48 adult mice by left anterior descending coronary artery occlusion for 1 h prior to reperfusion and randomisation to receive anti-CD14 antibody, isotype control (IgG2a) or saline weekly for four weeks (independently blinded). Outcome measures included functional assessments by 9.4T cardiac magnetic resonance (CMR) imaging and ultra-high-resolution echocardiography at 21- and 28-days post-STEMI, respectively. Results Multi-plane short axis CMR at 21-days post-STEMI revealed greater left ventricular (LV) ejection fraction in the anti-CD14 antibody-treated group compared with saline- and isotype-treated controls (30±1 vs 19±1 and 20±1 %, respectively, p<0.001; Figure 1A). CMR also revealed reduced LV dilatation with anti-CD14-treatment compared to control groups (76±5 vs 102±8 and 107±7 µl EDV in saline- and isotype-treated controls, respectively, p<0.05). Parasternal long axis echocardiography at 28 days post-STEMI confirmed that anti-CD14 antibody treatment was associated with greater LV systolic function compared to saline and isotype controls (30±1 vs 24±1 and 25±2 % EF, respectively, p<0.05; Figure 1B). Preservation of LV end-diastolic (86±4 vs 111±6 and 101±5 µl, p<0.01) and end-systolic (61±3 vs 84±5 and 77±5 µl, p<0.01) volumes was also observed with anti-CD14 treatment. Global longitudinal strain (GLS) of the LV was also significantly greater at 28 days post-STEMI with anti-CD14 treatment relative to saline and isotype controls (-8.5±0.4 vs -6.8±0.3 and -6.6±0.4 %, respectively, p<0.05). Conclusion(s) The findings of this fully blinded and randomised preclinical study provide evidence of a clinically practicable strategy of CD14 blockade initiated at reperfusion to mitigate progressive LV systolic dysfunction and dilatation post-STEMI.Figure 1.Functional outcome measures

The alternative splicing landscape of infarcted mouse heart identifies isoform level therapeutic targets

Alternative splicing is an important process that contributes to highly diverse transcripts and protein products, which can affect the development of disease in various organisms. Cardiovascular disease (CVD) represents one of the greatest global threats to humans, particularly acute myocardial infarction (MI) and subsequent ischemic reperfusion (IR) injury, which involve complex transcriptomic changes in heart tissues associated with metabolic reshaping and immunological response. In this study, we used a newly developed ONT full-length transcriptomic approach and performed transcript-resolved differential expression profiling in murine models of MI and IR. We built an analytical pipeline to reliably identify and quantify alternative splicing products (isoforms), expanding on the currently available catalog of isoforms described in mice. The updated alternative splicing landscape included transcripts, genes, and pathways that were differentially regulated during IR and MI. Our study establishes a pipeline to profile highly diverse isoforms using state-of-the-art long-read sequencing, builds a landscape of alternative splicing in the mouse heart during MI and IR.

Non-invasive in vivo imaging of changes in Collagen III turnover in myocardial fibrosis

Heart failure (HF) affects 64 million people globally with enormous societal and healthcare costs. Myocardial fibrosis, characterised by changes in collagen content drives HF. Despite evidence that collagen type III (COL3) content changes during myocardial fibrosis, in vivo imaging of COL3 has not been achieved. Here, we discovered the first imaging probe that binds to COL3 with high affinity and specificity, by screening candidate peptide-based probes. Characterisation of the probe showed favourable magnetic and biodistribution properties. The probe’s potential for in vivo molecular cardiac magnetic resonance imaging was evaluated in a murine model of myocardial infarction. Using the new probe, we were able to map and quantify, previously undetectable, spatiotemporal changes in COL3 after myocardial infarction and monitor response to treatment. This innovative probe provides a promising tool to non-invasively study the unexplored roles of COL3 in cardiac fibrosis and other cardiovascular conditions marked by changes in COL3.

Abstract We104: A New Small-Molecule ErbB4 Agonist Attenuates Adverse Ventricular Remodeling After Myocardial Infarction In a Sex-Specific Manner

Background: The beneficial effects of the neuregulin/ErbB pathway on the diseased heart have been well-established. The cardioprotective aspects of neuregulin are largely attributable to the activation of the ErbB4 receptor. We recently developed a small-molecule selective ErbB4 agonist, which reduced collagen production in fibroblasts and prevented oxidative-stress induced cardiomyocyte death in vitro . Hypothesis: We hypothesized that the ErbB4 agonist (referred to as compound, Cpd), would attenuate left ventricular remodeling, reduce interstitial cardiac fibrosis and preserve cardiac function in a murine model of myocardial infarction (MI). Since there is a potential interaction between the ErbB4 receptor and the estrogen receptor, we further hypothesized these effects would be sex-dependent. Methods: Mice were divided by sex and then further divided into three experimental groups: a sham group, an MI group receiving Cpd (MI/Cpd), and a control MI group receiving vehicle only (MI/Veh). Mice started treatment with either Cpd (2mg/kg/d) or its vehicle through subcutaneous osmotic minipumps seven days after MI. Cardiac ultrasound was performed weekly until mice were sacrificed after four weeks of treatment. Hearts were excised and stained with Masson’s trichrome to assess interstitial cardiac fibrosis in myocardium remote from the infarction scar. Results: In females (n=25), Cpd significantly decreased left ventricular end-diastolic volume compared to vehicle (Fig.1A, 80±23 µl vs.108±29µl, p=0.045) and induced a slightly increased left ventricular ejection fraction (Fig.2A, 32±12% vs. 22±9%, p=0.086). In addition, Cpd significantly reduced reactive interstitial fibrosis (fibrotic area, Fig.3A, 1.4±1.0% vs. vehicle: 3.8±3.1%, p=0.036) in females. In males (n=30), Cpd had no significant effects on either of the parameters mentioned above (Fig.1B, 2B, 3B). Conclusion: Treatment with the novel small-molecule ErbB4 agonist attenuates left ventricular dilation and reactive interstitial fibrosis after MI in female mice, but not in males. These data support the premise that small molecule-induced ErbB4 activation is a potential new therapy for heart failure, especially in female patients. The mechanisms underlying the sex-dependent effects warrant further exploration.

RNF149 Destabilizes IFNGR1 in Macrophages to Favor Postinfarction Cardiac Repair.

Macrophage-driven inflammation critically involves in cardiac injury and repair following myocardial infarction (MI). However, the intrinsic mechanisms that halt the immune response of macrophages, which is critical to preserve homeostasis and effective infarct repair, remain to be fully defined. Here, we aimed to determine the ubiquitination-mediated regulatory effects on averting exaggerated inflammatory responses in cardiac macrophages. We used transcriptome analysis of mouse cardiac macrophages and bone marrow-derived macrophages to identify the E3 ubiquitin ligase RNF149 (ring finger protein 149) as a modulator of macrophage response to MI. Employing loss-of-function methodologies, bone marrow transplantation approaches, and adenovirus-mediated RNF149 overexpression in macrophages, we elucidated the functional role of RNF149 in MI. We explored the underlying mechanisms through flow cytometry, transcriptome analysis, immunoprecipitation/mass spectrometry analysis, and functional experiments. RNF149 expression was measured in the cardiac tissues of patients with acute MI and healthy controls. RNF149 was highly expressed in murine and human cardiac macrophages at the early phase of MI. Knockout of RNF149, transplantation of Rnf149-/- bone marrow, and bone marrow macrophage-specific RNF149-knockdown markedly exacerbated cardiac dysfunction in murine MI models. Conversely, overexpression of RNF149 in macrophages attenuated the ischemia-induced decline in cardiac contractile function. RNF149 deletion increased infiltration of proinflammatory monocytes/macrophages, accompanied by a hastened decline in reparative subsets, leading to aggravation of myocardial apoptosis and impairment of infarct healing. Our data revealed that RNF149 in infiltrated macrophages restricted inflammation by promoting ubiquitylation-dependent proteasomal degradation of IFNGR1 (interferon gamma receptor 1). Loss of IFNGR1 rescued deleterious effects of RNF149 deficiency on MI. We further demonstrated that STAT1 (signal transducer and activator of transcription 1) activation induced Rnf149 transcription, which, in turn, destabilized the IFNGR1 protein to counteract type-II IFN (interferon) signaling, creating a feedback control mechanism to fine-tune macrophage-driven inflammation. These findings highlight the significance of RNF149 as a molecular brake on macrophage response to MI and uncover a macrophage-intrinsic posttranslational mechanism essential for maintaining immune homeostasis and facilitating cardiac repair following MI.

Energy Metabolism Dysregulation in Myocardial Infarction: An Integrative Analysis of Ischemic Cardiomyopathy.

Myocardial metabolism is closely related to functional changes after myocardial infarction (MI). This study aimed to present an integrative examination of human ischemic cardiomyopathy. We used both GSE121893 single-cell suspension sequencing and GSE19303 transcription microarray data sets from the GEO database, along with a murine MI model for full-spectrum metabolite detection. Through a systematic investigation that involved differential metabolite identification and functional enrichment analysis, we shed light on the pivotal role of energy metabolism dysregulation in the progression of MI. Our findings revealed an association between the core regulatory genes CDKN1A, FOS, ITGB4, and MAP2K1 and the underlying pathophysiology of the disease. These genes are identified as critical elements in the complex landscape of myocardial ischemic disorder, highlighting novel insights into therapeutic targets and the intricate biological mechanisms involved. This analysis provides a framework for future research on the metabolic alterations associated with MI.

The effect of ageing in an experimental model of myocardial infarction in senescent mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Spanish Ministry of Science and Innovation (ISCIII) PI19/01714 and PI22/1083 co-financed by the European Regional Development Fund (ERDF), and by the Generalitat Valenciana. Introduction The murine experimental model of myocardial infarction allows the study of underlying molecular and cellular mechanisms in cardiac function. Specifically, intercellular communication mediated by small non-coding RNAs, microRNAs (miRNAs) which can modulate gene expression post-transcriptionally, is one of the fundamental processes in the investigation of this pathology that needs to be explored in animal models. The Senescence-Accelerated Mouse Prone (SAMP8) and its control strain SAM Resistant (SAMR1) have been established as an experimental model to study ageing-associated vascular dysfunction but scarcely used in epigenetic studies. Purpose To assess the suitability of the SAMR1/SAMP8 ageing model for the study of miRNAs expression following an acute myocardial infarction (AMI), and to determine the optimal time after AMI to measure circulating miRNA levels. Methods Six-month old male and female SAMR1 and SAMP8 (n = 6 per group) underwent AMI by ligation of the left anterior descending coronary artery and sham surgery. All interventions were performed under full anesthesia and analgesia. Animals were euthanized at different times (1h, 4h and 24h) and blood were collected. AMI was confirmed by immediate discoloration of the left ventricle, by ST segment elevation in the electrocardiogram, and by triphenyltetrazolium chloride staining of the non-ischemic areas of heart sections. RNA was obtained from non-hemolyzed serum. Circulating miRNA previously considered as possible AMI biomarkers, miR-1-3p, miR-133-3p, miR-208-3p and miR-499-5p, were measured by qRT-PCR. Results are shown as mean ± SEM of the relative expression (2-ΔΔCt), using U6 snRNA as endogenous control. p-values were calculated using ANOVA and statistical significance was considered when p<0.05. The investigation complies with ethical standards and was approved by the Ethics Committee for Animal Welfare of University of Valencia (2020/VSC/PEA/0128). Results miR-1-3p, miR-133-3p, miR-208-3p and miR-499-5p were significantly increased 4 hours after AMI when compared with sham group in both SAMR1 and SAMP8. It was noteworthy that only with the sham surgery all biomarkers exhibited a time-dependent decrease (p < 0.05). While miR-1-3p rose at 4 hours after AMI (6.46 ± 1.32, p<0.001), miR-133-3p, miR-208-3p and miR-499-5p showed a time-dependent upregulation, being maximal at 24 hours after AMI (p < 0.001 vs. sham group). When comparing samples from SAMR1 and SAMP8, the expression of the analyzed miRNAs was higher in SAMP8 4 hours after AMI (p < 0.05), to a greater extent than in SAMR1. Conclusion Through the use of the SAMR1/SAMP8 mice model, the impact of ageing on miRNA expression following an AMI was notably confirmed, particularly at 4 hours after AMI. Consequently, this study proposes the SAMR1/SAMP8 mouse model as suitable for studying the effects of both AMI and ageing on circulating miRNA expression.

Novel method for induction of myocardial infarction in rats by ultrasound guided electrocoagulation of the left anterior descending artery

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Minerva Imaging Background Murine models of acute myocardial infarction (MI) are commonly established through ligation of the left anterior descending artery (LAD). Despite inherent procedural challenges, including high mortality rates, limited improvements of the induction procedure have been made. Ultrasound-guided electrocoagulation of the LAD could be advantageous by improving animal welfare and decreasing procedural mortality. Purpose To investigate the feasibility of MI induction by ultrasound guided electrocoagulation in rats in comparison to traditional LAD ligation. Methods Thirty-eight female Sprague Dawley rats were randomized to MI by either of the two induction methods leading to 19 rats in each group. Infarct size and left ventricular ejection fraction (LVEF) were evaluated using positron emission tomography (PET) with [18F]fluorodeoxyglucose ([18F]FDG) 9 days post-MI. Myocardial infarct size was categorized as non-infarcted, mild, moderate, or severe based on [18F]FDG uptake. Animal welfare was assessed twice daily for the initial 48h post-surgery using a behavioural scoring system. Results There was no significant difference in the mortality rate of rats with MI induced by electrocoagulation compared to LAD ligation (16% vs 37%, P=0.27). Among the surviving animals both methods produced an equal number of myocardial infarcts (n=5). Electrocoagulation resulted in 1 mild, 2 moderate, and 2 severe MIs, while LAD ligation produced 1 mild, 0 moderate, and 4 severe MIs. Infarct size was similar in the electrocoagulation group compared to the LAD ligation group (mean±SD electrocoagulation: 15±5% vs ligation: 28±13%, P=0.13). The number of non-infarcted surviving rats in the electrocoagulation group was 11 compared to 7 in the LAD ligation group (P=0.33). The LVEF of infarcted rats showed no significant difference between groups (electrocoagulation: 47±8% vs ligation: 38±10%, P=0.14). The LVEF for non-infarcted rats was 53±5%. Post-surgical animal welfare appeared to be improved in the electrocoagulation group, however, the accumulated behavioural score did not reveal a significant difference (median [interquartile range] electrocoagulation: 0 [0-1] vs ligation 2 [0-2], P=0.19). Conclusion Induction of myocardial infarction by ultrasound-guided electrocoagulation is feasible in rats and should be considered as an alternative to LAD ligation. Further development is needed to improve the rate of success and consistency in infarct size.

Cathelicidin Antimicrobial Peptide Levels in Atherosclerosis and Myocardial Infarction in Mice and Human.

Obesity represents a worldwide health challenge, and the condition is accompanied by elevated risk of cardiovascular diseases caused by metabolic dysfunction and proinflammatory adipokines. Among those, the immune-modulatory cathelicidin antimicrobial peptide (human: CAMP; murine: CRAMP) might contribute to the interaction of the innate immune system and metabolism in these settings. We investigated systemic CAMP/CRAMP levels in experimental murine models of atherosclerosis, myocardial infarction and cardiovascular patients. Atherosclerosis was induced in low-density lipoprotein receptor-deficient (Ldlr-/-) mice by high-fat diet (HFD). C57BL/6J wild-type mice were subjected to myocardial infarction by permanent or transient left anterior descending (LAD)-ligation. Cramp gene expression in murine organs and tissues was investigated via real-time PCR. Blood samples of 234 adult individuals with or without coronary artery disease (CAD) were collected. Human and murine CAMP/CRAMP serum levels were quantified by ELISA. Atherosclerotic mice exhibited significantly increased CRAMP serum levels and induced Cramp gene expression in the spleen and liver, whereas experimental myocardial infarction substantially decreased CRAMP serum levels. Human CAMP serum quantities were not significantly affected by CAD while being correlated with leukocytes and pro-inflammatory cytokines. Our data show an influence of cathelicidin in experimental atherosclerosis, myocardial infarction, as well as in patients with CAD. Further studies are needed to elucidate the pathophysiological mechanism.

ALK1 Deficiency Impairs the Wound-Healing Process and Increases Mortality in Murine Model of Myocardial Infarction.

The functional role of TGFβ type I receptor, activin-like kinase (ALK)-1 in post-myocardial infarction (MI) cardiac remodeling is unknown. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after MI. MI was induced in wild-type (WT), and ALK+/- mice by left coronary ligation. After 14 days ALK1+/- mice had reduced survival with a higher rate of cardiac rupture compared to WT mice. ALK1+/- left ventricles (LVs) had increased volumes at the end of systole and at the end of diastole. After MI ALK1+/- LVs had increased profibrotic SMAD3 signaling, type 1 collagen, and fibrosis as well as increased levels of TGFβ1 co-receptor, endoglin, VEGF, and ALK1 ligands BMP9 and BMP10. ALK1+/- LVs had decreased levels of stromal-derived factor 1α. These data identify the critical role of ALK1 in post-MI survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after MI.

Endothelial cells adopt a pro-reparative immune responsive signature during cardiac injury.

Modulation of the heart's immune microenvironment is crucial for recovery after ischemic events such as myocardial infarction (MI). Endothelial cells (ECs) can have immune regulatory functions; however, interactions between ECs and the immune environment in the heart after MI remain poorly understood. We identified an EC-specific IFN responsive and immune regulatory gene signature in adult and pediatric heart failure (HF) tissues. Single-cell transcriptomic analysis of murine hearts subjected to MI uncovered an EC population (IFN-ECs) with immunologic gene signatures similar to those in human HF. IFN-ECs were enriched in regenerative-stage mouse hearts and expressed genes encoding immune responsive transcription factors (Irf7, Batf2, and Stat1). Single-cell chromatin accessibility studies revealed an enrichment of these TF motifs at IFN-EC signature genes. Expression of immune regulatory ligand genes by IFN-ECs suggests bidirectional signaling between IFN-ECs and macrophages in regenerative-stage hearts. Our data suggest that ECs may adopt immune regulatory signatures after cardiac injury to accompany the reparative response. The presence of these signatures in human HF and murine MI models suggests a potential role for EC-mediated immune regulation in responding to stress induced by acute injury in MI and chronic adverse remodeling in HF.

Extracellular vesicles derived from different tissues attenuate cardiac dysfunction in murine MI models

BackgroundExtracellular vesicles (EVs) derived from various cell sources exert cardioprotective effects during cardiac ischemic injury. Our previous study confirmed that EVs derived from ischemic-reperfusion injured heart tissue aggravated cardiac inflammation and dysfunction. However, the role of EVs derived from normal cardiac tissue in myocardial ischemic injury remains elusive.ResultsIn the present study, normal heart-derived EVs (cEVs) and kidney-derived EVs (nEVs) were isolated and intramyocardially injected into mice after myocardial infarction (MI). We demonstrated that administration of both cEVs and nEVs significantly improved cardiac function, reduced the scar size, and alleviated inflammatory infiltration into the heart. In addition, cardiomyocyte apoptosis was inhibited, whereas angiogenesis was enhanced in the hearts receiving cEVs or nEVs treatment. Moreover, intramyocardial injection of cEVs displayed much better cardiac protective efficacy than nEVs in murine MI models. RNA-seq and protein-protein interaction (PPI) network analysis revealed the protective mRNA clusters in both cEVs and nEVs. These mRNAs were involved in multiple signaling pathways, which may synergistically orchestrate to prevent the heart from further damage post MI.ConclusionsCollectively, our results indicated that EVs derived from normal heart tissue may represent a promising strategy for cardiac protection in ischemic heart diseases.

Abstract 17130: Metabolic and Epigenetic Coupled Regulation in Angiogenic Transdifferentiation and Vascular Recovery

Recent studies from our group and others have characterized the phenomenon of "angiogenic transdifferentiation” from fibroblasts to endothelial cells as a novel endogenous mechanism for microvasculature expansion and perfusion restoration in murine models of hindlimb ischemia and myocardial infarction. Metabolic regulation of epigenetic reprogramming has emerged as a key regulatory process controlling cell fate transition. To study the comprehensive role of metabolism in this healing process, we conducted liquid chromatography-mass spectrometry (LC-MS) based untargeted and targeted metabolomics during transdifferentiation. Our analysis revealed the upregulation of key metabolites, including UDP-GlcNAc (substrate for post-translational modification O-GlcNAcylation) and acetyl-CoA (substrate for histone acetylation). With our in vitro transdifferentiation protocol, we found that pharmacologic or genetic inhibition of OGT (O-GlcNAc transferase, the enzyme responsible for protein O-GlcNAcylation) or ACL (ATP citrate lyase, the enzyme converting citrate to acetyl-CoA) in human BJ fibroblasts impairs transdifferentiation. Employing a preclinical peripheral arterial disease (PAD) hindlimb ischemia model with fibroblast lineage tracing mice, we discovered that O-GlcNAcylation and ACL were accumulated in fibroblast lineage cells in ischemic limbs, and pharmacologic inhibition of OGT or ACL diminished the transdifferentiation cell population in the ischemic tissue. Furthermore, we identified fibroblast-specific OGT or ACL KO impaired vascular recovery post femoral artery ligation in the mice. Finally, we showed that metabolism-dependent post-translational modifications on histones (acetylation of H3K9, H3K27, H3K14) or histone chaperons (O-GlcNAcylation of HIRA, a histone H3.3 chaperone) mechanistically enhanced chromatin openness, thereby facilitating cell fate plasticity and vascular regeneration. In conclusion, our studies demonstrate the significant regulatory potential of metabolic factors in angiogenic transdifferentiation which presents an innovative avenue to enhance vascular recovery in ischemic vascular diseases such as PAD.

Abstract 18810: Mitochondria-Rich Extracellular Vesicles From Stem Cell-Derived Cardiomyocytes Restore Bioenergetic Metabolism of Ischemic Myocardium in a Porcine Chronic Heart Failure Model

Introduction: The disruption of mitochondrial energy metabolism underlies the pathophysiology of ischemic heart failure. We previously demonstrated that intramyocardial injection of mitochondria-rich extracellular vesicles (M-EVs) derived from iPSC-derived cardiomyocytes (iCMs) transfers mitochondria and non-mitochondrial biologics into recipient cardiomyocytes. This process stimulates mitochondrial biogenesis and enhances functional capacities in murine and porcine acute myocardial infarction (MI) models. However, the therapeutic potential and underlying mechanism in chronic heart failure models remain to be assessed. Method: M-EVs were isolated from the conditioned medium of human iCMs using differential centrifugation. A total of 11 Yorkshire pigs underwent myocardial ischemia for one hour via balloon occlusion of the left anterior descending artery (Week-0). At week-4, 1.0 x 10 11 M-EVs or PBS control were administered transendocardially to the peri-infarct region (PIR) using a percutaneous catheter delivery system (Biocardia, Inc, CA). Cardiac functions and scar size were analyzed by cardiac MRI with gadolinium contrast at week-4 (pre-injection) and -8 (post-injection). Myocardial tissue samples were collected from the PIR and remote non-ischemic areas at week-8. Proteomic analysis and single-nucleus RNA sequencing were performed to investigate comprehensive protein and gene expression profiles of the myocardium. Results: Left ventricular (LV) ejection fractions were significantly decreased at Week-4 (32.7±5.4%). M-EV treatment significantly improved absolute LVEF from week-4 to -8 in the treatment vs. non-treatment groups, +3.15% vs. -4.99%, respectively, (p<0.0001). No significant difference was observed in the scar size. Multiomic analysis of proteomes and single-nucleus transcriptomes suggested restoration of dysregulated cardiometabolic protein profile and mitochondrial energy metabolism in the myocardium. Conclusion: M-EV-mediated transfer of mitochondria and enriched proteins augmented LV dysfunction in the pig chronic MI model. M-EV therapy may represent a novel biologic approach that directly targets cardiac metabolism by restoring mitochondrial function and cellular bioenergetics.

Abstract 11966: Translational Cardioinformatics: Neutrophil-Derived Translocator Protein as a Novel Regulator of Myocarditis and Coronary Artery Aneurysm in Kawasaki Disease and Multisystem Inflammatory Syndrome in Children

Introduction: Cardiovascular disease (CVD) in Kawasaki disease (KD) and multisystem inflammatory syndrome in children (MIS-C) lead to increased mortality through myocardial infarction. Hypothesis: Since KD and MIS-C are shaped by the same immune continuum, the cardiac pathologies and lethal complications may be averted by targeting a key regulator controlling both diseases; however, the pathogenic drivers are largely undefined. Methods: Through integrated, multi-cohort, single-cell meta-analysis of KD and MIS-C PBMC scRNA-seq profiles, we investigated the immunological drivers associated with systemic damage and cardiac pathologies, compared with other pediatric diseases, and validated across independent cohorts of KD, MIS-C, and myocardial infarction. Via cheminformatics approaches, we identified FDA-approved drugs for repurposing. Results: We found the expansion and hyperactivation of CD177 + neutrophils in KD and MIS-C, but not in other pediatric diseases. CD177 + neutrophils overexpressed markers and causal mediators of CVD development, supported by activation of prognostic CVD gene signatures (acute CVD, major adverse cardiovascular and limb events, and myocardial infarction). CD177 + neutrophils’ overactivated effector repertoire may mediate systemic inflammation and molecular damage via a vascular homing mechanism. We identified a dysfunctional SPI1-dependent regulatory network shared by KD and MIS-C during neutrophil hyperactivation, as well as key regulators which may induce cardiac disorders. Importantly, we found that TSPO is associated with myocarditis and coronary artery aneurysm, disease severity, IVIG treatment, and patient recovery. Via independent cohorts analyses, we validated TSPO as a prime target against KD and MIS-C pathologies. Murine myocardial infarction model confirmed the cardiac hyper-infiltration of hyperactivated CD177 + neutrophils. Lastly, we identified FDA-approved drugs against KD and MIS-C neutrophil hyperactivation. Conclusions: Collectively, CD177 + neutrophils have a pivotal role in KD/MIS-C systemic pathologies, clinical severity, and prognosis. We suggest drugs that can be developed as a single therapeutic strategy against either KD or MIS-C for clinical translation.

Danlou Tablet Protects Against Myocardial Infarction Through Promoting eNOS-Dependent Endothelial Protection and Angiogenesis.

Myocardial infarction (MI) is one of the leading causes of death worldwide. Danlou tablet (Dan) is an effective traditional Chinese medicine for cardiac protection, although the underlying mechanism was not fully understood. In this study, we used a murine MI model and demonstrated that Dan administration effectively attenuated myocardial apoptosis, cardiac remodeling, and heart failure post MI. Dan increased CD31-positive capillaries in MI hearts, and reduced the apoptosis and oxidative stress in human umbilical vein endothelial cells after oxygen-glucose deprivation stress, simultaneously with the activated HIF-1α/VEGFA/eNOS signaling. Moreover, inhibition of eNOS by L-NAME attenuated Dan-induced protection against MI, and abolished its effect in promoting angiogenesis and reducing endothelial apoptosis and oxidative stress. Collectively, Dan is beneficial to promote eNOS-dependent endothelial protection and angiogenesis thus protecting against MI. A deep understanding of Dan-induced protection might help promote clinical usage of Dan in MI treatment.

Exosomes secreted by endothelial cells derived from human induced pluripotent stem cells improve recovery from myocardial infarction in mice

BackgroundHuman induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) exhibit the potential to repair the injured heart after myocardial infarction (MI) by promoting neovascularization and cardiomyocyte survival. However, because of the low cellular retention and poor engraftment efficacy, cell therapy of MI is partly mediated by exosomes secreted from the transplanted cells. In this study, we investigated whether exosomes secreted from hiPSC-ECs could become a promising acellular approach to repair the infarcted heart after MI and elucidated the underlying protective mechanism.MethodsThe hiPSC-ECs were differentiated, and exosomes were isolated in vitro. Then, hiPSC-EC exosomes were delivered by intramyocardial injection in a murine MI model in vivo. Echocardiography, combined with hemodynamic measurement, histological examination, Ca2+ transient and cell shortening assessment, and Western blot, was used to determine the protective effects of hiPSC-EC exosomes on the infarcted heart. Furthermore, microRNA sequencing, luciferase activity assay, and microRNA gain–loss function experiments were performed to investigate the enriched microRNA and its role in exosome-mediated effects.ResultsIn vitro, the hiPSC-EC exosomes enhanced intracellular Ca2+ transients, increased ATP content, and improved cell survival to protect cardiomyocytes from oxygen–glucose deprivation injury. Congruously, hiPSC-EC exosome administration in vivo improved the myocardial contractile function and attenuated the harmful left ventricular remodeling after MI without increasing the frequency of arrhythmias. Mechanistically, the hiPSC-EC exosomes notably rescued the protein expression and function of the sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA-2a) and ryanodine receptor 2 (RyR-2) to maintain intracellular Ca2+ homeostasis and increase cardiomyocyte contraction after MI. The microRNA sequencing showed that miR-100-5p was the most abundant microRNA in exosomes. miR-100-5p could target protein phosphatase 1β (PP-1β) to enhance phospholamban (PLB) phosphorylation at Ser16 and subsequent SERCA activity, which contributes to the hiPSC-EC exosome-exerted cytoprotective effects on maintaining intracellular Ca2+ homeostasis and promoting cardiomyocyte survival.ConclusionThe hiPSC-EC exosomes maintain cardiomyocyte Ca2+ homeostasis to improve myocardial recovery after MI, which may provide an acellular therapeutic option for myocardial injury.

Amplified inhibition of atherosclerotic plaque-induced platelet activation by glenzocimab with dual antiplatelet therapy

BackgroundAspirin and platelet P2Y12 inhibitors, such as ticagrelor, suboptimally inhibit microvascular thrombosis during ST-elevation myocardial infarction. Glycoprotein (GP) IIb/IIIa inhibitors may further inhibit this but cause excessive bleeding. ObjectivesWe investigated whether combination of glenzocimab, a GPVI inhibitor, with aspirin and ticagrelor provides additional antithrombotic effects, as GPVI has a critical role in atherothrombosis but minimal involvement in hemostasis. MethodsWe investigated the effects of glenzocimab (monoclonal antibody Fab fragment) using blood from healthy donors and patients with acute coronary syndrome treated with aspirin and ticagrelor. Platelets were stimulated with multiple agonists, including atherosclerotic plaque, from patients undergoing carotid endarterectomy. ResultsAspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation by 48% compared with control (34 ± 3 vs 65 ± 4 U; P < .001). Plaque-induced platelet aggregation, adhesion, secretion, and activation were critically dependent on GPVI activation. Glenzocimab alone reduced plaque-induced aggregation by 75% compared with control (16 ± 4 vs 65 ± 4 U; P < .001) and by >95% when combined with aspirin and ticagrelor (3 ± 1 vs 65 ± 4 U; P < .001). Glenzocimab reduced platelet aggregation, adhesion, and thrombin generation when added to blood of aspirin- and ticagrelor-treated patients with acute coronary syndrome. Glenzocimab shared several antithrombotic effects with the GPIIb/IIIa inhibitor eptifibatide with less effect on general hemostasis assessed by rotational thromboelastometry. In a murine intravital model of ST-elevation myocardial infarction, genetic depletion of GPVI reduced microvascular thrombosis. ConclusionAddition of glenzocimab to aspirin and ticagrelor enhances platelet inhibition via multiple mechanisms of atherothrombosis. Compared with a GPIIb/IIIa inhibitor, glenzocimab shares multiple antithrombotic effects, with less inhibition of mechanisms involved in general hemostasis.