DYRK激酶家族的抑制剂在心血管疾病领域的应用或潜在治疗策略

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) play a large variety of cellular functions and are involved in several diseases (cognitive disorders, diabetes, cancers, etc.). There is, thus, growing interest in pharmacological inhibitors as chemical probes and potential drug candidates. This study presents an unbiased evaluation of the kinase inhibitory activity of a library of 56 reported DYRK/CLK inhibitors on the basis of comparative, side-by-side, catalytic activity assays on a panel of 12 recombinant human kinases, enzyme kinetics (residence time and Kd), in-cell inhibition of Thr-212-Tau phosphorylation, and cytotoxicity. The 26 most active inhibitors were modeled in the crystal structure of DYRK1A. The results show a rather large diversity of potencies and selectivities among the reported inhibitors and emphasize the difficulties to avoid "off-targets" in this area of the kinome. The use of a panel of DYRKs/CLKs inhibitors is suggested to analyze the functions of these kinases in cellular processes.

Backgrounds: Coronary heart disease (CHD) is a leading cause of morbidity and mortality worldwide, with myocardial infarction (MI) representing its most prevalent type. Previous studies have suggested an association between the dual-specificity tyrosine-regulated kinases ( DYRK) gene family and various cardiovascular disease risks. However, the specific role of DYRK4 in CHD, particularly in MI, remains unclear. Objective: We hereby investigated the association between blood-based DYRK4 methylation and CHD, MI and non-MI related CHD. Methods: A total of 88 CHD cases (23 MI and 65 non-MI) were diagnosed by coronary angiography and clinical presentation at Chinese People's Liberation Army General Hospital from 2018 to 2019. 93 persons who participated in the annual health examination were randomly selected as controls. The DYRK4 methylation, which contains three measurable CpG sites, was quantified by mass spectrometry of nucleic acid. Covariates-adjusted odds ratios (ORs) for -10% methylation was calculated by binary logistic regression, and receiver operating characteristic (ROC) curves were generated to evaluate the discriminatory power. Results: Decreased methylation of DYRK4_CpG_2 and DYRK4_CpG_3 in peripheral blood was significantly associated with increased CHD risk (OR: 1.49 and 1.20; p = 0.044 and 0.049). Notably, these two CpG sites were only significant in non-MI related CHD (OR: 1.71 and 1.33; p = 0.015 and 0.009) rather than MI (OR: 1.11 and 0.99; p = 0.759 and 0.968). The combination of DYRK4_CpG_2 and DYRK4_CpG_3 methylation levels could efficiently discriminate CHD, MI and non-MI related CHD patients from controls (area under curve (AUC) = 0.771, 0.795 and 0.784, respectively). Conclusion: Our study suggests that hypomethylation of DYRK4_CpG_2, DYRK4_CpG_3 in blood is associated with CHD, and DYRK4 methylation model may provide a new strategy for detection of CHD, especially non-MI related CHD.

Summary Background While the adult mammalian heart undergoes only modest renewal through cardiomyocyte proliferation, boosting this process is considered a promising therapeutic strategy to repair cardiac injury. This study explored the role and mechanism of dual-specificity tyrosine regulated kinase 1A (DYRK1A) in regulating cardiomyocyte cell cycle activation and cardiac repair after myocardial infarction (MI). Methods DYRK1A-knockout mice and DYRK1A inhibitors were used to investigate the role of DYRK1A in cardiomyocyte cell cycle activation and cardiac repair following MI. Additionally, we explored the underlying mechanisms by combining genome-wide transcriptomic, epigenomic, and proteomic analyses. Findings In adult mice subjected to MI, both conditional deletion and pharmacological inhibition of DYRK1A induced cardiomyocyte cell cycle activation and cardiac repair with improved cardiac function. Combining genome-wide transcriptomic and epigenomic analyses revealed that DYRK1A knockdown resulted in robust cardiomyocyte cell cycle activation (shown by the enhanced expression of many genes governing cell proliferation) associated with increased deposition of trimethylated histone 3 Lys4 (H3K4me3) and acetylated histone 3 Lys27 (H3K27ac) on the promoter regions of these genes. Mechanistically, via unbiased mass spectrometry, we discovered that WD repeat-containing protein 82 and lysine acetyltransferase 6A were key mediators in the epigenetic modification of H3K4me3 and H3K27ac and subsequent pro-proliferative transcriptome and cardiomyocyte cell cycle activation. Interpretation Our results reveal a significant role of DYRK1A in cardiac repair and suggest a drug target with translational potential for treating cardiomyopathy. Funding This study was supported in part by grants from the National Natural Science Foundation of China (81930008, 82022005, 82070296, 82102834), National Key R&D Program of China (2018YFC1312700), Program of Innovative Research Team by the National Natural Science Foundation (81721001), and National Institutes of Health (5R01DK039308-31, 7R37HL023081-37, 5P01HL074940-11).

Background: DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. Methods: We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. Results: We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10−3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10−2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10−2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. Conclusions: The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Cardiac fibroblasts (CFs) are a critical cell population responsible for myocardial extracellular matrix homeostasis. After stimulation by myocardial infarction (MI), CFs transdifferentiate into cardiac myofibroblasts (CMFs) and play a fundamental role in the fibrotic healing response. Transient receptor potential ankyrin 1 (TRPA1) channels are cationic ion channels with a high fractional Ca2+ current, and they are known to influence cardiac function after MI injury; however, the molecular mechanisms regulating CMF transdifferentiation remain poorly understood. TRPA1 knockout mice, their wild-type littermates, and mice pretreated with the TRPA1 agonist cinnamaldehyde (CA) were subjected to MI injury and monitored for survival, cardiac function, and fibrotic remodeling. TRPA1 can drive myofibroblast transdifferentiation initiated 1 week after MI injury. In addition, we explored the underlying mechanisms via in vitro experiments through gene transfection alone or in combination with inhibitor treatment. TRPA1 overexpression fully activated CMF transformation, while CFs lacking TRPA1 were refractory to transforming growth factor β- (TGF-β-) induced transdifferentiation. TGF-β enhanced TRPA1 expression, which promoted the Ca2+-responsive activation of calcineurin (CaN). Moreover, dual-specificity tyrosine-regulated kinase-1a (DYRK1A) regulated CaN-mediated NFAT nuclear translocation and TRPA1-dependent transdifferentiation. These findings suggest a potential therapeutic role for TRPA1 in the regulation of CMF transdifferentiation in response to MI injury and indicate a comprehensive pathway driving CMF formation in conjunction with TGF-β, Ca2+ influx, CaN, NFATc3, and DYRK1A.

The adult mammalian heart has a limited ability to regenerate lost myocardium following myocardial infarction (MI), largely due to the poor proliferative capacity of cardiomyocytes. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a known regulator of cell quiescence, though the mechanisms underlying its function remain unclear. Previous studies have shown that pharmacological inhibition of DYRK1A using harmine induces cardiomyocyte cell cycle re-entry after ischemia/reperfusion (I/R) MI. Here, we developed a computational network model of DYRK1A-mediated regulation of the cell cycle, which predicts how DYRK1A inhibition promotes cardiomyocyte re-entry. To validate these predictions, we tested selective DYRK1A inhibitors and observed robust induction of cell cycle activity in neonatal rat cardiomyocytes (NRCMs). Integrating our network model with bulk RNA-sequencing data from DYRK1A inhibitor-treated NRCMs, we identified E2F1 as a key transcriptional driver of cell cycle gene expression. Finally, we demonstrate that both pharmacological and post-developmental inhibition of DYRK1A enhances heart function and increases cardiomyocyte cycling following I/R MI. Our findings suggest that functional recovery induced by small molecule inhibitor of DYRK1A is mediated by the induction of cycling cardiomyocytes. One Sentence Summary Inhibition of DYRK1A through LCTB-92 induces cardiomyocyte cycling and improved heart function in a mouse model of ischemic/reperfusion injury.

No abstract available

Background Recent evidence highlights the epidemiological value of blood DNA methylation (DNAm) as surrogate biomarker for exposure to risk factors for non-communicable diseases (NCD). DNAm surrogate of exposures predicts diseases and longevity better than self-reported or measured exposures in many cases. Consequently, disease prediction models based on blood DNAm surrogates may outperform current state-of-the-art prediction models. This study aims to develop novel DNAm surrogates for cardiovascular diseases (CVD) risk factors and develop a composite biomarker predictive of CVD risk. We compared the prediction performance of our newly developed risk score with the state-of-the-art DNAm risk scores for cardiovascular diseases, the ‘next-generation’ epigenetic clock DNAmGrimAge, and the prediction model based on traditional risk factors SCORE2. Results Using data from the EPIC Italy cohort, we derived novel DNAm surrogates for BMI, blood pressure, fasting glucose and insulin, cholesterol, triglycerides, and coagulation biomarkers. We validated them in four independent data sets from Europe and the USA. Further, we derived a DNAmCVDscore predictive of the time-to-CVD event as a combination of several DNAm surrogates. ROC curve analyses show that DNAmCVDscore outperforms previously developed DNAm scores for CVD risk and SCORE2 for short-term CVD risk. Interestingly, the performance of DNAmGrimAge and DNAmCVDscore was comparable (slightly lower for DNAmGrimAge, although the differences were not statistically significant). Conclusions We described novel DNAm surrogates for CVD risk factors useful for future molecular epidemiology research, and we described a blood DNAm-based composite biomarker, DNAmCVDscore , predictive of short-term cardiovascular events. Our results highlight the usefulness of DNAm surrogate biomarkers of risk factors in epigenetic epidemiology to identify high-risk populations. In addition, we provide further evidence on the effectiveness of prediction models based on DNAm surrogates and discuss methodological aspects for further improvements. Finally, our results encourage testing this approach for other NCD diseases by training and developing DNAm surrogates for disease-specific risk factors and exposures.

Background DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. Results Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1–5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. Conclusions The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.

No abstract available

ABSTRACT Heart failure (HF) is a major global health challenge, contributing to over 18 million deaths annually. While the roles of genetic and environmental factors are widely studied, the role of DNA methylation in HF pathogenesis is not fully understood. This study leverages the Hybrid Mouse Diversity Panel (HMDP) to investigate the relationship between DNA methylation, gene expression, and HF phenotypes under isoproterenol-induced cardiac stress. Using reduced representational bisulfite sequencing, we analyzed DNA methylation profiles in the left ventricles of 90 HMDP strains. Epigenome-wide association studies identified 56 CpG loci linked to HF phenotypes, with 18 loci predicting HF progression. Key genes, including Prkag2, Anks1a, and Mospd3, were implicated through integration with gene expression and phenotypic data. In vitro validation confirmed the roles of Anks1aand Mospd3 in attenuating isoproterenol-induced hypertrophy. Additionally, treatment with the DNA methyltransferase inhibitor RG108 mitigated cardiac hypertrophy, preserved ejection fraction, and restored methylation-sensitive gene expression, underscoring the therapeutic potential of targeting DNA methylation in HF. This study highlights the interplay between DNA methylation, gene expression, and HF progression, offering new insights into its molecular underpinnings. The findings emphasize the role of epigenetic regulation in HF and suggest DNA methylation as a promising target for therapeutic intervention. Key policy highlights The epigenetics of Heart Failure has been difficult to study in humans, and consequently we have performed a study in mice where cardiac tissue is far more readily available Epigenome-wide association studies of heart failure traits across the lines of the Hybrid Mouse Diversity Panel uncovered 56 genome-wide significant loci for cardiac phenotypes, including loci in healthy animals that predicted later heart failure prognosis. Study of candidate genes using cell models revealed numerous genes with functionally relevant effects on cardiac cell size and abundance Use of a DNA methyltransferase inhibitor in certain mouse strains had a strongly protective effect against heart failure progression, suggesting future therapeutic potential.

BACKGROUND AND AIMS Metals serve as co-factors for a host of metalloenzymes involved in mitochondrial metabolic reprogramming. Modifications in metal homeostasis are linked to epigenetic mechanisms. However, the epigenetic mechanisms through which metal affects cardiac fibrosis (CF) remain poorly understood. METHODS The metal content of mouse heart samples was measured using inductively coupled plasma mass spectrometry. Cardiac fibroblast-specific MeCP2-deficient mice and control mice were treated with isoprenaline/angiotensin II to induce CF. AAV9 carrying POSTN promoter-driven small hairpin RNA targeting MeCP2, YTHDF1, or SLC31A1 and the copper-chelating agent tetrathiomolybdate were administered to investigate their vital roles in CF. Histological and biochemical analyses were performed to determine how YTHDF1/MeCP2 regulated SLC31A1 expression in CF. The reconstitution of SLC31A1 in YTHDF1/MeCP2-deficient cardiac fibroblasts and mouse hearts was performed to study its effect on mitochondrial copper depletion and fibrosis. Human heart tissues from atrial fibrillation patients were used to validate the findings. RESULTS Lower copper concentrations are accompanied by SLC31A1 down-regulation and mitochondrial copper depletion in CF. Fibroblast-specific SLC31A1 deficiency enhances mitochondrial copper depletion, augments glycolysis, promotes fibroblast proliferation and triggers CF. SLC31A1 inhibition due to increased MeCP2-recognized methylating CpG islands of SLC31A1 in the promoter region restrains its transcription. Conversely, MeCP2 knockdown rescued SLC31A1 expression, resulting in contradictory effects. MeCP2 up-regulation is associated with elevated m6A mRNA levels. Mechanistically, YTHDF1 recognizes target MeCP2 mRNA and induces its translation. In human heart tissues from atrial fibrillation patients, reduced copper concentrations and SLC31A1 expression, along with elevated levels of YTHDF1 and MeCP2, were observed. These changes were associated with mitochondrial copper depletion, enhanced glycolysis, and CF. CONCLUSIONS A novel epigenetic mechanism was demonstrated through which copper deficiency increases mitochondrial copper depletion and impairs CF. Findings provide new insights for the development of preventive measures for CF.

BACKGROUND Diabetic cardiomyopathy (DCM) represents an important concern associated with diabetes, inhibiting pyroptosis has shown promising results in alleviating DCM symptoms. The objective of this work is to investigate the role and underlying mechanism of hydrogen sulfide (H2S) in the suppression of pyroptosis in the context of diabetic myocardial fibrosis (MF). METHODS The effect of H2S on pyroptosis was detected using CCK-8, ELISA, and flow cytometry. The expression of Sestrin2 and its DNA methylation modification, as well as the quantification of pyroptosis-related proteins and the activation of the TLR4/MyD88/NF-κB signaling pathway, were measured using Western blot, ChIP, Immunofluorescence, and methylation-specific quantitative PCR. To establish a type 2 diabetes rat model, a high-fat diet was administered, followed by injection of streptozotocin (HFD/STZ). After five weeks, the rats received H2S treatment for four weeks, either with or without sh-Sestrin2. The effects of H2S treatment on myocardial function, tissue structure, and myocardial cell apoptosis were assessed. Furthermore, the CCK-8 assay was used to detect the cell viability induced by TGF-β. Cellular oxidative stress levels were measured by the ELISA method. Western blotting was applied to determine the protein expression levels of Collagen I, Fibronectin, α-SMA, NLRP3, caspase-1, GSDMD-N, and Sestrin2. RESULTS H2S ameliorates HG-induced fibrosis, injury, pyroptosis, and inflammatory response of cardiac fibroblasts (CFs). H2S inhibits Sestrin2 methylation and up-regulates its expression through DNMT3a. H2S ameliorates HG-induced CFs pyroptosis and fibrosis through Sestrin2. Sestrin2 regulates HG-induced CFs pyroptosis and fibrosis through the TLR4/MyD88/NF-κB pathway. The myocardial tissue injury was improved after H2S treatment, and the results of the H2S treatment were reversed after knockdown of Sestrin2. In addition, TGF-β1 can induce an increase in the activity of cardiac fibroblasts and enhance cellular oxidative stress. Meanwhile, it significantly upregulates the expression of Collagen I, Fibronectin, α-SMA, as well as NLRP3, caspase-1, and GSDMD-N, and remarkably downregulates the expression of Sestrin2. However, H2S intervention can reverse the aforementioned phenomena. CONCLUSION H2S can suppress pyroptosis and ameliorate diabetic MF by inhibiting DNMT3a-mediated DNA methylation of Sestrin2 promoter through the TLR4/MyD88/NF-κB pathway.

Background Cardiovascular disease (CVD) is the leading cause of death worldwide and considered one of the most environmentally driven diseases. The role of DNA methylation in response to the individual exposure for the development and progression of CVD is still poorly understood and a synthesis of the evidence is lacking. Results A systematic review of articles examining measurements of DNA cytosine methylation in CVD was conducted in accordance with PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines. The search yielded 5,563 articles from PubMed and CENTRAL databases. From 99 studies with a total of 87,827 individuals eligible for analysis, a database was created combining all CpG-, gene- and study-related information. It contains 74,580 unique CpG sites, of which 1452 CpG sites were mentioned in ≥ 2, and 441 CpG sites in ≥ 3 publications. Two sites were referenced in ≥ 6 publications: cg01656216 (near ZNF438 ) related to vascular disease and epigenetic age, and cg03636183 (near F2RL3 ) related to coronary heart disease, myocardial infarction, smoking and air pollution. Of 19,127 mapped genes, 5,807 were reported in ≥ 2 studies. Most frequently reported were TEAD1 (TEA Domain Transcription Factor 1) and PTPRN2 (Protein Tyrosine Phosphatase Receptor Type N2) in association with outcomes ranging from vascular to cardiac disease. Gene set enrichment analysis of 4,532 overlapping genes revealed enrichment for Gene Ontology molecular function “DNA-binding transcription activator activity” ( q  = 1.65 × 10^–11) and biological processes “skeletal system development” ( q  = 1.89 × 10^–23). Gene enrichment demonstrated that general CVD-related terms are shared, while “heart” and “vasculature” specific genes have more disease-specific terms as PR interval for “heart” or platelet distribution width for “vasculature.” STRING analysis revealed significant protein–protein interactions between the products of the differentially methylated genes ( p  = 0.003) suggesting that dysregulation of the protein interaction network could contribute to CVD. Overlaps with curated gene sets from the Molecular Signatures Database showed enrichment of genes in hemostasis ( p  = 2.9 × 10^–6) and atherosclerosis ( p  = 4.9 × 10^–4). Conclusion This review highlights the current state of knowledge on significant relationship between DNA methylation and CVD in humans. An open-access database has been compiled of reported CpG methylation sites, genes and pathways that may play an important role in this relationship.

BACKGROUND Existing analyses with conventional assays have generated significant insights into static states of DNA methylation but were unable to visualize the dynamics of epigenetic regulation. MATERIALS & RESULTS We utilized a genomic DNA methylation reporter (GMR) system carrying Snrpn minimal promoter and CpG regions of Cdk1 (Cyclin-dependent kinase 1) or Sox2 (SRY-Box Transcription Factor 2). Mouse Sox2 GMR iPSCs rapidly lost fluorescent reporter signal upon the induction of cardiac differentiation. Cdk1 GMR reporter signal was strong in undifferentiated iPSCs, and gradually decreased during cardiomyocyte differentiation. RT-qPCR and pyrosequencing demonstrated that the reduction of Sox2 and Cdk1 was regulated by hypermethylation of their promoters' CpG regions during cardiac differentiation. CONCLUSION The GMR reporter system can be useful for monitoring real-time epigenetic DNA modification at single-cell resolution.

Anthracycline-induced cardiomyopathy is a leading cause of late morbidity in childhood cancer survivors. Aberrant DNA methylation plays a role in de novo cardiovascular disease. Epigenetic processes could play a role in anthracycline-induced cardiomyopathy but remain unstudied. We sought to examine if genome-wide differential methylation at ‘CpG’ sites in peripheral blood DNA is associated with anthracycline-induced cardiomyopathy. This report used participants from a matched case–control study; 52 non-Hispanic White, anthracycline-exposed childhood cancer survivors with cardiomyopathy were matched 1:1 with 52 survivors with no cardiomyopathy. Paired ChAMP (Chip Analysis Methylation Pipeline) with integrated reference-based deconvolution of adult peripheral blood DNA methylation was used to analyze data from Illumina HumanMethylation EPIC BeadChip arrays. An epigenome-wide association study (EWAS) was performed, and the model was adjusted for GrimAge, sex, interaction terms of age at enrollment, chest radiation, age at diagnosis squared, and cardiovascular risk factors (CVRFs: diabetes, hypertension, dyslipidemia). Prioritized genes were functionally validated by gene knockout in human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 technology. DNA-methylation EPIC array analyses identified 32 differentially methylated probes (DMP: 15 hyper-methylated and 17 hypo-methylated probes) that overlap with 23 genes and 9 intergenic regions. Three hundred and fifty-four differential methylated regions (DMRs) were also identified. Several of these genes are associated with cardiac dysfunction. Knockout of genes EXO6CB , FCHSD2, NIPAL2, and SYNPO2 in hiPSC-CMs increased sensitivity to doxorubicin. In addition, EWAS analysis identified hypo-methylation of probe ‘cg15939386’ in gene RORA to be significantly associated with anthracycline-induced cardiomyopathy. In this genome-wide DNA methylation profile study, we observed significant differences in DNA methylation at the CpG level between anthracycline-exposed childhood cancer survivors with and without cardiomyopathy, implicating differential DNA methylation of certain genes could play a role in pathogenesis of anthracycline-induced cardiomyopathy.

Maternal obesity during pregnancy significantly increases the offspring’s risk of later-life cardiovascular disease. This study investigated cardiometabolic perturbations by utilizing a mouse model of maternal high-fat diet (HFD)-induced obesity that recapitulates metabolic abnormalities observed in humans. We report that offspring of HFD-fed mothers (Off-HFD) exhibit a progression of obesity, hypertension, dyslipidemia, and metabolic inflexibility when compared with offspring of regular diet-fed mothers (Off-RD). Deeper investigation of cardiac function further identified significant functional, metabolic, and immune perturbations in adult offspring of mothers on HFD. Specifically, Off-HFD mice presented progressing cardiac hypertrophy with reduced ejection fraction, increased accumulation of fibrotic tissue, mitochondrial dysfunction, and altered immune complexity including increases in cardiac resident and infiltrated macrophages, and decreases in CD4+ and CD8+ T-cell subpopulations. While these alterations may not be immediately catastrophic, they likely predispose the offspring to heightened sensitivity to nutritional, psychological, or environmental stressors. Analysis of DNA methylation in the hearts of newly-weaned offspring from RD and HFD mothers revealed numerous differentially methylated CpGs and regions within genes associated with cardiac development, hypertrophy, mitochondrial function, and immune response. Thus, our study shows epigenetic remodeling early in development, which is likely responsible for the cardiovascular dysregulation observed in adult life. These findings uncover potential windows of opportunity for preventive therapy and early therapeutic interventions.

Ambient fine particulate matter (PM2.5) exposure is well-documented for cardiovascular risks, however, limited evidence regarding the underlying mechanisms on cardiac conduction in the elderly, especially regarding longitudinal impacts. We aimed to identify key toxic inorganic elemental constituents in PM2.5 linked to cardiac electrophysiological abnormalities, DNA methylation-mediated pathways and the complex interplay with gene expression. The associations of PM2.5 and its inorganic chemical constituents with electrocardiography (ECG) parameters were analyzed in 348 measurements of the healthy elderly by mixture exposure models. Epigenomic analyses and bidirectional mediation analyses were conducted to explore the effect patterns of epigenetic changes. Integrated analysis were performed to identify the potential biological pathways. Mixture exposure models identified S and Pb as major contributors to prolonged QRS duration and QTc interval. Bidirectional mediation analysis combined high-dimension mediating analysis revealed 43 cytosine-phosphoguanine sites (CpGs) significantly mediated the association between exposure to PM2.5, Pb, and S and ECG parameters, with about 90% showing weaker reverse mediation. Cis-eQTM analysis showed that PM2.5 and S elements associated DMPs modulated downstream gene expression, influencing ECG parameters. Specifically, four CpGs and their annotated gene expressions (HMBOX1, C1orf109, ATAD3A, and RINL) showed consistent effects on these associations, collectively involving multiple pathways, including the antigen presentation, ceramide accumulation, and circadian rhythm. This study elucidates that the incorporation of integrated epigenomic and transcriptomic profiles may provide novel insights into environmental origins and promising biomarkers for the clinical treatment of cardiovascular diseases.

Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure.

Untreated fresh cardiac tissue is the optimal tissue material for investigating DNA methylation patterns of cardiac biology and diseases. However, fresh tissue is difficult to obtain. Therefore, tissue stored as frozen or formalin-fixed, paraffin-embedded (FFPE) is widely used for DNA methylation studies. It is unknown whether storage conditions alter the DNA methylation in cardiac tissue. In this study, we compared the DNA methylation patterns of fresh, frozen, and FFPE cardiac tissue to investigate if the storage method affected the DNA methylation results. We used the Infinium MethylationEPIC assay to obtain genome-wide methylation levels in fresh, frozen, and FFPE tissues from nine individuals. We found that the DNA methylation levels of 21.4% of the examined CpG sites were overestimated in the FFPE samples compared to that of fresh and frozen tissue, whereas 5.7% were underestimated. Duplicate analyses of the DNA methylation patterns showed high reproducibility (precision) for frozen and FFPE tissues. In conclusion, we found that frozen and FFPE tissues gave reproducible DNA methylation results and that frozen and fresh tissues gave similar results.

Rett syndrome (RTT) is a rare and severe neurological disorder mainly affecting females, usually linked to methyl-CpG-binding protein 2 (MECP2) gene mutations. Manifestations of RTT typically include loss of purposeful hand skills, gait and motor abnormalities, loss of spoken language, stereotypic hand movements, epilepsy, and autonomic dysfunction. Patients with RTT have a higher incidence of sudden death than the general population. Literature data indicate an uncoupling between measures of breathing and heart rate control that could offer insight into the mechanisms that lead to greater vulnerability to sudden death. Understanding the neural mechanisms of autonomic dysfunction and its correlation with sudden death is essential for patient care. Experimental evidence for increased sympathetic or reduced vagal modulation to the heart has spurred efforts to develop quantitative markers of cardiac autonomic profile. Heart rate variability (HRV) has emerged as a valuable non-invasive test to estimate the modulation of sympathetic and parasympathetic branches of the autonomic nervous system (ANS) to the heart. This review aims to provide an overview of the current knowledge on autonomic dysfunction and, in particular, to assess whether HRV parameters can help unravel patterns of cardiac autonomic dysregulation in patients with RTT. Literature data show reduced global HRV (total spectral power and R-R mean) and a shifted sympatho-vagal balance toward sympathetic predominance and vagal withdrawal in patients with RTT compared to controls. In addition, correlations between HRV and genotype and phenotype features or neurochemical changes were investigated. The data reported in this review suggest an important impairment in sympatho-vagal balance, supporting possible future research scenarios, targeting ANS.

Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.

No abstract available

Background: Inflammaging, a chronic low-grade inflammation as individuals age, is measured by elevated levels of circulating markers of inflammation (e.g., C-reactive protein [CRP] and interleukin-6 [IL-6]) and is associated with age-related chronic health conditions (CHCs). We aim to evaluate epigenetic inflammation scores (EIS) for CRP and IL-6 and their associations with cardiometabolic and cardiac-based CHCs and epigenetic age acceleration (EAA) in childhood cancer survivors (CCS). Methods: Among 2,052 CCS (47.2% female; mean [SD] = 33.7 [9.3] years of age at blood draw) from the St Jude Lifetime Cohort, EIS of CRP and IL-6 were calculated as the weighted sum of the DNA methylation (DNAm) levels of CpGs that are associated with circulating levels of CRP or IL-6, where blood DNAm data were generated with Infinium EPIC BeadChip, and weights are regression coefficients from previously published EWAS studies. CHCs with a CTCAE (Common Terminology Criteria for Adverse Events) grade ≥2 were considered except for myocardial infarction (MI) where a minimum CTCAE grade of 3 was applied. Residuals of EIS were calculated by regressing EIS on age at blood draw and sex. A multivariable Cox proportional hazard regression model evaluated the associations between the EIS residuals and each CHC adjusting for sex and cancer treatments. The timeframe was years since age at blood draw. All participants alive at the last follow-up were censored. EAA was calculated as residuals from regressing epigenetic age (both GrimAge and PhenoAge) on age at blood draw. A linear regression model evaluated the association between the EIS residual and EAA adjusting for sex and treatments. Results: The incidence of cardiometabolic conditions (i.e., after the blood draw) included: abnormal glucose metabolism (AGM, 5.1%), hypertension (HTN, 16.6%), hypercholesterolemia (HCL, 3.9%), hypertriglyceridemia (HTG, 6.0%), and cardiomyopathy (CMP, 9.0%), MI (4.4%), and obesity (24.2%). CRP-EIS and IL6-EIS were correlated (r=0.62). The CRP-EIS residual was significantly associated with increased risk of AGM (Hazard Ratio [95% CI] = 1.33 [1.01-1.75], P=0.04) and CMP (1.44 [1.19-1.74], P=1.58 × 10−4) and suggestively associated with HTN (1.16 [0.99-1.36], P=0.07) and MI (1.55 [0.94-2.57], P=0.09). IL6-EIS residual was associated with CMP (1.25 [1.04-1.49], P=0.02). In addition, both CRP- and IL6-EIS were positively associated with PhenoAge-EAA (β = 0.53 and 0.50 years per SD increase of EIS) and GrimAge-EAA (β = 0.30 and 0.36 per SD increase of EIS). Conclusion: The CRP- and IL6-EIS are associated with increased risk of cardiometabolic and cardiac-based CHCs and EAA among CCS, suggesting that EIS may be used as clinical tools to identify survivors at higher risk of such CHCs and inflammation may be targeted for intervention to reduce the burden of CHCs. Citation Format: Qian Dong, Yan Chen, Xijun Zhang, John Easton, Heather Mulder, Emily Walker, Geoffrey Neale, Kyla Shelton, Stephanie B. Dixon, Jinghui Zhang, Gregory T. Armstrong, Melissa M. Hudson, Kirsten K. Ness, Zhaoming Wang. Epigenetic inflammation scores for C-Reactive protein and interleukin-6 associate with adverse cardiometabolic and cardiac outcomes among survivors of childhood cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2235.

Heart failure (HF) treatment advances have improved outcomes but HF heterogeneity leads to ~50% response to standard therapies, emphasizing a need to further study mechanisms of disparate outcomes. One serious disparity is increased cardiovascular disease risk and mortality in African Americans (AA) compared to Caucasian Americans (CA). Race is a social construct and thus poor proxy for physiology; hence, our lab studies epigenetics, stable yet reversible changes to DNA serving as a possible interface of how environment impacts gene regulation. We’ve previously implicated cardiac DNA methylation as a new indicator of socio-economic disparities in HF outcomes. Related to HF, US adult obesity rates are 40% and similar disparities exist with AA having higher rates, and studies on obesity show how inequity leads to disparate rates. Thus, our secondary analysis investigates the role of obesity, through its impact on cardiac epigenetics and transcription, as an underlying environment/molecular driver within HF disparities. Multi-omics data of left ventricular assist device placement biopsies of age-matched males were from our previous publication’s public dataset. Cardiac DNA methylation (850k-array) analyzed via minfi, and differential gene expression (RNA-seq) via DESeq2. Two-year survival is decreased in non-obese (NO) vs. obese (OB) patients (p<0.01); specifically, decreased survival in AA-NO patients vs. CA-NO patients (Hazard Ratio=0.26, 0.08-0.91). Subgroup analyses reveal 2632 gene-associated differentially methylated CpG-probes between AA-NO vs. OB, and 5309 between CA-NO vs. OB (p<0.05, |methyl-change|>5%). Additionally, 19 differentially expressed genes between AA-NO vs. OB, and 236 between CA-NO vs. OB (q<0.1, |Log FC|>1.5); one key gene, TNFa induced protein 8 ( TNFAIP8 ), is increased in AA-NO vs. OB, but decreased in CA-NO vs. OB. Furthermore, the differential expression in each group correlates with canonical significant inverse DNA methylation at TNFAIP8 . Our survival data supports the literature of an “Obesity Paradox” where obese HF patients have better prognoses than non-obese patients. Through our analyses, we characterized impacts of obesity status on DNA methylation and gene expression within disparate HF outcomes and identified TNFAIP8 as a marker related to increased apoptosis in pathology progression. Thus, our work contributes molecular characterization of a possible effector of the differences seen in these specific patient populations.

Background Sodium-glucose co-transporters (SGLT) inhibitors (SGLT2i) showed many beneficial effects at the cardiovascular level. Several mechanisms of action have been identified. However, no data on their capability to act via epigenetic mechanisms were reported. Therefore, this study aimed to investigate the ability of SGLT2 inhibitors (SGLT2i) to induce protective effects at the cardiovascular level by acting on DNA methylation. Methods To better clarify this issue, the effects of empagliflozin (EMPA) on hyperglycemia-induced epigenetic modifications were evaluated in human ventricular cardiac myoblasts AC16 exposed to hyperglycemia for 7 days. Therefore, the effects of EMPA on DNA methylation of NF-κB, SOD2, and IL-6 genes in AC16 exposed to high glucose were analyzed by pyrosequencing-based methylation analysis. Modifications of gene expression and DNA methylation of NF-κB and SOD2 were confirmed in response to a transient SGLT2 gene silencing in the same cellular model. Moreover, chromatin immunoprecipitation followed by quantitative PCR was performed to evaluate the occupancy of TET2 across the investigated regions of NF-κB and SOD2 promoters. Results Seven days of high glucose treatment induced significant demethylation in the promoter regions of NF-kB and SOD2 with a consequent high level in mRNA expression of both genes. The observed DNA demethylation was mediated by increased TET2 expression and binding to the CpGs island in the promoter regions of analyzed genes. Indeed, EMPA prevented the HG-induced demethylation changes by reducing TET2 binding to the investigated promoter region and counteracted the altered gene expression. The transient SGLT2 gene silencing prevented the DNA demethylation observed in promoter regions, thus suggesting a role of SGLT2 as a potential target of the anti-inflammatory and antioxidant effect of EMPA in cardiomyocytes. Conclusions In conclusion, our results demonstrated that EMPA, mainly acting on SGLT2, prevented DNA methylation changes induced by high glucose and provided evidence of a new mechanism by which SGLT2i can exert cardio-beneficial effects. Graphical Abstract

Background The present study investigates whether epigenetic differences emerge in the heart of patients undergoing cardiac surgery for an aortic valvular replacement (AVR) or coronary artery bypass graft (CABG). An algorithm is also established to determine how the pathophysiological condition might influence the human biological cardiac age. Results Blood samples and cardiac auricles were collected from patients who underwent cardiac procedures: 94 AVR and 289 CABG. The CpGs from three independent blood-derived biological clocks were selected to design a new blood- and the first cardiac-specific clocks. Specifically, 31 CpGs from six age-related genes, ELOVL2, EDARADD, ITGA2B, ASPA, PDE4C, and FHL2, were used to construct the tissue-tailored clocks. The best-fitting variables were combined to define new cardiac- and blood-tailored clocks validated through neural network analysis and elastic regression. In addition, telomere length (TL) was measured by qPCR. These new methods revealed a similarity between chronological and biological age in the blood and heart; the average TL was significantly higher in the heart than in the blood. In addition, the cardiac clock discriminated well between AVR and CABG and was sensitive to cardiovascular risk factors such as obesity and smoking. Moreover, the cardiac-specific clock identified an AVR patient's subgroup whose accelerated bioage correlated with the altered ventricular parameters, including left ventricular diastolic and systolic volume. Conclusion This study reports on applying a method to evaluate the cardiac biological age revealing epigenetic features that separate subgroups of AVR and CABG.

Neuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system. Harmine is a tricyclic β-carboline alkaloid isolated from the harmal plant that exhibits both cytostatic and cytotoxic effects. Harmine is capable of blocking the activities of dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins and mitogen-activated protein kinase. These kinases promote proliferation and inhibit apoptosis. Four human NB cell lines were used to study the effects of harmine treatment: SKNBE and KELLY (MYCN-amplified) as well as SKNAS and SKNFI (MYCN non-amplified). The anti-cancer properties of harmine were examined by RealTime-Glo MT cell viability assays, caspase activity assays, PARP cleavage using Western blot analysis, and flow cytometry-based Annexin V detection. A molecular interaction model of harmine bound to the DYRK2 family kinase was generated by computational docking using X-ray structures. NB tumors from human patients were profiled for DYRK mRNA expression patterns and clinical correlations using the R2 platform. The IC50 values for harmine after 72 h treatment were 169.6, 170.8, and 791.7 μM for SKNBE, KELLY, and SKNFI, respectively. Exposure of these NB cell lines to 100 μM of harmine resulted in caspase-3/7 and caspase-9 activation as well as caspase-mediated PARP cleavage and Annexin V-positive stained cells, as early as 24 h after treatment, clearly suggesting apoptosis induction, especially in MYCN-amplified cell lines. Elevated DYRK2 mRNA levels correlated with poor prognosis in a large cohort of NB tumors. Harmine is a known inhibitor of DYRK family kinases. It can induce apoptosis in NB cell lines, which led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that DYRK inhibition by harmine and the subsequent triggering of caspase-mediated apoptosis might present a novel approach to NB therapy.

No abstract available

Background The treatment effects on hospitalization for heart failure (hHF) from sodium-glucose cotransporter 2 (SGLT2) inhibitors may vary among type 2 diabetes (T2D) patients depending on whether or not they have established atherosclerotic cardiovascular diseases (ASCVD). We aimed to examine differences in hHF outcomes after dapagliflozin or empagliflozin use between T2D patients with and without a history of established ASCVD. Methods We conducted a retrospective multi-institutional cohort study in Taiwan. We included T2D patients newly receiving dapagliflozin or empagliflozin during 2016–2019, and followed them up until December 31, 2020. We implemented 1:1 propensity score matching to create homogenous groups for comparisons. We generated Cox proportional hazard models to compare the risk of hHF between dapagliflozin and empagliflozin (reference group). We included interaction terms of SGLT2 inhibitor and ASCVD history in the regression models to examine effect modification by ASCVD. Results We included a total cohort of 9,586 dapagliflozin new users and 9,586 matched empagliflozin new users. The overall hHF risks were similar for dapagliflozin and empagliflozin (HR: 0.90, 95% CI 0.74–1.09). However, differential hHF risks between dapagliflozin and empagliflozin were observed only in the subgroup without ASCVD (HR: 0.67, 95% CI 0.49–0.90), while not in the subgroup with ASCVD (HR: 1.12, 95% 0.87–1.45), and the p-value for examining interaction was 0.0097. Conclusion In this study, history of established ASCVD was associated with different hHF risks among SGLT2 inhibitors. For T2D patients without ASCVD, dapagliflozin may offer a more favorable hHF reduction effect, compared to empagliflozin, in clinical practice. Future prospective studies should be conducted to validate our findings.

Background Whether sodium glucose co-transporter 2 inhibitors (SGLT2i) are associated with a lower risk of cardiovascular as well as adverse lower limb events in patients with type-2 diabetes mellitus (T2DM) and concomitant peripheral artery disease (PAD) is unclear. We aimed to evaluate the risk of cardiovascular and limb events, and death associated with the use of SGLT2i compared with dipeptidyl peptidase-4 inhibitors (DPP4i) among a longitudinal and national cohort of patients with T2DM. Methods In this nationwide retrospective cohort study based on the Taiwan National Health Insurance Research Database, we identified a total of 11,431 and 93,972 consecutive T2DM patients with PAD taking SGLT2i and DPP4i, respectively, from May 1, 2016, to December 31, 2017. We used 1:1 propensity score matching (PSM) to balance covariates across study groups. Patients were followed from the drug index date until the occurrence of clinical outcomes, death, discontinuation of the index drug, or the end of the study period, whichever occurred first. Results Overall, 56% and 44% of the patients were treated with dapagliflozin and empagliflozin, respectively. The use of SGLT2i had comparable risks of ischemic stroke and acute myocardial infarction, and was associated with lower risks of congestive heart failure (CHF) [hazard ratio (HR): 0.66; 95% confidence interval (CI) 0.49–0.89; p  = 0.0062], lower limb ischemia requiring revascularization (HR: 0.73; 95% CI 0.54–0.98; p  = 0.0367) or amputation (HR: 0.43; 95% CI 0.30–0.62; p  < 0.0001), and cardiovascular death (HR: 0.67; 95% CI 0.49–0.90; p  = 0.0089) when compared with the DDP4i group after PSM. The subgroup analysis revealed consistent results for CHF and major adverse limb outcomes for SGLT2i versus DPP4i among patients aged ≥ 75 years, the presence of chronic kidney disease and established cardiovascular disease was consistent with the main analysis. Conclusions SGLT2i were associated with lower risks of CHF and adverse lower limb events compared with DPP4i among patients with T2DM and PAD in real-world practice.

BackgroundSodium-glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1 RA) improve cardiovascular and renal outcomes in patients with type 2 diabetes through distinct mechanisms. However, evidence on clinical outcomes in patients treated with both GLP-1 RA and SGLT2i is lacking. We aim to provide insight into the effects of open-label SGLT2i use in parallel with or shortly after once-weekly GLP-1 RA exenatide (EQW) on cardiorenal outcomes.MethodsIn the EXSCEL cardiovascular outcomes trial EQW arm, SGLT2i drop-in occurred in 8.7% of participants. These EQW+SGLT2i users were propensity-matched to: (1) placebo-arm participants not taking SGLT2i (n = 572 per group); and to (2) EQW-arm participants not taking SGLT2i (n = 575), based on their last measured characteristics before SGLT2i initiation, and equivalent study visit in comparator groups. Time-to-first major adverse cardiovascular event (MACE) and all-cause mortality (ACM) were compared using Cox regression analyses. eGFR slopes were quantified using mixed model repeated measurement analyses.ResultsIn adjusted analyses, the risk for MACE with combination EQW+SGLT2i use was numerically lower compared with both placebo (adjusted hazard ratio 0.68, 95% CI 0.39–1.17) and EQW alone (0.85, 0.48–1.49). Risk of ACM was nominally significantly reduced compared with placebo (0.38, 0.16–0.90) and compared with EQW (0.41, 0.17–0.95). Combination EQW+SGLT2i use also nominally significantly improved estimated eGFR slope compared with placebo (+ 1.94, 95% CI 0.94–2.94 mL/min/1.73 m2/year) and EQW alone (+ 2.38, 1.40–3.35 mL/min/1.73 m2/year).ConclusionsThis post hoc analysis supports the hypothesis that combinatorial EQW and SGLT2i therapy may provide benefit on cardiovascular outcomes and mortality.Trial registration Clinicaltrials.gov, Identifying number: NCT01144338, Date of registration: June 15, 2010.