Critical Role of the cGAS-STING Pathway in Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Doxorubicin is an effective chemotherapy drug for treating various types of cancer. However, lethal cardiotoxicity severely limits its clinical use. Recent evidence has indicated that aberrant activation of the cytosolic DNA-sensing cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING (stimulator of interferon genes) pathway plays a critical role in cardiovascular destruction. Here, we investigate the involvement of this mechanism in doxorubicin-induced cardiotoxicity (DIC). METHODS: Mice were treated with low-dose doxorubicin to induce chronic DIC. The role of the cGAS-STING pathway in DIC was evaluated in cGAS-deficiency (cGAS-/-), Sting-deficiency (Sting-/-), and interferon regulatory factor 3 (Irf3)-deficiency (Irf3-/-) mice. Endothelial cell (EC)-specific conditional Sting deficiency (Stingflox/flox/Cdh5-CreERT) mice were used to assess the importance of this pathway in ECs during DIC. We also examined the direct effects of the cGAS-STING pathway on nicotinamide adenine dinucleotide (NAD) homeostasis in vitro and in vivo. RESULTS: In the chronic DIC model, we observed significant activation of the cGAS-STING pathway in cardiac ECs. Global cGAS, Sting, and Irf3 deficiency all markedly ameliorated DIC. EC-specific Sting deficiency significantly prevented DIC and endothelial dysfunction. Mechanistically, doxorubicin activated the cardiac EC cGAS-STING pathway and its target, IRF3, which directly induced CD38 expression. In cardiac ECs, the cGAS-STING pathway caused a reduction in NAD levels and subsequent mitochondrial dysfunction via the intracellular NAD glycohydrolase (NADase) activity of CD38. Furthermore, the cardiac EC cGAS-STING pathway also regulates NAD homeostasis and mitochondrial bioenergetics in cardiomyocytes through the ecto-NADase activity of CD38. We also demonstrated that pharmacological inhibition of TANK-binding kinase 1 or CD38 effectively ameliorated DIC without compromising the anticancer effects of doxorubicin. CONCLUSIONS: Our findings indicate a critical role of the cardiac EC cGAS-STING pathway in DIC. The cGAS-STING pathway may represent a novel therapeutic target for preventing DIC.

Soluble guanylate cyclase (sGC) stimulator vericiguat alleviates myocardial ischemia-reperfusion injury by improving microcirculation.

Background: This study aimed to verify the effect of soluble guanylate cyclase (sGC) stimulator vericiguat on myocardial ischemia-reperfusion injury and explore its mechanism. Methods: A myocardial ischemia-reperfusion injury model of mice was established and intravenous administration was performed 2 minutes before reperfusion. Triphenyltetrazolium chloride (TTC) staining and echocardiography were used to verify the effect of vericiguat on myocardial ischemia-reperfusion injury in the infarct area, and immunofluorescence was used to observe myocardial pathological changes at different time points after reperfusion. Quantitative proteomics was conducted to analysis the main differentially expressed proteins after drug intervention. The distribution of endothelial cells and sGC after myocardial ischemia-reperfusion injury in mice was observed by immunofluorescence. RNA sequencing of endothelial cells was used to search for differentially expressed molecules. Thioflavin-S staining was used to observe the effect of vericiguat on improving the nonrecurrence phenomenon and reducing the infarct size after reperfusion. Results: The effect of the sGC stimulator vericiguat on myocardial ischemia-reperfusion injury was verified, and myocardial microcirculation significantly increased after drug intervention. Quantitative proteomics found that the protein expression of myocardial tissue in the ischemia-reperfusion area was not significantly different in the drug intervention group, except for increased adenosine triphosphate (ATP) activity. Vericiguat, nitroglycerin, and nitrite did not directly affect apoptosis or cell viability. RNA sequencing of human umbilical vein endothelial cells screened the upregulated antioxidant response. Conclusions: SGC stimulator vericiguat ameliorated myocardial ischemia-reperfusion injury through indirect pathways of improving microcirculation.

Impact and potential mechanism of effects of chronic moderate alcohol consumption on cardiac function in aldehyde dehydrogenase 2 gene heterozygous mice.

BACKGROUND: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme in alcohol metabolism. The ALDH2*2 mutations are found in approximately 45% of East Asians, with 40% being heterozygous (HE) ALDH2*1/*2 and 5% homozygous (HO) ALDH2*2/*2. Studies have shown that HO mice lack cardioprotective effects induced by moderate alcohol consumption. However, the impact of moderate alcohol consumption on cardiac function in HE mice is unknown. METHODS: In this study, HO, HE, and wild-type (WT) mice were subjected to a 6-week moderate alcohol drinking protocol, following which myocardial tissue and cardiomyocytes of the mice were extracted. RESULTS: We found that moderate alcohol exposure did not increase mortality, myocardial fibrosis, apoptosis, or inflammation in HE mice, which differs from the effects observed in HO mice. After exposure to the 6-week alcohol drinking protocol, there was impaired cardiac function, cardiomyocyte contractility, and intracellular Ca2+ homeostasis and mitochondrial function in both HE and HO mice as compared to WT mice. Moreover, these animals showed overt oxidative stress production and increased levels of the activated forms of calmodulin-dependent protein kinase II (CaMKII) and ryanodine receptor type 2 (RYR2) phosphorylation protein. CONCLUSION: We found that moderate alcohol exposure impaired cardiac function in HE mice, possibly by increasing reactive oxygen species (ROS)/CaMKII/RYR2-mediated Ca2+ handling abnormalities. Hence, we advocate that people with ALDH2*1/*2 genotypes rigorously avoid alcohol consumption to prevent potential cardiovascular harm induced by moderate alcohol consumption.

The critical roles of m6A modification in metabolic abnormality and cardiovascular diseases.

N6-methyladenosine (m6A) RNA methylation is an emerging area of epigenetics, which is a reversible and dynamic modification mediating by 'writers' (methylase, adding methyl groups, METTL3, METTL14, and WTAP), 'erasers' (demethylase, deleting methyl groups, FTO and ALKBH5), and 'readers' (YTHDF1-3, YTHDC1 and YTHDC2). Recent studies in human, animal models and cell levels have disclosed a critical role of m6A modification in regulating the homeostasis of metabolic processes and cardiovascular function. Evidence from these studies identify m6A as a candidate of biomarker and therapeutic target for metabolic abnormality and cardiovascular diseases (CVD). Comprehensive understanding of the complexity of m6A regulation in metabolic diseases and CVD will be helpful for us to understand the pathogenesis of CVD. In this review, we discuss the regulatory role of m6A in metabolic abnormality and CVD. We will emphasize the clinical relevance of m6A dysregulation in CVD.

Loss of m6A demethylase ALKBH5 promotes post-ischemic angiogenesis via post-transcriptional stabilization of WNT5A.

BACKGROUND: Post-ischemic angiogenesis is critical for blood flow recovery and ischemic tissue repair. N6-methyladenosine (m6A) plays essential roles in numerous biological processes. However, the impact and connected mechanism of m6A on post-ischemic angiogenesis are not fully understood. METHODS: AlkB homolog 5 (ALKBH5) was screened out among several methyltransferases and demethylases involved in dynamic m6A regulation. Cardiac microvascular endothelial cells (CMECs) angiogenesis and WNT family member 5A (WNT5A) stability were analyzed upon ALKBH5 overexpression with adenovirus or knockdown with small interfering RNAs in vitro. The blood flow recovery, capillary, and small artery densities were evaluated in adeno-associated virus (AAV)-ALKBH5 overexpression or ALKBH5 knockout (KO) mice in a hind-limb ischemia model. The same experiments were conducted to explore the translational value of transient silencing of ALKBH5 with adenovirus. RESULTS: ALKBH5 was significantly upregulated in hypoxic CMECs and led to a global decrease of m6A level. ALKBH5 overexpression further reduced m6A level in normoxic and hypoxic CMECs, impaired proliferation, migration, and tube formation only in hypoxic CMECs. Conversely, ALKBH5 knockdown preserved m6A levels and promoted angiogenic phenotypes in hypoxic but not in normoxic CMECs. Mechanistically, ALKBH5 regulated WNT5A expression through post-transcriptional mRNA modulation in an m6A-dependent manner, which decreased its stability and subsequently impeded angiogenesis in hypoxic CMECs. Furthermore, ALKBH5 overexpression hindered blood flow recovery and reduced CD31 and alpha-smooth muscle actin expression in hind-limb ischemia mice. As expected, ALKBH5-KO mice exhibited improved blood flow recovery, increased capillary, and small artery densities after hind-limb ischemia, and similar beneficial effects were observed in mice with transient adenoviral ALKBH5 gene silencing. CONCLUSION: We demonstrate that ALKBH5 is a negative regulator of post-ischemic angiogenesis via post-transcriptional modulation and destabilization of WNT5A mRNA in an m6A-dependent manner. Targeting ALKBH5 may be a potential therapeutic option for ischemic diseases, including peripheral artery disease.

Alteration of m6A RNA Methylation in Heart Failure With Preserved Ejection Fraction.

Background: Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous disease, in which its pathogenesis is very complex and far from defined. Here, we explored the N6-methyladenosine (m6A) RNA methylation alteration in patients with HFpEF and mouse model of HFpEF. Methods: In this case-control study, peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood samples obtained from 16 HFpEF patients and 24 healthy controls. The change of m6A regulators was detected by quantitative real-time PCR (RT-PCR). A "two-hit" mouse model of HFpEF was induced by a high-fat diet and drinking water with 0.5 g/L of N ω-nitro-l-arginine methyl ester (L-NAME). MeRIP-seq was used to map transcriptome-wide m6A in control mice and HFpEF mice, and the gene expression was high-throughput detected by RNA-seq. Results: The expression of m6A writers METTL3, METTL4, and KIAA1429; m6A eraser FTO; and reader YTHDF2 was up-regulated in HFpEF patients, compared with health controls. Furthermore, the expression of FTO was also elevated in HFpEF mice. A total of 661 m6A peaks were significantly changed by MeRIP-seq. Gene Ontology (GO) analysis revealed that protein folding, ubiquitin-dependent ERAD pathway, and positive regulation of RNA polymerase II were the three most significantly altered biological processes in HFpEF. The pathways including proteasome, protein processing in the endoplasmic reticulum, and PI3K-Akt signaling pathway were significantly changed in HFpEF by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Conclusions: The expression pattern of m6A regulators and m6A landscape is changed in HFpEF. This uncovers a new transcription-independent mechanism of translation regulation. Therefore, our data suggest that the modulation of epitranscriptomic processes, such as m6A methylation, might be an interesting target for therapeutic interventions.

Exercise improves cardiac function and glucose metabolism in mice with experimental myocardial infarction through inhibiting HDAC4 and upregulating GLUT1 expression.

This study aims to determine the effect of exercise on the cardiac function, metabolic profiles and related molecular mechanisms in mice with ischemic-induced heart failure (HF). HF was induced by myocardial infarction (MI) in C57BL6/N mice. Cardiac function and physical endurance were improved in HF mice after exercise. Micro-PET/CT scanning revealed enhanced myocardial glucose uptake in vivo in HF mice after exercise. Exercise reduced mitochondrial structural damage in HF mice. Cardiomyocytes isolated from HF + exercise mice showed increased glycolysis capacity, respiratory function and ATP production. Both mRNA and protein expression of glucose transporter 1 (GLUT1) were upregulated after exercise. Results of ChIP-PCR revealed a novel interaction between transcription factor myocyte enhancer factor 2a (MEF2a) and GLUT1 in hearts of HF + exercise mice. Exercise also activated myocardial AMP-activated protein kinase (AMPK), which in turn phosphorylated histone deacetylase 4 (HDAC4), and thereby modulated the GLUT1 expression through reducing its inhibition on MEF2a in HF mice. Inhibition of HDAC4 also improved cardiac function in HF mice. Moreover, knockdown of GLUT1 impaired the systolic and diastolic function of isolated cardiomyocytes. In conclusion, exercise improves cardiac function and glucose metabolism in HF mice through inhibiting HDAC4 and upregulating GLUT1 expression.

Insights from Exercise-induced Cardioprotection-from Clinical Application to Basic Research.

Exercise has long been recognized as a beneficial living style for cardiovascular health. It has been applied to be a central component of cardiac rehabilitation for patients with chronic heart failure (CHF), coronary heart disease (CHD), post-acute coronary syndrome (ACS) or primary percutaneous coronary intervention (PCI), post cardiac surgery or transplantation. Although the effect of exercise is multifactorial, in this review, we focus on the specific contribution of regular exercise on the heart and vascular system. We will summarize the known result of clinical findings and possible mechanisms of chronic exercise on the cardiovascular system.

Berberine alleviates oxidized low-density lipoprotein-induced macrophage activation by downregulating galectin-3 via the NF-κB and AMPK signaling pathways.

Macrophage activation plays a central role in neoatherosclerosis and in-stent restenosis after percutaneous coronary intervention (PCI). Galectin-3, mainly expressed on macrophages, is an important regulator of inflammation. This study aimed to investigate the effects of berberine (BBR) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage activation and galectin-3 expression and their underlying mechanisms. THP-1-derived macrophages were pretreated with BBR prior to stimulation with ox-LDL. Galectin-3 expression was measured by real-time PCR, Western blotting, and confocal microscopy. Macrophage activation was assessed by lipid accumulation, expression of inflammatory cytokines, and CD11b and CD86. Plasma galectin-3 levels were measured in patients undergoing PCI at baseline and after BBR treatment for 3 months. BBR suppressed ox-LDL-induced upregulation of galectin-3 and macrophage activation. Overexpression of galectin-3 intervened the inhibitory effect of BBR on macrophage activation. BBR activated phospho-AMPK and inhibited phospho-NF-κB p65 nuclear translocation. AMPK inhibition and NF-κB activation abolished the inhibitory effects of BBR on galectin-3 expression and macrophage activation. Combination of BBR and rosuvastatin exerted greater effects than BBR or rosuvastatin alone. However, BBR treatment did not further reduce plasma galectin-3 after PCI in patients receiving standard therapy. In conclusion, BBR alleviates ox-LDL-induced macrophage activation by downregulating galectin-3 via the NF-κB and AMPK signaling pathways.

Iso-Osmolar Iodixanol Induces Less Increase in Circulating Endothelial Microparticles In Vivo and Less Endothelial Apoptosis In Vitro Compared with Low-Osmolar Iohexol.

Background and Aims: There is no consensus on whether iodixanol is superior to iohexol. This study aimed to compare the effects of iodixanol and iohexol on circulating endothelial microparticles (EMPs) in stable coronary artery disease (CAD) patients with diabetes mellitus (DM), and also their cytotoxic effects on human umbilical vein endothelial cells (HUVECs) in vitro. Methods: 100 CAD patients with DM were randomly assigned to receive iso-osmolar contrast medium iodixanol (group I) or low-osmolar iohexol (group II) during coronary angioplasty. An additional 49 CAD patients without DM receiving iohexol were recruited as group III. Circulating CD31+/CD41a- EMPs, CD62E+ EMPs, and CD31+/CD41a+ platelet microparticles (PMPs) were determined by flow cytometry. In vitro, the cytotoxic effects of iodixanol and iohexol on HUVECs were determined. Results: Circulating CD31+/CD41a- EMPs and PMPs were significantly increased after angioplasty in all 3 groups, while CD62E+ EMPs significantly decreased in group I. CD31+/CD41a- EMPs and PMPs were significantly higher in group II than group I or III. In vitro, both contrast media induced EMP release and inhibited the viability and induced apoptosis of HUVECs, as well as increasing Bax and cleaved caspase-3 and decreasing Bcl-2. The above effects were less evident in iodixanol than in iohexol. Conclusions: Compared with iohexol, iodixanol induces less release of EMPs in both CAD patients with DM during angioplasty and in vitro HUVEC culture, which is associated with less pronounced proapoptotic effects of iodixanol on HUVECs. Clinical Study Registration Number: This study is registered with ChiCTR-TRC-14005183.

High Resting Heart Rate and High BMI Predicted Severe Coronary Atherosclerosis Burden in Patients With Stable Angina Pectoris by SYNTAX Score.

We investigated the independent and combined association of resting heart rate (RHR expressed as beats/min, bpm) and body mass index (BMI) with SYNTAX score (SS) in patients with stable angina. We divided 312 patients into 4 groups according to RHR quartiles: Q1 (<65 bpm), Q2 (65-69 bpm), Q3 (70-79 bpm), and Q4 (≥80 bpm). The SS (12.0 ± 9.0, 16.0 ± 15.5, 18.0 ± 16.5, and 20.0 ± 27.5; P < .001) was significantly higher for those in Q4 than for those in Q1, Q2, and Q3. Multivariate logistic regression analysis indicated that each 10-bpm increment in RHR was significantly associated with SS (odds ratio [OR] 1.62, 95% confidence interval [CI] 1.27-2.06). Patients with high RHR and high BMI had significantly greater odds ratio (OR) of high SS (4.03, 95% CI 2.00-8.14), compared to participants with low RHR and low BMI. Both RHR and BMI were independent predictors of coronary atherosclerosis as assessed by SS. RHR in combination with BMI and multivariate logistic regression analysis emphasized the importance of the correlation between RHR and SS in patients with stable angina pectoris.