Phosphoproteomic and proteomic profiling in post-infarction chronic heart failure.

Background: Post-infarction chronic heart failure is the most common type of heart failure. Patients with chronic heart failure show elevated morbidity and mortality with limited evidence-based therapies. Phosphoproteomic and proteomic analysis can provide insights regarding molecular mechanisms underlying post-infarction chronic heart failure and explore new therapeutic approaches. Methods and results: Global quantitative phosphoproteomic and proteomic analysis of left ventricular tissues from post-infarction chronic heart failure rats were performed. A total of 33 differentially expressed phosphorylated proteins (DPPs) and 129 differentially expressed proteins were identified. Bioinformatic analysis indicated that DPPs were enriched mostly in nucleocytoplasmic transport and mRNA surveillance pathway. Bclaf1 Ser658 was identified after construction of Protein-Protein Interaction Network and intersection with Thanatos Apoptosis Database. Predicted Upstream Kinases of DPPs based on kinase-substrate enrichment analysis (KSEA) app showed 13 kinases enhanced in heart failure. Proteomic analysis showed marked changes in protein expression related to cardiac contractility and metabolism. Conclusion: The present study marked phosphoproteomics and proteomics changes in post-infarction chronic heart failure. Bclaf1 Ser658 might play a critical role in apoptosis in heart failure. PRKAA1, PRKACA, and PAK1 might serve as potential therapeutic targets for post-infarction chronic heart failure.

Physiological ischemic training improves cardiac function through the attenuation of cardiomyocyte apoptosis and the activation of the vagus nerve in chronic heart failure.

Purpose: This study investigated the functional outcomes of patients with chronic heart failure (CHF) after physiological ischemic training (PIT), identified the optimal PIT protocol, evaluated its cardioprotective effects and explored the underlying neural mechanisms. Methods: Patients with CHF were randomly divided into experimental group (n = 25, PIT intervention + regular treatment) and control group (n = 25, regular treatment). The outcomes included the left ventricular ejection fraction (LVEF), brain natriuretic peptide (BNP) and cardiopulmonary parameters. LVEF and cardiac biomarkers in CHF rats after various PIT treatments (different in intensity, frequency, and course of treatment) were measured to identify the optimal PIT protocol. The effect of PIT on cardiomyocyte programmed cell death was investigated by western blot, flow cytometry and fluorescent staining. The neural mechanism involved in PIT-induced cardioprotective effect was assessed by stimulation of the vagus nerve and muscarinic M2 receptor in CHF rats. Results: LVEF and VO2max increased while BNP decreased in patients subjected to PIT. The optimal PIT protocol in CHF rats was composed of five cycles of 5 min ischemia followed by 5 min reperfusion on remote limbs for 8 weeks. LVEF and cardiac biomarker levels were significantly improved, and cardiomyocyte apoptosis was inhibited. However, these cardioprotective effects disappeared after subjecting CHF rats to vagotomy or muscarinic M2 receptor inhibition. Conclusion: PIT improved functional outcomes in CHF patients. The optimal PIT protocol required appropriate intensity, reasonable frequency, and adequate treatment course. Under these conditions, improvement of cardiac function in CHF was confirmed through cardiomyocyte apoptosis reduction and vagus nerve activation.

Anti-angiogenic effect of exo-LncRNA TUG1 in myocardial infarction and modulation by remote ischemic conditioning.

The successful use of exosomes in therapy after myocardial infarction depends on an improved understanding of their role in cardiac signaling and regulation. Here, we report that exosomes circulating after myocardial infarction (MI) carry LncRNA TUG1 which downregulates angiogenesis by disablement of the HIF-1α/VEGF-α axis and that this effect can be counterbalanced by remote ischemic conditioning (RIC). Rats with MI induced through left coronary artery ligation without (MI model) and with reperfusion (ischemia/reperfusion I/R model) were randomized to RIC, or MI (I/R) or sham-operated (SO) control. Data from one cohort study and one randomized-controlled trial of humans with MI were also utilized, the former involving patients who had not received percutaneous coronary intervention (PCI) and the latter patients with PCI. Exosome concentrations did not differ between intervention groups (RIC vs. control) in rats (MI and I/R model) as well as humans (with and without PCI). However, MI and I/R exosomes attenuated HIF-1α, VEGF-α, and endothelial function. LncRNA TUG1 was increased in MI and I/R exosomes, but decreased in SO and RIC exosomes. HIF-1α expression was downregulated with MI and I/R exosomes but increased with RIC exosomes. Exosome inhibition suppressed HIF-1α upregulation through RIC exosomes. VEGF-α was identified as HIF-1α-regulated target gene. Knockdown of HIF-1α decreased VEGF-α, endothelial cell capability, and tube formation. Overexpression of HIF-1α exerted opposite effects. Transfection and co-transfection of 293 T cells with exosome-inhibitor GW4869 and HIF-1α inhibitor si-HIF-1α confirmed the exosomal-LncRNA TUG1/HIF-1α/VEGF-α pathway. LncRNA TUG1 is a potential therapeutic target after MI with or without reperfusion through PCI.

Novel lncRNA-miRNA-mRNA Competing Endogenous RNA Triple Networks Associated Programmed Cell Death in Heart Failure.

Objective: Increasing evidence has uncovered the roles of lncRNA-miRNA-mRNA regulatory networks in cardiovascular diseases. However, the crosstalk between ceRNA networks and development of heart failure (HF) remains unclear. This study was to investigate the role of lncRNA-mediated ceRNA networks in the pathophysiological process of HF and its potential regulatory functions on programmed cell death. Methods: We firstly screened the GSE77399, GSE52601 and GSE57338 datasets in the NCBI GEO database for screening differentially expressed lncRNAs, miRNAs and mRNAs. lncRNA-miRNA-mRNA regulatory networks based on the ceRNA theory were subsequently constructed. GO and KEGG enrichment analysis was conducted to predict potential biological functions of mRNAs in ceRNA networks. Differentially expressed mRNAs were then interacted with programmed cell death related genes. lncRNA-mediated ceRNA regulatory pathways on programmed cell death were validated with qRT-PCR testing. Results: Based on our bioinformatic analysis, two lncRNAs, eight miRNAs and 65 mRNAs were extracted to construct two lncRNAs-mediated ceRNA networks in HF. Biological processes and pathways were enriched in extracellular matrix. Seven lncRNA-mediated ceRNA regulatory pathways on programmed cell death, GAS5/miR-345-5p/ADAMTS4, GAS5/miR-18b-5p/AQP3, GAS5/miR-18b-5p/SHISA3, GAS5/miR-18b-5p/C1orf105, GAS5/miR-18b-5p/PLIN2, GAS5/miR-185-5p/LPCAT3, and GAS5/miR-29b-3p/STAT3, were finally validated. Conclusions: Two novel ceRNA regulatory networks in HF were discovered based on our bioinformatic analysis. Based on the interaction and validation analysis, seven lncRNA GAS5-mediated ceRNA regulatory pathways were hypothesized to impact programmed cell death including seven for apoptosis, three for ferroptosis, and one for pyroptosis. Upon which, we provided novel insights and potential research plots for bridging ceRNA regulatory networks and programmed cell death in HF.

Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis.

Objectives: N6-methyladenosine (m6A) is hypothesized to play a role in the regulation of pathogenesis of myocardial infarction (MI). This study was designed to compare m6A-tagged transcript profiles to identify mRNA-specific changes on pathophysiological variations after MI. Methods: N6-methyladenosine methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were interacted to select m6A-modified mRNAs with samples collected from sham operated and MI rat models. m6A methylation regulated mRNAs were interacted with apoptosis/angiogenesis related genes in GeneCards. Afterwards, MeRIP-quantitative real-time PCR (MeRIP-qRT-PCR) was performed to measure m6A methylation level of hub mRNAs. m6A methylation variation was tested under different oxygen concentration or hypoxic duration in H9c2 cells and HUVECs. In addition, Western blot and qRT-PCR were employed to detect expression of hub mRNAs and relevant protein level. Flow cytometry and Tunel assay were conducted to assess apoptotic level. CCK-8, EdU, and tube formation assay were performed to measure cell proliferation and tube formation ability. Results: Upregulation of Mettl3 was firstly observed in vivo and in vitro, followed by upregulation of m6A methylation level. A total of 567 significantly changed m6A methylation peaks were identified, including 276 upregulated and 291 downregulated peaks. A total of 576 mRNAs were upregulated and 78 were downregulated. According to combined analysis of MeRIP-seq and RNA-seq, we identified 26 significantly hypermethylated and downregulated mRNAs. Based on qRT-PCR and interactive analysis, Hadh, Kcnn1, and Tet1 were preliminarily identified as hub mRNAs associated with apoptosis/angiogenesis. MeRIP-qRT-PCR assay confirmed the results from MeRIP-seq. With the inhibition of Mettl3 in H9c2 cells and HUVECs, downregulated m6A methylation level of total RNA and upregulated expression of hub mRNAs were observed. Increased m6A level was verified in the gradient context in terms of prolonged hypoxic duration and decreased oxygen concentration. Under simulated hypoxia, roles of Kcnn1 and Tet1 in angiogenesis and Hadh, Tet1, and Kcnn1 in apoptosis were further confirmed with our validation experiments. Conclusion: Roles of m6A-modified mRNA transcripts in the context of MI were preliminarily verified. In the context of m6A methylation, three hub mRNAs were validated to impact the process of apoptosis/angiogenesis. Our study provided theoretical basis and innovative targets for treatment of MI and paved the way for future investigations aiming at exploring upstream epigenetic mechanisms of pathogenesis after MI.

Protective Role of Fecal Microbiota Transplantation on Colitis and Colitis-Associated Colon Cancer in Mice Is Associated With Treg Cells.

Colitis-associated cancer (CAC) is the most serious outcome of inflammatory bowel disease, which has an alteration of commensal intestinal microbiota. However, the role of intestinal microbiota on CAC progression is not well-understood. Fecal microbiota transplantation (FMT) was used for treating murine azoxymethane-dextran sodium sulfate (AOM-DSS) model of CAC. Composition of gut microbiota during FMT treatment was analyzed. RT-PCR and ELISA were used to detect the inflammatory factors, and immunofluorescence was applied to examine the phospho-nuclear factor (NF)-κB p65/p100 and Ki67-positive cells in the colons. In addition, flow cytometry was performed to analyze the immune cell after FMT treatment. Rehabilitation of the intestinal microbiota by FMT restored both the ratio and diversity of microbiota during CAC progression. Remarkably, a favorable morphometric outcome characterized by decreased tumor load and size was observed in CAC mice with FMT treatment. In addition, an anti-inflammatory function of FMT was demonstrated by decreasing pro-inflammatory factors but increasing anti-inflammatory factors through inhibiting canonical NF-κB activity and cellular proliferation in colons of CAC mice. The expression of CD4+CD25+Foxp3+ regulatory T cells (Tregs) was significantly increased after FMT treatment in CAC mice, but not T helper (Th)1/2/17 cells. Our study aids in the understanding of CAC pathogenesis and reveals a previously unrecognized role for FMT in the treatment of CAC through restoring the intestinal microbiota and inducing regulatory T cells.