Immune-associated pivotal biomarkers identification and competing endogenous RNA network construction in post-operative atrial fibrillation by comprehensive bioinformatics and machine learning strategies.

Background: Atrial fibrillation (AF) is the most common arrhythmia. Previous studies mainly focused on identifying potential diagnostic biomarkers and treatment strategies for AF, while few studies concentrated on post-operative AF (POAF), particularly using bioinformatics analysis and machine learning algorithms. Therefore, our study aimed to identify immune-associated genes and provide the competing endogenous RNA (ceRNA) network for POAF. Methods: Three GSE datasets were downloaded from the GEO database, and we used a variety of bioinformatics strategies and machine learning algorithms to discover candidate hub genes. These techniques included identifying differentially expressed genes (DEGs) and circRNAs (DECs), building protein-protein interaction networks, selecting common genes, and filtering candidate hub genes via three machine learning algorithms. To assess the diagnostic value, we then created the nomogram and receiver operating curve (ROC). MiRNAs targeting DEGs and DECs were predicted using five tools and the competing endogenous RNA (ceRNA) network was built. Moreover, we performed the immune cell infiltration analysis to better elucidate the regulation of immune cells in POAF. Results: We identified 234 DEGs (82 up-regulated and 152 down-regulated) of POAF via Limma, 75 node genes were visualized via PPI network, which were mainly enriched in immune regulation. 15 common genes were selected using three CytoHubba algorithms. Following machine learning selection, the nomogram was created based on the four candidate hub genes. The area under curve (AUC) of the nomogram and individual gene were all over 0.75, showing the ideal diagnostic value. The dysregulation of macrophages may be critical in POAF pathogenesis. A novel circ_0007738 was discovered in POAF and the ceRNA network was eventually built. Conclusion: We identified four immune-associated candidate hub genes (C1QA, C1R, MET, and SDC4) for POAF diagnosis through the creation of a nomogram and evaluation of its diagnostic value. The modulation of macrophages and the ceRNA network may represent further therapy methods.

Integrating Evidence of the Traditional Chinese Medicine Collateral Disease Theory in Prevention and Treatment of Cardiovascular Continuum.

Cardiovascular disease has become a major public health problem. The concept of "cardiovascular continuum" refers to the continuous process from the risk factors that lead to arteriosclerosis, vulnerable plaque rupture, myocardial infarction, arrhythmia, heart failure, and death. These characteristics of etiology and progressive development coincide with the idea of "preventing disease" in traditional Chinese medicine (TCM), which corresponds to the process of systemic intervention. With the update of the understanding via translational medicine, this article reviews the current evidence of the TCM collateral disease theory set prescriptions in both mechanical and clinical aspects, which could lead to the development of new therapeutic strategies for prevention and treatment.

ADAR2 increases in exercised heart and protects against myocardial infarction and doxorubicin-induced cardiotoxicity.

Exercise training benefits the heart. The knowledge of post-transcription regulation, especially RNA editing, in hearts remain rare. ADAR2 is an enzyme that edits adenosine to inosine nucleotides in double-stranded RNA, and RNA editing is associated with many human diseases. We found that ADAR2 was upregulated in hearts during exercise training. AAV9-mediated cardiac-specific ADAR2 overexpression attenuated acute myocardial infarction (AMI), MI remodeling, and doxorubicin (DOX)-induced cardiotoxicity. In vitro, overexpression of ADAR2 inhibited DOX-induced cardiomyocyte (CM) apoptosis. but it could also induce neonatal rat CM proliferation. Mechanistically, ADAR2 could regulate the abundance of mature miR-34a in CMs. Regulations of miR-34a or its target genes (Sirt1, Cyclin D1, and Bcl2) could affect the pro-proliferation and anti-apoptosis effects of ADAR2 on CMs. These data demonstrated that exercise-induced ADAR2 protects the heart from MI and DOX-induced cardiotoxicity. Our work suggests that ADAR2 overexpression or a post-transcriptional associated RNA editing via ADAR2 may be a promising therapeutic strategy for heart diseases.

Traditional Chinese Medication Qiliqiangxin Attenuates Diabetic Cardiomyopathy via Activating PPARγ.

Background: Diabetic cardiomyopathy is the primary complication associated with diabetes mellitus and also is a major cause of death and disability. Limited pharmacological therapies are available for diabetic cardiomyopathy. Qiliqiangxin (QLQX), a Chinese medication, has been proven to be beneficial for heart failure patients. However, the role and the underlying protective mechanisms of QLQX in diabetic cardiomyopathy remain largely unexplored. Methods: Primary neonatal rat cardiomyocytes (NRCMs) were treated with glucose (HG, 40 mM) to establish the hyperglycemia-induced apoptosis model in vitro. Streptozotocin (STZ, 50 mg/kg/day for 5 consecutive days) was intraperitoneally injected into mice to establish the diabetic cardiomyopathy model in vivo. Various analyses including qRT-PCR, western blot, immunofluorescence [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining] histology (hematoxylin-eosin and Masson's trichrome staining), and cardiac function (echocardiography) were performed in these mice. QLQX (0.5 µg/ml in vitro and 0.5 g/kg/day in vivo) was used in this study. Results: QLQX attenuated hyperglycemia-induced cardiomyocyte apoptosis via activating peroxisome proliferation-activated receptor γ (PPARγ). In vivo, QLQX treatment protected mice against STZ-induced cardiac dysfunction and pathological remodeling. Conclusions: QLQX attenuates diabetic cardiomyopathy via activating PPARγ.

Low-intensity pulsed ultrasound prevents prolonged hypoxia-induced cardiac fibrosis through HIF-1α/DNMT3a pathway via a TRAAK-dependent manner.

Hypoxia-induced cardiac fibrosis is an important pathological process in cardiovascular disorders. This study aimed to determine whether low-intensity pulsed ultrasound (LIPUS), a novel and safe apparatus, could alleviate hypoxia-induced cardiac fibrosis, and to elucidate the underlying mechanisms. Hypoxia (1% O2 ) and transverse aortic constriction (TAC) were performed on neonatal rat cardiac fibroblasts and mice to induce cardiac fibrosis, respectively. LIPUS irradiation was applied for 20 minutes every 6 hours for a total of 2 times in vitro, and every 2 days from 1 week before surgery to 4 weeks after surgery in vivo. We found that LIPUS dose-dependently attenuated hypoxia-induced cardiac fibroblast phenotypic conversion in vitro, and ameliorated TAC-induced cardiac fibrosis in vivo. Hypoxia significantly upregulated the nuclear protein expression of hypoxia-inducible factor-1α (HIF-1α) and DNA methyltransferase 3a (DNMT3a). LIPUS pre-treatment reversed the elevated expression of HIF-1α, and DNMT3a. Further experiments revealed that HIF-1α stabilizer dimethyloxalylglycine (DMOG) hindered the anti-fibrotic effect of LIPUS, and hampered LIPUS-mediated downregulation of DNMT3a. DNMT3a small interfering RNA (siRNA) prevented hypoxia-induced cardiac fibrosis. Results also showed that the mechanosensitive protein-TWIK-related arachidonic acid-activated K+ channel (TRAAK) messenger RNA (mRNA) expression was downregulated in hypoxia-induced cardiac fibroblasts, and TAC-induced hearts. TRAAK siRNA impeded LIPUS-mediated anti-fibrotic effect and downregulation of HIF-1α and DNMT3a. Above results indicated that LIPUS could prevent prolonged hypoxia-induced cardiac fibrosis through TRAAK-mediated HIF-1α/DNMT3a signalling pathway.

Citri Reticulatae Pericarpium protects against isoproterenol-induced chronic heart failure via activation of PPARγ.

BACKGROUND: Accumulated clinical trials and animal studies showed that Qiliqiangxin (QLQX), a traditional Chinese medicine formula containing extracts of 11 herbs, exerts beneficial effects on chronic heart failure (HF). Citri Reticulatae Pericarpium (CRP), one herbal medicine in QLQX, has been widely used in treatment against digestive, respiratory and cardiovascular diseases (CVDs) in China. However, the cardiac protective effects and mechanisms of CRP are still unclear. METHODS: The effects of CRP were investigated in isoproterenol (ISO)-induced chronic HF mice model and neonatal rat ventricular cardiomyocytes (NRVMs) treated with ISO. Echocardiography was used to determine cardiac function. Hematoxylin-eosin (HE) staining and α-actinin immunofluorescent staining were used to measure cardiomyocyte size. Cardiac fibrosis was evaluated by Masson's trichrome staining. The expression of atrial natriuretic polypeptide (ANP) and brain natriuretic polypeptide (BNP) were determined by quantitative real time PCR (qRT-PCR). Western blot was applied to examine the expression of peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coactivator-1α (PGC-1α), fibrosis-related and apoptosis-related proteins. RESULTS: We found that CRP could significantly attenuate ISO-induced cardiac dysfunction, inhibit cardiac pathological hypertrophy and alleviate myocardial fibrosis and apoptosis. Mechanistically, the downregulation of PPARγ and PGC-1α in ISO-injected mice hearts and ISO-treated NRVMs could be reversed by CRP treatment. The beneficial effects of CRP against ISO-induced HF were abolished by PPARγ inhibitor (T0070907), suggesting that CRP-mediated PPARγ upregulation was essential for the preventive effect of CRP on ISO-induced cardiac dysfunction. CONCLUSIONS: In conclusion, our study demonstrated that CRP attenuates ISO-induced cardiac remodeling via PPARγ activation, which represents a new application for CRP in the prevention of chronic HF.

Citri reticulatae Pericarpium attenuates Ang II-induced pathological cardiac hypertrophy via upregulating peroxisome proliferator-activated receptors gamma.

BACKGROUND: Pathological cardiac hypertrophy is a major risk factor for cardiovascular diseases, including heart failure. However, limited pharmacological therapies are available for reversing the maladaptive process and restoring cardiac function. Citri reticulatae Pericarpium (CRP) has been used in traditional Chinese medicine prescriptions for clinical treatment. Previous studies have shown that CRP and its ingredients have beneficial effects on the cardiovascular system. However, whether CRP has a protective effect against pathological cardiac hypertrophy remains unknown. METHODS: Primary neonatal rat cardiomyocytes (NRCMs) were treated with angiotensin II (Ang II) to induce pathological hypertrophy in vitro. Immunofluorescent staining and quantitative real-time PCR (qRT-PCR) were used to determine the cell size and the expression of hypertrophic gene markers (Anp and Bnp), respectively. Male C57BL/6 mice were subjected to the investigation of cardiac hypertrophy induced by Ang II (2.5 mg/kg/d for 4 weeks). CRP (0.5 g/kg/d for 4 weeks) was administrated to treat mice with or without peroxisome proliferator-activated receptors gamma (PPARγ) inhibitor T0070907 (1 mg/kg/d for 4 weeks treatment) infused with Ang II. Cardiac hypertrophy (hematoxylin-eosin staining and qRT-PCR), fibrosis (Masson's Trichrome staining, qRT-PCR, and western blot), and cardiac function (echocardiography) were examined in these mice. Western blot was used to determine the protein level of PPARγ and PGC-1α both in NRCMs and in mice. RESULTS: We found that CRP could prevent Ang II-induced pathological cardiac hypertrophy evidenced by improving cardiac function, decreasing hypertrophic growth and reducing cardiac fibrosis. Also, we demonstrated that PPARγ was upregulated by CRP both in NRCMs and in hearts. Moreover, PPARγ inhibitor could abolish the inhibitory effects of CRP on Ang II-induced pathological cardiac hypertrophy. CONCLUSIONS: CRP attenuates Ang II-induced pathological cardiac hypertrophy by activating PPARγ.

MicroRNA-221 promotes proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) by targeting tissue inhibitor of metalloproteinases-3 (TIMP3).

BACKGROUND: Aberrant vascular smooth muscle cell (VSMC) proliferation and migration play an important role in the development of cardiovascular diseases including pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs, miRs) have been considered to be implicated in the progression of PAH pathogenesis. In this study, we aim to clarify the role of miR-221 on proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and identify the target genes involved in this biological process. METHODS: PASMCs were isolated from the pulmonary arteries of male Sprague-Dawley (SD) rats. Cell proliferation of PASMCs was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell migration was determined by a scratch wound assay. Quantitative real-time PCR was used to determine the expression of miR-221 while western blot analysis was used to determine the expression of TIMP3. Luciferase assay was used to confirm that TIMP3 was a direct target gene of miR-221. Monocrotaline (MCT) induced-PAH rat model was established and miR-221 and TIMP3 levels were checked in lung tissue and PASMCs from PAH rats. RESULTS: miR-221 was able to promote the proliferation and migration PASMCs. TIMP3 were negatively regulated by miR-221 at the protein level in PASMCs. In addition, TIMP3 was identified to be a direct target gene of miR-221 in PASMCs based on luciferase assays. TIMP3 knockdown abolished the inhibitory effect of miR-221 inhibitor on PASMCs proliferation and migration, suggesting TIMP3 mediated the effects of miR-221 in PASMCs. Finally, we found that miR-221 was increased while TIMP3 was down-regulated in PASMCs in MCT-treated rats. CONCLUSIONS: In conclusion, miR-221 promotes PASMCs proliferation and migration by targeting TIMP3. MiR-221 and TIMP3 could be potential therapeutic targets for the treatment of PAH.

Prognostic Value of N-Terminal Pro-B-Type Natriuretic Peptide and Glomerular Filtration Rate in Patients With Acute Heart Failure.

Aims: To investigate the relationship between N-terminal pro-B-type natriuretic peptide (NT-proBNP), Glomerular Filtration Rate (GFR), and outcomes in patients hospitalized with acute heart failure (AHF). Methods: The trial was registered at http://www.chictr.org/cn/. (ChiCTR - ONC - 12001944). A total of 493 patients hospitalized for AHF in cardiology department of the First Affiliated Hospital of Nanjing Medical University from March 2012 to October 2016 were enrolled into registry. The end event was the occurrence of all-cause death within an 18-month follow-up. The data collected from the participants in admission were used to calculate the GFR by chronic kidney disease epidemiology collaboration equation (CKD-EPI) and performed the according statistical analysis. Results: There were 74 participants (13.8%) dropped out and 91 (21.7%) passed away within the 18-month follow up. Comparison of clinical indicators between survival and death group were analyzed for the long-term prognosis of patients with AHF. In the single factor analysis, both NT-proBNP and GFR were statistically significant (P < 0.001). Combined NT-proBNP and GFR in multi-factor COX regression analysis showed significant predictive value (P < 0.001). In receiver operator characteristics (ROC) analyses, the area under the curves (AUC) for NT-proBNP was 0.648 [95%CI: 0.598-0.695, P < 0.001] and for GFR was 0.677 [95%CI: 0.627-0.723, P < 0.001]. According to the Youden index, the best prediction point of NT-proBNP was 2,137 pg/ml and GFR was 61.7 ml/(min·1.73 m2). After using the Binary Logistic Regression to combine the two indicators, the AUC was 0.711, which was significantly compared to the AUC of either single factor. The sensitivity of the combined indicators were 0.535, the specificity were 0.853. According to the cut-off point, these two indexes were separated into four groups for further analysis by Kaplan-Meier survival curve comparison (log-rank test), which showed that patients in the group with higher NT-proBNP and lower GFR had the worst prognosis. Conclusions: In patients with NT-proBNP > 2,137 pg/ml and GFR < 61.7 ml/(min·1.73 m2), the risk of death was significantly higher. The combination of GFR and NT-proBNP improved the predictive value for the long-term prognosis of AHF patients.

The anti-microbial peptide LL-37/CRAMP levels are associated with acute heart failure and can attenuate cardiac dysfunction in multiple preclinical models of heart failure.

Rationale: Biomarkers for the diagnosis of heart failure (HF) are clinically essential. Circulating antimicrobial peptides LL-37 has emerged as a novel biomarker in cardiovascular disease, however, its relevance as a biomarker for acute HF are undetermined. Methods: Acute HF patients were enrolled in this study and the serum levels of LL-37/CRAMP (cathelicidin-related antimicrobial peptide) were measured by ELISA. The receiver-operator characteristic (ROC) curve was used to determine if serum LL-37 could be a biomarker for acute HF. Mouse CRAMP (mCRAMP, mouse homolog for human LL-37) was also determined in both heart and serum samples of, transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced HF mice models, and phenylephrine (PE) and angiotensin II (AngII)-induced neonatal mouse cardiomyocytes (NMCMs) hypertrophic models, both intracellular and secreted, by ELISA. The protective effects of mCRAMP were determined in TAC, ISO, and AngII-induced HF in mice while whether HF was exacerbated in AngII-infused animals were checked in mCRAMP knockout mice. The underlying mechanism for protective effects of CARMP in pathological hypertrophy was determined by using a NF-κB agonist together with rCRAMP (rat homolog for human LL-37) in AngII or PE treated neonatal rat cardiomyocytes (NRCMs). Results: Serum levels of LL-37 were significantly decreased in acute HF patients (area under the curve (AUC) of 0.616), and negatively correlated with NT-proBNP. We further confirmed that mCRAMP was decreased in both heart and serum samples of TAC- and ISO-induced HF mice models. Moreover, in PE and AngII-induced NMCMs hypertrophic models, both intracellular and secreted mCRAMP levels were reduced. Functionally, mCRAMP could attenuate TAC, ISO, and AngII-induced HF in mice while CRAMP deficiency exacerbated HF. Mechanistically, the anti-hypertrophy effects of CRAMP were mediated by NF-κB signaling. Conclusions: Collectively, serum LL-37 is associated with acute HF and increasing CRAMP is protective against deleterious NF-κB signaling in the rodent.