ABHD15 promotes cell viability, glycolysis, and inhibits apoptosis in cardiomyocytes under hypoxia.

BACKGROUND AND AIMS: Myocardial infarction (MI) has been an important heart disease affecting human health. The aim of this study was to investigate the regulatory effect of abhydrolase domain containing 15 (ABHD15) on hypoxic cardiomyocytes. METHODS AND RESULTS: Hypoxic cardiomyocytes are commonly used as an vitro model for the study of MI. We found that cardiomyocyte viability was decreased under hypoxia, but cell glucose uptake, insulin receptor phosphorylation level and apoptosis were increased. Interestingly, ABHD15 expression was up-regulated in hypoxia-induced cardiomyocytes. Then, we identified the function of ABHD15 in hypoxic cardiomyocytes by using ABHD15 overexpression vector or short interfering RNA (siRNA) against ABHD15. The results showed that overexpression of ABHD15 promoted hypoxic cardiomyocyte viability, glucose uptake and IR phosphorylation (p-IR), and inhibited cell apoptosis. However, knockdown of ABHD15 attenuated hypoxic cardiomyocyte viability, glucose uptake and IR phosphorylation, and promoted apoptosis. Moreover, we found that ABHD15 promoted glucose transporter 4 (GLUT4) expression, translocation and enhance rate-limiting enzyme activation of glycolysis, thereby affecting glucose uptake. Furthermore, our study suggested that ABHD15 may affect the viability and apoptosis of hypoxic cardiomyocytes through IR/Ras/Raf/ERK/MEK and IR/PI3K/AKT/Bcl2/Bad/caspase9 signaling pathways, respectively. When the phosphorylation of IR, Raf or ERK was blocked by inhibitors, the protective effect of overexpressing ABHD15 on the viability of hypoxic cardiomyocytes was eliminated. Furthermore, inhibiting the phosphorylation of IR, AKT or Bcl2 abolished the inhibitory effect of overexpressing ABHD15 on hypoxic cardiomyocyte apoptosis. CONCLUSION: ABHD15 regulated myocardial cell viability, glycolysis, and apoptosis under hypoxia, providing a novel potential therapeutic strategy for MI.

Interleukin-5 deletion promotes sepsis-induced M1 macrophage differentiation, deteriorates cardiac dysfunction, and exacerbates cardiac injury via the NF-κB p65 pathway in mice.

Inflammation plays a crucial role in sepsis-induced cardiac injury. The purpose of this study was to determine whether interleukin-5 (IL-5) affected lipopolysaccharide (LPS)-induced cardiac injury by regulating the inflammatory response. First, the expression level and source of cardiac IL-5 were examined, and the results showed that LPS treatment and cecal ligation decreased cardiac IL-5 expression in macrophages. In addition, LPS was used to establish a mouse sepsis model, and the effects of IL-5 deletion on cardiac injury, M1 macrophage differentiation and myocardial cell apoptosis were analyzed. The results showed that IL-5 deficiency significantly increased cardiac injury marker expression, worsened cardiac dysfunction, promoted M1 macrophage differentiation and exacerbated myocardial cell apoptosis in LPS-induced septic mice. The nuclear factor-kappa B (NF-κB) p65 pathway was inhibited by JSH-23, and the results showed that treatment with JSH-23 inhibited M1 macrophage differentiation and alleviated cardiac injury in LPS-treated IL-5-knockout mice. Furthermore, the effects of IL-5 deficiency on M1 macrophage differentiation and myocardial cell apoptosis were measured in vitro. The IL-5-mediated promotion of M1 macrophage differentiation was also reversed by S31-201, and the pro-apoptotic effect of IL-5 knockout on macrophage-mediated myocardial cell apoptosis was also reversed by JSH-23. In conclusion, we found that IL-5 knockout may exacerbate sepsis-induced cardiac injury by promoting M1 macrophage differentiation in mice. IL-5 may be a potential target for the clinical prevention of sepsis-related cardiac injury.

Advances in Research on the circRNA-miRNA-mRNA Network in Coronary Heart Disease Treated with Traditional Chinese Medicine.

There has been an increase in morbidity and mortality related to coronary heart disease (CHD) in China in recent years. Numerous clinical experiences and studies have shown that traditional Chinese medicine (TCM) plays an important role in the prevention, treatment, and prognosis of CHD. However, the mechanism of TCM in the treatment of CHD has not yet been elucidated. The circRNA-miRNA-mRNA network consists of miRNA that is competitively bound by circRNA, and miRNA regulates the transcription level of mRNA. Through literature review, we found that the circRNA-miRNA-mRNA network acts to contribute to certain effects to CHD such as myocardial hypertrophy, myocardial fibrosis, and heart failure. TCM contains constituents that act against CHD by antiatherosclerosis and apoptosis inhibition action, cardiac and cardiomyocyte protection, and these components also promote cell growth and protection of the vascular system by regulating miRNAs. Therefore, we consider that the circRNA-miRNA-mRNA network may be a new regulatory mechanism for the effective treatment of CHD by TCM.

Early expressed circulating long noncoding RNA CHAST is associated with cardiac contractile function in patients with acute myocardial infarction.

BACKGROUND: The mortality rate during the acute myocardial infarction (AMI) phase has substantially decreased, but post-AMI cardiac remodeling remains an important factor affecting patient prognosis. Several circulating long noncoding RNAs (lncRNAs) are reportedly involved in the chronic pathological process of cardiac function and remodeling in cardiovascular diseases. However, the potential roles of these circulating lncRNAs as biomarkers of cardiac function and remodeling during early-stage AMI remain unclear. METHODS: Fifty-three patients with AMI and 90 controls without AMI were consecutively enrolled in this study. Clinical parameters and blood samples at different time points (i.e., 24 h and 3 days) were collected. RESULTS: Compared with the controls, the circulating levels of cardiac hypertrophy-associated transcript (CHAST) significantly increased in AMI patients, and the CHAST levels obviously decreased at 3 days. In AMI patients, the expression levels of CHAST at 24 h were positively associated with cardiac contractile function and measured as left ventricular ejection fraction and left ventricular short-axis shortening rate (all P < 0.050). Multivariate regression analysis indicated that the expression level of CHAST at 24 h was an independent predictor of cardiac contractile function (standardized ß = 0.319, P =0.034). When grouped according to the quartile values of the CHAST in the AMI population, patients with the highest quartiles of CHAST expression level showed better cardiac contractile function than all the other quartiles (all P < 0.050). CONCLUSION: CHAST was an independent predictor of cardiac contractile function at early-stage AMI and may serve as a candidate biomarker for cardiac remodeling.

circRNA­miRNA association for coronary heart disease.

Coronary heart disease (CHD) is a major cause of morbidity and mortality and an important public health problem globally, but the mechanism of CHD is still complex and unclear. The purpose of the current study was to explore the mechanism underlying CHD using high­throughput technology. The study participants were patients with coronary angiography (CAG)­proven severity of coronary artery stenosis. Patients were divided into control and test group based on specific inclusion criteria, and data were collected regarding the results of routine inspection and the Gensini score (GS). We explored the mechanism underlying CHD with high­throughput integration of circular RNA (circRNA)­microRNA (miRNA) data. Through the expression of circRNA­miRNA, we discovered a total of 110 circRNAs to be differentially expressed in the two groups. Of these, 73 were upregulated and 37 downregulated in the CHD (fold ≥2.0 and P<0.05). Among 18 miRNAs, 13 were upregulated and 5 were downregulated in the CHD group (fold ≥2.0 and P<0.05). Enrichment analysis showed that circRNAs participate in a variety of disease development processes, biological processes, molecular functions, cellular components, and pathways (P<0.05). The mechanism underlying CHD may be closely related to up­ or downregulated circRNA and miRNA and co­expression of circRNA­miRNA specifically involved regulate multiple pathways and multiple cellular and molecular biological processes.

NDUFA4L2 protects against ischaemia/reperfusion-induced cardiomyocyte apoptosis and mitochondrial dysfunction by inhibiting complex I.

Myocardial ischaemia/reperfusion (I/R) injury may cause the apoptosis of cardiomyocytes as well as mitochondrial dysfunction. The aims of the present study were to investigate whether NADH dehydrogenase 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2) on myocardial ischaemia-reperfusion (I/R) injury and the underlying molecular mechanism. The hypoxia-reperfusion (H/R) model was established in vitro using H9c2 cells to simulate I/R injury. NDUFA4L2 and complex I expression levels were detected using RT-PCR and western blot. The apoptosis of H9c2 cells was evaluated by flow cytometry and the expression of Bax and Bcl-2 was detected by western blot. The mitochondrial function was assessed by ATP concentration, mPTP opening and cytochrome c (cyto C) expression. Our data indicated that NDUFA4L2 expression was significantly down-regulated in myocardial H/R injury. Overexpression of NDUFA4L2 led to a dramatic prevention of H/R-induced apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl-2. Meanwhile, augmentation of NDUFA4L2 dramatically prevented mitochondrial dysfunction caused by H/R as reflecting in the increased ATP concentration, delayed mPTP opening, as well as down-regulated cyto C expression. Moreover, complex I activation was heightened and negatively regulated by NDUFA4L2. Silencing complex I conspicuously attenuated cell apoptosis and mitochondrial dysfunction. Taken together, our findings demonstrated that NDUFA4L2 protects against H/R injury by preventing myocardium apoptosis and mitochondrial dysfunction via the complex I, and may be a potential therapeutic approach for attenuating myocardial I/R injury.

Downregulation of miR-122 attenuates hypoxia/reoxygenation (H/R)-induced myocardial cell apoptosis by upregulating GATA-4.

MicroRNA-122 has been reported to play a potential role in the apoptosis of myocardial cells. However, the effect of miR-122 in regulating myocardial ischemic injury has not been previously addressed. This study aimed to investigate the effect and the molecular basis of miR-122 on myocardial ischemic injury. Using the hypoxia/reoxygenation (H/R) model of rat cardiomyocytes H9C2 in vitro, we found that miR-122 was highly expressed in H9C2 cells after H/R treatment. Overexpression of miR-122 by recombinant adeno-associated viral vector infection markedly promoted the apoptosis of H9C2 cells induced by H/R treatment, whereas miR-122 inhibition significantly decreased cell apoptosis. Dual-luciferase reporter assay and western blot assay revealed that GATA-4 was a direct target gene of miR-122, and miR-122 suppressed the expression of GATA-4 via binding to its 3'-UTR. We further identified that overexpression of miR-122 inhibited the expression of GATA-4 at the mRNA and protein levels, whereas the inhibition of miR-122 upregulated the expression of GATA-4. Moreover, GATA-4 was poorly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the decrease in GATA-4 expression. Importantly, silencing of GATA-4 apparently abrogated the inhibitory effect of anti-miR-122 on H/R-induced cell apoptosis. In conclusion, these findings indicate that downregulation of miR-122 alleviates cardiomyocyte H/R injury through upregulation of GATA-4 expression, supplying a novel molecular target for myocardial ischemic injury.

Knockdown of FSTL1 inhibits oxLDL-induced inflammation responses through the TLR4/MyD88/NF-κB and MAPK pathway.

Activation of inflammation by oxidized low-density lipoprotein (oxLDL) has been implicated in the development of atherosclerosis. Follistatin-like protein 1 (FSTL1) play a central role in the inflammation process and modulate cardiovascular disorders. However, little is known about the effects of FSTL1 on the inflammation induced by oxLDL. The aim of this study was to evaluate the anti-inflammatory effect of FSTL1 and investigate potential mechanisms in cultured endothelial cells. A model of oxLDL-induced injury in human coronary artery endothelial cells (HCAECs) was established to evaluate the protective role of FSTL1. The mRNA transcription and secretion of TNF-α, IL-6, IL-8, ICAM-1, VCAM-1 and MCP-1 were assayed using RT-PCR and ELISA, respectively. We also investigated the effects of FSTL1 on the TLR4/MyD88/NF-κB and MAPK signaling pathways. OxLDL increased the expression and release of TLR4, IL-6, IL-8, ICAM-1, VCAM-1 and MCP-1 in a dose- and time-dependent manner. The effects of oxLDL on the production of inflammatory cytokines by endothelial cells were completely inhibited after depletion of FSTL1. Moreover, down-regulation of FSTL1 resulted in a significant reduction in the expression of TLR4 and its downstream proteins MyD88 and p-p65, along with p-p38, p-JNK and p-ERK. However, FSTL1 had no effect on the JAK/STAT signaling pathway. These findings provide strong evidence that FSTL1 displays anti-inflammatory effects against oxLDL-induced pro-inflammatory cytokine production via a mechanism that involves the TLR4/MyD88/NF-κB and MAPK signaling pathways.

Upregulated microRNA-214 enhances cardiac injury by targeting ITCH during coxsackievirus infection.

Viral myocarditis (VM), a severe clinical condition characterized by cardiac inflammation, is most frequently induced as a result of coxsackievirus infection. Evidence suggests that microRNAs may have significant roles in the progression of cardiac injury during coxsackievirus infection. Concurrently, microRNA (miR)-214 was found to be upregulated in the plasma and myocardial cells during this process. In the present study, eight candidate miRNAs, the functions of which are associated with myocarditis, were selected and their expression levels were evaluated by reverse transcription-quantitative polymerase chain reaction. miR-146b and miR-214 were found to have significantly upregulated expression levels in the heart tissues of patients with VM compared with those of the control subjects. Predictions via the use of online bioinformatics tools and confirmed by dual-luciferase assay and western blot analysis, revealed that ITCH, an NF-κB signaling suppressor, was a target gene of miR-214. To investigate the biological function of miR-214, tumor necrosis factor-α and interleukin-6 expression levels were evaluated in HeLa cell culture supernatant. The results revealed that miR-214 overexpression enhanced the expression of the two cytokines. In addition, the function of miR-214 was partially rescued by ITCH overexpression. Subsequently, concurrent results were obtained following experiments in murine cardiac myocytes. In conclusion, the results of the present study demonstrated that miR-214 contributed to the adverse inflammatory response to viral infection of the heart during coxsackievirus infection and is therefore a potential therapeutic target for the treatment of viral myocarditis.

An Egr-1-specific DNAzyme regulates Egr-1 and proliferating cell nuclear antigen expression in rat vascular smooth muscle cells.

The aim of the present study was to transfect rat aortic smooth muscle cells with an early growth response factor-1 (Egr-1)-specific DNAzyme (ED5), to observe its effect on Egr-1 and proliferating cell nuclear antigen (PCNA) expression and to elucidate the mechanism of ED5-mediated inhibition of vascular smooth muscle cell (VSMC) proliferation. VSMCs in primary culture obtained by tissue block adhesion were identified by morphological observation and α smooth muscle actin (α-SM-actin) immunocytochemistry. The cells were then transfected with ED5 or scrambled ED5 (ED5SCR). The three groups of cells used in the present study were the control group, ED5 group and ED5SCR group. The expression levels of Egr-1 and PCNA protein were detected following transfection by analyzing and calculating the integral optical density value in each group. Primary culture of VSMCs and transfection of ED5 and ED5SCR were successfully accomplished. Following stimulation with 10% fetal calf serum, the Egr-1 protein was expressed most strongly at 1 h and demonstrated a declining trend over time; the expression of PCNA protein began at 4 h, peaked at 24 h and then demonstrated a slightly declining trend over time. Compared with the control group and the ED5SCR group, ED5 inhibited the expression of Egr-1 and PCNA (P<0.05). ED5 was able to inhibit the expression of Egr-1 and PCNA proteins in VSMCs to a certain extent and VSMC proliferation in vitro. DNAzyme gene therapy may be useful as a new method for treating vascular proliferative diseases, including atherosclerosis and restenosis.