Jie-Ning Zhu

miR-1/miR-206 regulate Hsp60 expression contributing to glucose-mediated apoptosis in cardiomyocytes

Hsp60 is an important component of defense mechanisms against diabetic myocardial injury; however, the cause of Hsp60 reduction in the diabetic myocardium remains unknown. After stimulation of cardiomyocytes with high glucose in vivo and in vitro, significant up‐regulation of miR‐1/miR‐206 and post‐transcriptional modulation of Hsp 60 were observed. Serum response factor (SRF) and the MEK1/2 pathway were involved in miR‐1 and miR‐206 expression in cardiomyocytes. miR‐1 and miR‐206 regulated Hsp60 expression post‐transcriptionally and accelerated cardiomyocyte apoptosis through Hsp60. These results revealed that miR‐1 and miR‐206 regulate Hsp60 expression, contributing to high glucose‐mediated apoptosis in cardiomyocytes.

Panax notoginseng saponins inhibit ischemia-induced apoptosis by activating PI3K/Akt pathway in cardiomyocytes.

AIM OF THIS STUDY The panax notoginseng saponins (PNS) have been clinically used for the treatment of cardiovascular diseases and stroke in China. Evidences demonstrated that PNS could protect cardiomyocytes from injury induced by ischemia, but the underlying molecular mechanisms of this protective effect are still unclear. This study was aimed to investigate the protective effect and potential molecular mechanisms of PNS on apoptosis in H9c2 cells in vitro and rat myocardial ischemia injury model in vivo. MATERIALS AND METHODS H9c2 cells subjected to serum, glucose and oxygen deprivation (SGOD) were used as in vitro models and SD rats subjected to left anterior descending (LAD) coronary artery ligation were used as in vivo models. The anti-apoptotic effect of PNS was evaluated by Annexin V/PI analysis or TUNEL assay. Mitochondrial membrane potential (Δψm) was detected by JC-1 analysis. The expression of Akt and phosphorylated Akt (p-Akt) were detected by western blot assay. RESULTS PNS exhibited anti-apoptotic effect both in H9c2 cells and in ischemic myocardial tissues. However, the effect was blocked in vitro by LY294002, a specific PI3K inhibitor. The anti-apoptotic effect of PNS was mediated by stabilizing Δψm in H9c2 cells. Furthermore the indices of the left ventricular ejection fractions (EF), left ventricular fractional shortening (FS), left ventricular dimensions at end diastole (LVDd) and left ventricular dimensions at end systole (LVDs) suggested that PNS improved rats cardiac function. PNS significantly increased p-Akt both in H9c2 cells and in ischemic myocardial tissues and this effect was also blocked by LY294002 in H9c2 cells. CONCLUSION Results of this study suggested that PNS could protect myocardial cells from apoptosis induced by ischemia in both the in vitro and in vivo models through activating PI3K/Akt signaling pathway.

Smad3 inactivation and MiR-29b upregulation mediate the effect of carvedilol on attenuating the acute myocardium infarction-induced myocardial fibrosis in rat.

Carvedilol, a nonselective β-adrenoreceptor antagonist, protects against myocardial injury induced by acute myocardium infarction (AMI). The mechanisms underlying the anti-fibrotic effects of carvedilol are unknown. Recent studies have revealed the critical role of microRNAs (miRNAs) in a variety of cardiovascular diseases. This study investigated whether miR-29b is involved in the cardioprotective effect of carvedilol against AMI-induced myocardial fibrosis. Male SD rats were randomized into several groups: the sham surgery control, left anterior descending (LAD) surgery-AMI model, AMI plus low-dose carvedilol treatment (1 mg/kg per day, CAR-L), AMI plus medium-dose carvedilol treatment (5 mg/kg per day, CAR-M) and AMI plus high-dose carvedilol treatment (10 mg/kg per day, CAR-H). Cardiac remodeling and impaired heart function were observed 4 weeks after LAD surgery treatment; the observed cardiac remodeling, decreased ejection fraction, and fractional shortening were rescued in the CAR-M and CAR-H groups. The upregulated expression of Col1a1, Col3a1, and α-SMA mRNA was significantly reduced in the CAR-M and CAR-H groups. Moreover, the downregulated miR-29b was elevated in the CAR-M and CAR-H groups. The in vitro study showed that Col1a1, Col3a1, and α-SMA were downregulated and miR-29b was upregulated by carvedilol in a dose-dependent manner in rat cardiac fibroblasts. Inhibition of ROS-induced Smad3 activation by carvedilol resulted in downregulation of Col1a1, Col3a1, and α-SMA and upregulation of miR-29b derived from the miR-29b-2 precursor. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA expression. Taken together, we found that smad3 inactivation and miR-29b upregulation contributed to the cardioprotective activity of carvedilol against AMI-induced myocardial fibrosis.

MicroRNA 16 enhances differentiation of human bone marrow mesenchymal stem cells in a cardiac niche toward myogenic phenotypes in vitro

AIM Upregulation of microRNA 16 (miR-16) contributed to the differentiation of human bone marrow mesenchymal stem cells (hMSCs) toward myogenic phenotypes in a cardiac niche, the present study aimed to determine the role of miR-16 in this process. MAIN METHODS hMSCs and neonatal rat ventricular myocytes were co-cultured indirectly in two chambers to set up a cardiac microenvironment (niche). miRNA expression profile in cardiac-niche-induced hMSCs was detected by miRNA microarray. Cardiac marker expression and cell cycle analysis were determined in different treatment hMSCs. Quantitative real-time PCR and Western blot were used to identify the expression of mRNA, mature miRNA and protein of interest. KEY FINDINGS miRNA dysregulation was shown in hMSCs after cardiac niche induction. miR-16 was upregulated in cardiac-niche-induced hMSCs. Overexpression of miR-16 significantly increased G1-phase arrest of the cell cycle in hMSCs and enhanced the expression of cardiac marker genes, including GATA4, NK2-5, MEF2C and TNNI3. Differentiation-inducing factor 3 (DIF-3), a G0/G1 cell cycle arrest compound, was used to induce G1 phase arrest in cardiac-niche-induced hMSCs, and the expression of cardiac marker genes was up-regulated in DIF-3-treated hMSCs. The expression of CCND1, CCND2 and CDK6 was suppressed by miR-16 in hMSCs. CDK6, CCND1 or CCND2 knockdown resulted in G1 phase arrest in hMSCs and upregulation of cardiac marker gene expression in hMSCs in a cardiac niche. SIGNIFICANCE miR-16 enhances G1 phase arrest in hMSCs, contributing to the differentiation of hMSCs toward myogenic phenotypes when in a cardiac niche. This mechanism provides a novel strategy for pre-modification of hMSCs before hMSC-based transplantation therapy for severe heart diseases.

High glucose induces apoptosis in AC16 human cardiomyocytes via macrophage migration inhibitory factor and c-Jun N-terminal kinase

1. It is known that high glucose can induce cardiomyocyte apoptosis and that macrophage migration inhibitory factor (MIF) may be involved in the development of diabetes. However, the relationship between high glucose and MIF in diabetic cardiomyopathy remains unclear.

Attenuation of microRNA-16 derepresses the cyclins D1, D2 and E1 to provoke cardiomyocyte hypertrophy

Cyclins/retinoblastoma protein (pRb) pathway participates in cardiomyocyte hypertrophy. MicroRNAs (miRNAs), the endogenous small non‐coding RNAs, were recognized to play significant roles in cardiac hypertrophy. But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy. This study investigates the potential role of microRNA‐16 (miR‐16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy. An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC), and in a mouse with transverse aortic constriction (TAC) and in a mouse with subcutaneous injection of phenylephrine (PE) respectively. In addition, a cell model of hypertrophy was also achieved based on PE‐promoted neonatal rat ventricular cardiomyocyte and based on Ang‐II‐induced neonatal mouse ventricular cardiomyocyte respectively. We demonstrated that miR‐16 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats and mice. Overexpression of miR‐16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes, and inhibition of miR‐16 induced a hypertrophic phenotype in cardiomyocytes. Expressions of cyclins D1, D2 and E1, and the phosphorylated pRb were increased in hypertrophic myocardium and hypertrophic cardiomyocytes, but could be reversed by enforced expression of miR‐16. Cyclins D1, D2 and E1, not pRb, were further validated to be modulated post‐transcriptionally by miR‐16. In addition, the signal transducer and activator of transcription‐3 and c‐Myc were activated during myocardial hypertrophy, and inhibitions of them prevented miR‐16 attenuation. Therefore, attenuation of miR‐16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2 and E1, and activating cyclin/Rb pathway, revealing that miR‐16 might be a target to manage cardiac hypertrophy.

The caspase-8 shRNA-modified mesenchymal stem cells improve the function of infarcted heart

The beneficial effects of mesenchymal stem cells (MSCs) in cardiac cell therapy are greatly limited due to poor survival after transplantation into ischemic hearts. Here, we investigated whether caspase 8 small hairpin RNA (shRNA) modification enhance human MSCs (hMSCs) survival and improve infarcted heart function. Recombinant adenovirus encoding pre-miRNA-155-designed caspase 8 shRNA was prepared to inhibit caspase 8 expression in hMSCs. The effect of caspase 8 shRNA modification on protecting hMSCs from apoptosis under the conditions of serum deprivation and hypoxia was tested by Annexin V/PI staining and caspase 8 activity assay. The caspase 8 shRNA-modified and superparamagnetic iron oxide (SPIO)-labeled hMSCs were injected into the border zone of the infarcted region of rat heart. Echocardiography and Masson trichrome staining were performed to assess heart function and cardiac fibrosis. Our results showed that adenovirus-mediated caspase 8 shRNA could efficiently inhibit caspase 8 expression in hMSCs. Knock-down of caspase 8 expression lead to inhibition of hMSCs apoptosis, reduction of caspase 8 activity and up-regulations of HGF, IGF-1 and Bcl-2. Transplantation of caspase 8 shRNA-modified hMSCs could significantly improve infracted heart function, attenuate cardiac fibrosis. Consistently, the rate of cardiomyocyte apoptosis and caspase 8 activity were significantly decreased, and the survival rate of transplanted hMSCs was markedly elevated in the myocardium receiving caspase 8 shRNA-modified hMSCs transplantation. Together, our findings implicated the therapeutic potential of caspase 8 shRNA-modified hMSCs in improving the infarcted heart function.

Targeting EZH1 and EZH2 contributes to the suppression of fibrosis-associated genes by miR-214-3p in cardiac myofibroblasts

The role of microRNA-214-3p (miR-214-3p) in cardiac fibrosis was not well illustrated. The present study aimed to investigate the expression and potential target of miR-214-3p in angiotensin II (Ang-II)-induced cardiac fibrosis. MiR-214-3p was markedly decreased in the fibrotic myocardium of a mouse Ang-II infusion model, but was upregulated in Ang-II-treated mouse myofibroblasts. Cardiac fibrosis was shown attenuated in Ang-II-infused mice received tail vein injection of miR-214-3p agomir. Consistently, miR-214-3p inhibited the expression of Col1a1 and Col3a1 in mouse myofibroblasts in vitro. MiR-214-3p could bind the 3′-UTRs of enhancer of zeste homolog 1 (EZH1) and −2, and suppressed EZH1 and −2 expressions at the transcriptional level. Functionally, miR-214-3p mimic, in parallel to EZH1 siRNA and EZH2 siRNA, could enhance peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and inhibited the expression of Col1a1 and Col3a1 in myofibroblasts. In addition, enforced expression of EZH1 and −2, and knockdown of PPAR-γ resulted in the increase of Col1a1 and Col3a1 in myofibroblasts. Moreover, the NF-κB signal pathway was verified to mediate Ang-II-induced miR-214-3p expression in myofibroblasts. Taken together, our results revealed that EZH1 and −2 were novel targets of miR-214-3p, and miR-214-3p might be one potential miRNA for the prevention of cardiac fibrosis.

Macrophage migration inhibitory factor promotes expression of GLUT4 glucose transporter through MEF2 and Zac1 in cardiomyocytes

OBJECTIVE Evidence shows that both macrophage migration inhibitory factor (MIF) and GLUT4 glucose transporter are involved in diabetic cardiomyopathy (DCM), but it remains largely unknown whether and how MIF regulates GLUT4 expression in cardiomyocytes. The present study aims to investigate the mechanism underlying the modulation of GLUT4 by MIF in cardiomyocytes. MATERIAL AND METHODS Activations of AKT and AMPK signaling, and expressions of MIF, GLUT4 and the candidate GLUT4 regulation associated transcription factors in the diabetic mouse myocardium were determined. The screened transcription factors mediating MIF-promoted GLUT4 expression were verified by RNA interference (RNAi) and electrophoretic mobility shift assay (EMSA), respectively. RESULTS MIF was increased, but GLUT4 was decreased in the diabetic mouse myocardium. MIF could enhance glucose uptake and up-regulate GLUT4 expression in NMVCs. Expressions of transcription factor MEF2A, -2C, -2D and Zac1 were significantly up-regulated in MIF-treated neonatal mouse ventricular cardiomyocytes (NMVCs), and markedly reduced in the diabetic myocardium. Knockdown of MEF2A, -2C, -2D and Zac1 could significantly inhibit glucose uptake and GLUT4 expression in cardiomyocytes. Moreover, EMSA results revealed that transcriptional activities of MEF2 and Zac1 were significantly increased in MIF-treated NMVCs. AMPK signaling was activated in MIF-stimulated NMVCs, and AMPK activator AICAR could enhance MEF2A, -2C, -2D, Zac1 and GLUT4 expression. Additionally, MIF effects were inhibited by an AMPK inhibitor compound C and siRNA targeting MIF receptor CD74, suggesting the involvement of CD74-dependent AMPK activation. CONCLUSIONS Transcription factor MEF2 and Zac1 mediate MIF-induced GLUT4 expression through CD74-dependent AMPK activation in cardiomyocytes.

CDK6 mediates the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy

MicroRNA-1 (miR-1) is approved involved in cardiac hypertrophy, but the underlying molecular mechanisms of miR-1 in cardiac hypertrophy are not well elucidated. The present study aimed to investigate the potential role of miR-1 in modulating CDKs-Rb pathway during cardiomyocyte hypertrophy. A rat model of hypertrophy was established with abdominal aortic constriction, and a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocytes (NRVCs). We demonstrated that miR-1 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes. Dual luciferase reporter assays revealed that miR-1 interacted with the 3′UTR of CDK6, and miR-1 was verified to inhibit CDK6 expression at the posttranscriptional level. CDK6 protein expression was observed increased in hypertrophic myocardium and hypertrophic cardiomyocytes. Morover, miR-1 mimic, in parallel to CDK6 siRNA, could inhibit PE-induced hypertrophy of NRVCs, with decreases in cell size, newly transcribed RNA, expressions of ANF and β-MHC, and the phosphorylated pRb. Taken together, our results reveal that derepression of CDK6 and activation of Rb pathway contributes to the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.