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To address the problem of heat removal by phase change material (PCM) wall at nighttime in the summer season, a new cooling wall that makes use of the high latent heats of PCMs, the high heat conductivities of micro-channel heat pipes (MHPs), and the passive cooling of sky radiative cooling (RC) is introduced, and is named the MHP-RC-PCM wall. In this study, preliminary experiments were first conducted to determine the emissivity of the radiative plate and the properties of PCMs (paraffin, RT28HC). Next, numerical models of the MHP-RC-PCM wall were established to simulate the thermal behavior, and the model was validated with the experimental results. The parameters that affect the thermal behavior of the MHP-RC-PCM wall, including the phase transition temperature, latent heat of the PCM, number of MHPs, and year-round thermal behavior were investigated. The results showed that the phase transition temperature (Tm) of the PCM had a significant influence on the interior surface temperature, liquid fraction and cooling load reduction ratio of the MHP-RC-PCM wall, whereas the PCM latent heat had little effect. The cooling load reduction ratio was approximately 4% for Tm = 31 °C, which was higher than that for Tm = 26 °C. In addition, it was determined that the year-round energy-saving of the MHP-RC-PCM wall were approximately 18.2% greater than that of the Brick wall with the same thickness, and 0.4% higher than that of PCM wall in Guangzhou City, China.

HYD-PEP06, an endostatin-modified polypeptide, has been shown to produce effective anti-colorectal carcinoma effects through inhibiting epithelial–mesenchymal transition (EMT). However, whether HYD-PEP06 has similar suppressive effect on hepatocellular carcinoma (HCC) remained unknown. In this study, HYD-PEP06 inhibited metastasis and EMT but not proliferation in vitro. Cignal finder pathway reporter array and Western blot analysis revealed that HYD-PEP06 suppressed HCCLM3 cell metastasis and EMT by inhibiting the PI3K/AKT pathway. Moreover, HYD-PEP06 exerted anti-metastasis effects in HepG2 cancer stem-like cells (CSCs) via suppressing the WNT/β-catenin signaling pathway. Finally, in HCCLM3 tumor-bearing BALB/c nu/nu nude mice, HYD-PEP06 substantially suppressed tumor growth, lung metastasis and HCC progress. Our results suggest that HYD-PEP06 inhibits the metastasis and EMT of HCC and CSCs as well, and thus has the potential as an agent for HCC treatment., Graphical abstract HYD-PEP06 suppresses hepatocellular carcinoma (HCC) metastasis, epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties by inhibiting PI3K/AKT and WNT/β-catenin signaling activation, which significantly inhibited progression of HCC.Image 1

RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2O2/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2O2/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.

Small ubiquitin-related modifiers (SUMO) represent a class of ubiquitin-like proteins that are conjugated, like ubiquitin, by a set of enzymes to form cellular regulatory proteins, and play key roles in the control of cell proliferation, differentiation, and apoptosis. We found that ginkgolic acid (GA) can significantly reduce cell vitality in a dose- and time-dependent manner and can also accelerate cyto-apoptosis in both Tca8113 and Cal-27 cells. Migration and wound-healing assays were executed to determine the anti-migration effect of GA in oral squamous cell carcinoma (OSCC) cell lines. GA represses transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) markers in OSCC cell lines. This investigation is the first evidence that GA suppresses TGF-β1-induced SUMOylation of SMAD4. We show that GA affects the phosphorylation of SMAD2/3 protein and the release of SMAD4. In the xenograft mouse model, the OSCC progression was reduced by GA, effectively suppressing the growth of tumors. In addition, siSMAD4 improved cell migration and viability, which was inhibited by GA in Tca8113 cells. GA suppresses tumorigenicity and tumor progression of OSCC through inhibition of TGF-β1-induced enhancement of SUMOylation of SMAD4. Thus, GA could be a promising therapeutic for OSCC., Graphical Abstract

Transforming growth factor-β (TGF-β) signaling pathway is involved in fibrosis in most, if not all forms of cardiac diseases. Here, we evaluate a positive feedback signaling the loop of TGF-β1/promyelocytic leukemia (PML) SUMOylation/Pin1 promoting the cardiac fibrosis. To test this hypothesis, the mice underwent transverse aortic constriction (3 weeks) were developed and the morphological evidence showed obvious interstitial fibrosis with TGF-β1, Pin1 upregulation, and increase in PML SUMOylation. In neonatal mouse cardiac fibroblasts (NMCFs), we found that exogenous TGF-β1 induced the upregulation of TGF-β1 itself in a time- and dose-dependent manner, and also triggered the PML SUMOylation and the formation of PML nuclear bodies (PML-NBs), and consequently recruited Pin1 into nuclear to colocalize with PML. Pharmacological inhibition of TGF-β signal or Pin1 with LY364947 (3 μM) or Juglone (3 μM), the TGF-β1-induced PML SUMOylation was reduced significantly with downregulation of the messenger RNA and protein for TGF-β1 and Pin1. To verify the cellular function of PML by means of gain- or loss-of-function, the positive feedback signaling loop was enhanced or declined, meanwhile, TGF-β-Smad signaling pathway was activated or weakened, respectively. In summary, we uncovered a novel reciprocal loop of TGF-β1/PML SUMOylation/Pin1 leading to myocardial fibrosis.

Background and purpose PEP06, a polypeptide modified from endostatin, was investigated for its antitumour effects on colorectal cancer (CRC) and the possible mechanisms of this antitumour activity were examined in in vitro and in vivo models. Experimental approach After PEP06 treatment, cell proliferation and migration assays were performed in CRC cells. Epithelial-mesenchymal transition (EMT) progression was determined by Western blotting, immunofluorescent staining and immunohistochemistry in vitro and in a residual xenograft model. MiRNAs regulated by PEP06 were identified by miRNA microarray and verified by in situ hybridization and quantitative real-time PCR. The interactions between PEP06 and integrin αvβ3 were determined with Biacore SA biochips. The cellular function of miR-146b-5p was validated by gain-of-function and loss-of-function approaches. A mouse model of lung metastasis was used to determine the effect of PEP06 on metastatic growth. Key results PEP06 did not affect cell viability but reduced migration and EMT in SW620 and HCT116 cells. PEP06 significantly repressed the expression of miR-146b-5p in these two cell lines through binding to integrin αvβ3. MiR-146b-5p was shown to increase EMT by targeting Smad4, and the miR-146b-5p-Smad4 cascade regulated EMT in CRC. PEP06 also suppressed CRC pulmonary metastasis, increased survival of mice and hampered residual tumour growth by inhibiting EMT through down-regulating miR-146b-5p. Conclusions and implications PEP06 is a polypeptide that inhibits the growth and metastasis of colon cancer through its RGD motif binding to integrin αvβ3, thereby down-regulating miR-146b-5p to inhibit EMT in vitro and in vivo. It might have potential as a therapeutic for CRC.

With the rapid development of power demand response in grid interactive building, it is urgent for regional energy system with great power flexibility potential to realize the interaction with grid. The power flexibility of regional energy system can be divided into direct power flexibility and power flexibility transformed from thermal flexibility. The former comes from power generation system and power storage equipment, whereas the latter comes from cold (heat) storage system. To improve the power flexibility and economy of regional energy stations, this paper proposes a TOU-price-based optimization model for the operation strategy for a grid interactive regional energy system. The model optimizes the hourly load distribution of the direct energy supply system and the energy storage system to achieve load transfer without affecting user comfort requirements, to maximize the power flexibility, and to improve the economy of the system. The proposed model can serve as reference for the operation strategy optimization and the system design of other existing or new regional energy systems. On the basis of experimental comparison and simulation analysis, the accuracy and rationality of the optimization strategy are verified.

// Yu Liu 1, * , Duo Li 3, * , Dan Nie 1, * , Shang-Kun Liu 1 , Fang Qiu 1 , Mei-Tong Liu 1 , Yuan-Yuan Li 1 , Jia-Xin Wang 1 , Yan-Xin Liu 1 , Chang-Jiang Dong 1 , Di Wu 1 , Wei Tian 1 , Jia Yang 1 , Wei Mu 1 , Jia-Tong Li 1 , Dan Zhao 2 , Xiao-Feng Wang 3 , Wen-Feng Chu 1 and Bao-Feng Yang 1 1 Department of Pharmacology, The State-Province Key Laboratories of Biomedicine Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, College of Pharmacy, Harbin Medical University at Harbin, Heilongjiang 150081, P. R. China 2 Department of Clinical Pharmacy, Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, The 2nd Affiliated Hospital, Harbin Medical University at Harbin, Heilongjiang 150081, P. R. China 3 Department of Oral and Maxillofacial Surgery, The 2nd Affiliated Hospital, Harbin Medical University at Harbin, Heilongjiang 150081, P. R. China * These authors contributed equally to this work Correspondence to: Wen-Feng Chu, email: cwf76928@aliyun.com Bao-Feng Yang, email: yangbf@ems.hrbmu.edu.cn Keywords: cardiotoxicity, human ether-a-go-go-related gene, transforming growth factor β1, PML nuclear body, SUMOylation Received: October 12, 2016 Accepted: April 17, 2017 Published: May 02, 2017 ABSTRACT The human ether-a-go-go-related gene (HERG) channel is a novel target for the treatment of drug-induced long QT syndrome, which causes lethal cardiotoxicity. This study is designed to explore the possible role of PML SUMOylation and its associated nuclear bodies (NBs) in the regulation of HERG protein expression. Both arsenic trioxide (ATO) and angiotensin II (Ang II) were able to significantly reduce HERG protein expression, while also increasing PML SUMOylation and accelerating the formation of PML-NBs. Pre-exposure of cardiomyocytes to a SUMOylation chemical inhibitor, ginkgolic acid, or the silencing of UBC9 suppressed PML SUMOylation, subsequently preventing the downregulation of HERG induced by ATO or Ang II. Conversely, knockdown of RNF4 led to a remarkable increase in PML SUMOylation and the function of PML-NBs, further promoting ATO- or Ang II-induced HERG protein downregulation. Mechanistically, an increase in PML SUMOylation by ATO or Ang II dramatically enhanced the formation of PML and Pin1 complexes in PML-NBs, leading to the upregulation of TGF-β1 protein, eventually inhibiting HERG expression through activation of protein kinase A. The present work uncovered a novel molecular mechanism underlying HERG protein expression and indicated that PML SUMOylation is a critical step in the development of drug-acquired arrhythmia.

Background/Aims: The present study investigated whether the transient receptor potential melastatin 4 (TRPM4) channel plays a role in high salt diet (HSD)-induced endothelial injuries. Methods: Western blotting and immunofluorescence were used to examine TRPM4 expression in the mesenteric endothelium of Dahl salt-sensitive (SS) rats fed a HSD. The MTT, TUNEL, and transwell assays were used to evaluate the cell viability, cell apoptosis, and cell migration, respectively, of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were used to determine the concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), and E-selectin. Carboxy-H2DCFDA, a membrane-permeable reactive oxygen species (ROS)-sensitive fluorescent probe, was used to detect intracellular ROS levels. Results: TRPM4 was mainly expressed near the plasma membrane of mesenteric artery endothelial cells, and its expression level increased in SS hypertensive rats fed a HSD. Its protein expression was significantly upregulated upon treatment with exogenous hydrogen peroxide (H2O2) and aldosterone in cultured HUVECs. Cell viability decreased upon treatment with both agents in a concentration-dependent manner, which could be partially reversed by 9-phenanthrol, a specific TRPM4 inhibitor. Exogenous H2O2 induced apoptosis, enhanced cell migration, and increased the release of adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, all of which were significantly attenuated upon treatment with 9-phenanthrol. Aldosterone and H2O2 induced the accumulation of intracellular ROS, which was significantly inhibited by 9-phenanthrol, suggesting that oxidative stress is one of the mechanisms underlying aldosterone-induced endothelial injury. Conclusions: Given the fact that oxidative stress and high levels of circulating aldosterone are present in hypertensive patients, we suggest that the upregulation of TRPM4 in the vascular endothelium may be involved in endothelial injuries caused by these stimuli.

Tongue cancer is one of the most common oral malignancies. Quisinostat is a histone deacetylase inhibitor with antitumor activity. The aim of this study was to evaluate the effects of quisinostat on the viability of tongue squamous cell carcinoma (TSCC) cells (CAL-27, TCA-8113) in vitro and in vivo. Cell viability, cell morphological observation, scratch wound-healing assay, transwell migration assay, transmission electron microscope, flow cytometry and cellular reactive oxygen species were assessed in vitro. The results showed that quisinostat can significantly inhibit the viability, growth and migration of TSCC cells. And quisinostat could significantly induce TSCC cells apoptosis, pyroptosis, and ferroptosis. Quisinostat significantly inhibited tumor tissue growth in animal experiments. Up-regulation of the expression of Bax, cleaved-caspase3, caspase-1, p53, phospho-p53 and down-regulated of the expression of caspase-3, Bcl-2, GPX4 in cell lines and tumor tissues of nude mice were observed by Western blotting analysis. Up-regulation of the expression of caspase-1, Bax, cleaved-caspase3, p53 and down-regulated of the expression of ki67, caspase-3, Bcl-2, GPX4 in tumor tissues of nude mice were observed by immunohistochemistry. TUNEL analysis showed that quisinostat could increase the apoptosis rate in the tumor tissues of nude mice. Up-regulation of the expression of p53 and down-regulated expression of GPX4 in cell lines were observed by immunofluorescent staining, and the expression locations of p53 and GPX4 proteins in TSCC cells were observed. Based on these findings, quisinostat may be a potential drug for the treatment of tongue squamous cell carcinoma.

Background/Aims: Deregulated myocardial fibrosis is associated with a wide spectrum of cardiac conditions, being considered one of the major causes for heart disease. Our study was designed to investigate the role of microRNA-328 (miR-328) in regulating cardiac fibrosis. Methods: We induced cardiac fibrosis following MI by occlusion of the left coronary artery in C57BL/6 mice. Real-time PCR was employed to evaluate the level of miR-328. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site in the TGFβRIII gene. Western blot analysis was used to examine TGFβRIII, p-smad2/3 and TGF-β1 at protein level. Results: In this study, we found that miR-328 was significantly upregulated in the border zone of infarcted myocardium of wild type (WT) mice; TGFβRIII was downregulated whereas TGF-β1 was upregulated along with increased cardiac fibrosis. And miR-328 stimulated TGF-β1 signaling and promoted collagen production in cultured fibroblasts. We further found that the pro-fibrotic effect of miR-328 was mediated by targeting TGFβRIII. Additionally, cardiac fibrosis was significantly reduced in infarcted heart when treated with miR-328 antisense. Conclusions: These data suggest that miR-328 is a potent pro-fibrotic miRNA and an important determinant of cardiac fibrosis in diseased heart.

The epithelial-mesenchymal transition (EMT) is a key event associated with metastasis and dissemination in breast tumor pathogenesis. Promyelocytic leukemia (PML) gene produces several isoforms due to alternative splicing; however, the biological function of each specific isoform has yet to be identified. In this study, we report a previously unknown role for PMLIV, the most intensely studied nuclear isoform, in transforming growth factor-β (TGF-β) signaling-associated EMT and migration in breast cancer. This study demonstrates that PMLIV overexpression promotes a more aggressive mesenchymal phenotype and increases the migration of MCF-7 cancer cells. This event is associated with activation of the TGF-β canonical signaling pathway through the induction of Smad2/3 phosphorylation and the translocation of phospho-Smad2/3 to the nucleus. In this study, we report a previously unknown role for PMLIV in TGF-β signaling-induced regulation of breast cancer-associated EMT and migration. Targeting this pathway may be therapeutically beneficial.

Background and Purpose Statins decrease heart disease risk, but their mechanisms are not completely understood. We examined the role of the TGF-β receptor III (TGFBR3) in the inhibition of cardiac fibrosis by simvastatin. Experimental Approach Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in mice given simvastatin orally for 7 days. Cardiac fibrosis was measured by Masson staining and electron microscopy. Heart function was evaluated by echocardiography. Signalling through TGFBR3, ERK1/2, JNK and p38 pathways was measured using Western blotting. Collagen content and cell viability were measured in cultures of neonatal mouse cardiac fibroblasts (NMCFs). Interactions between TGFBR3 and the scaffolding protein, GAIP-interacting protein C-terminus (GIPC) were detected using co-immunoprecipitation (co-IP). In vivo, hearts were injected with lentivirus carrying shRNA for TGFBR3. Key Results Simvastatin prevented fibrosis following MI, improved heart ultrastructure and function, up-regulated TGFBR3 and decreased ERK1/2 and JNK phosphorylation. Simvastatin up-regulated TGFBR3 in NMCFs, whereas silencing TGFBR3 reversed inhibitory effects of simvastatin on cell proliferation and collagen production. Simvastatin inhibited ERK1/2 and JNK signalling while silencing TGFBR3 opposed this effect. Co-IP demonstrated TGFBR3 binding to GIPC. Overexpressing TGFBR3 inhibited ERK1/2 and JNK signalling which was abolished by knock-down of GIPC. In vivo, suppression of cardiac TGFBR3 abolished anti-fibrotic effects, improvement of cardiac function and changes in related proteins after simvastatin. Conclusions and Implications TGFBR3 mediated the decreased cardiac fibrosis, collagen deposition and fibroblast activity, induced by simvastatin, following MI. These effects involved GIPC inhibition of the ERK1/2/JNK pathway.

Background and purpose Protein modification by small ubiquitin-like modifier (SUMO) plays a critical role in the pathogenesis of heart diseases. The present study was designed to determine whether ginkgolic acid (GA) as a SUMO-1 inhibitor exerts an inhibitory effect on cardiac fibrosis induced by myocardial infarction (MI). Experimental approach GA was delivered by osmotic pumps in MI mice. Masson staining, electron microscopy (EM) and echocardiography were used to assess cardiac fibrosis, ultrastructure and function. Expression of SUMO-1, PML, TGF-β1 and Pin1 was measured with Western blot or Real-time PCR. Collagen content, cell viability and myofibroblast transformation were measured in neonatal mouse cardiac fibroblasts (NMCFs). Promyelocytic leukemia (PML) protein was over-expressed by plasmid transfection. Key results GA improved cardiac fibrosis and dysfunction, and decreased SUMO-1 expression in MI mice. GA (>20 μM) inhibited NMCF viability in a dose-dependent manner. Nontoxic GA (10 μM) restrained angiotensin II (Ang II)-induced myofibroblast transformation and collagen production. GA also inhibited expression of TGF-β1 mRNA and protein in vitro and in vivo. GA suppressed PML SUMOylation and PML nuclear body (PML-NB) organization, and disrupted expression and recruitment of Pin1 (a positive regulator of TGF-β1 mRNA), whereas over-expression of PML reversed that. Conclusions and implications Inhibition of SUMO-1 by GA alleviated MI-induced heart dysfunction and fibrosis, and the SUMOylated PML/Pin1/TGF-β1 pathway is crucial for GA-inhibited cardiac fibrosis.

Background Myocardial infarction ( MI ) is often accompanied by cardiomyocyte apoptosis, which decreases heart function and leads to an increased risk of heart failure. The aim of this study was to examine the effects of transforming growth factor‐β receptor III ( TGF βR3) on cardiomyocyte apoptosis during MI . Methods and Results An MI mouse model was established by left anterior descending coronary artery ligation. Cell viability, apoptosis, TGF βR3, and mitogen‐activated protein kinase signaling were assessed by methylthiazolyldiphenyl‐tetrazolium bromide assay, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling assay, immunofluorescence, electron microscopy, and Western blotting. Our results demonstrated that TGF βR3 expression in the border region of the heart was dynamically changed during MI . After stimulation with H 2 O 2 , TGF βR3 overexpression in cardiomyocytes led to increased cell apoptosis and activation of p38 signaling, whereas TGF βR3 knockdown had the opposite effect. ERK 1/2 and JNK 1/2 signaling was not altered by TGF βR3 modulation, and p38 inhibitor ( SB 203580) reduced the effect of TGF βR3 on apoptosis, suggesting that p38 has a nonredundant function in activating apoptosis. Consistent with the in vitro observations, cardiac TGF βR3 transgenic mice showed augmented cardiomyocyte apoptosis, enlarged infarct size, increased injury, and enhanced p38 signaling upon MI . Conversely, cardiac loss of function of TGF βR3 by adeno‐associated viral vector serotype 9–TGFβR3 short hairpin RNA attenuated the effects of MI in mice. Conclusions TGF βR3 promotes apoptosis of cardiomyocytes via a p38 pathway–associated mechanism, and loss of TGF βR3 reduces MI injury, which suggests that TGF βR3 may serve as a novel therapeutic target for MI .

A long non-coding RNA (lncRNA), named myocardial infarction associated transcript (MIAT), has been documented to confer risk of myocardial infarction (MI). The aim of this study is to elucidate the pathophysiological role of MIAT in regulation of cardiac fibrosis. In a mouse model of MI, we found that MIAT was remarkably up-regulated, which was accompanied by cardiac interstitial fibrosis. MIAT up-regulation in MI was accompanied by deregulation of some fibrosis-related regulators: down-regulation of miR-24 and up-regulation of Furin and TGF-β1. Most notably, knockdown of endogenous MIAT by its siRNA reduced cardiac fibrosis and improved cardiac function and restored the deregulated expression of the fibrosis-related regulators. In cardiac fibroblasts treated with serum or angiotensin II, similar up-regulation of MIAT and down-regulation of miR-24 were consistently observed. These changes promoted fibroblasts proliferation and collagen accumulation, whereas knockdown of MIAT by siRNA or overexpression of miR-24 with its mimic abrogated the fibrogenesis. Our study therefore has identified MIAT as the first pro-fibrotic lncRNA in heart and unraveled the role of MIAT in the pathogenesis of MI. These findings also promise that normalization of MIAT level may prove to be a therapeutic option for the treatment of MI-induced cardiac fibrosis and the associated cardiac dysfunction.

The promyelocytic leukemia protein (PML) is essential in the assembly of dynamic subnuclear structures called PML nuclear bodies (PML-NBs), which are involved in regulating diverse cellular functions. However, the possibility of PML being involved in cardiac disease has not been examined. In mice undergoing transverse aortic constriction (TAC) and arsenic trioxide (ATO) injection, transforming growth factor β1 (TGF-β1) was upregulated along with dynamic alteration of PML SUMOylation. In cultured neonatal mouse cardiac fibroblasts (NMCFs), ATO, angiotensin II (Ang II), and fetal bovine serum (FBS) significantly triggered PML SUMOylation and the assembly of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, the unique SUMO E2-conjugating enzyme, reduced the development of cardiac fibrosis and partially improved cardiac function in TAC mice. In contrast, enhancing SUMOylated PML accumulation, by silencing RNF4, a poly-SUMO-specific E3 ubiquitin ligase, accelerated the induction of cardiac fibrosis and promoted cardiac function injury. PML colocalized with Pin1 (a positive regulator for TGF-β1 mRNA expression in PML-NBs) and increased TGF-β1 activity. These findings suggest that the UBC9/PML/RNF4 axis plays a critical role as an important SUMO pathway in cardiac fibrosis. Modulating the protein levels of the pathway provides an attractive therapeutic target for the treatment of cardiac fibrosis and heart failure.

Objectives A great number of studies indicate that cardiac fibroblasts are essential for maintaining the structure and function of heart. Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study examined whether over-expression of HIF-1α could prevent hypoxia-induced injury in neonatal rat cardiac fibroblasts and, if so, its possible molecular targets. Methods Western blotting was used to detect protein level. MTT, electron microscopy, TUNEL staining and confocal microscopy were used to identify cell viability, cell apoptosis and intracellular calcium ([Ca2+]i) in cardiac fibroblasts, respectively. Results When cardiac fibroblasts were exposed to hypoxia, HIF-1α protein in nuclei was transiently accumulated at 1 h, and then gradually degraded within 24 h of hypoxia exposure. Over-expression of HIF-1α enhanced nucleus expression of HIF-1α in cardiac fibroblasts, and significantly abolished the decrease of cell viability and cell apoptosis caused by 24-h hypoxia. Accordingly, hypoxia-induced Bax up-regulation, Bcl-2 down-regulation, caspase-3 activation and overload of [Ca2+]i in cardiac fibroblasts were reversed by HIF-1α over-expression, but were promoted by 30 μmol/l SC205346, a specific HIF-1α blocker. Conclusions Our results indicate that HIF-1α may act as a protective factor in the apoptotic process of cardiac fibroblasts and represent a potential therapeutic target for heart remodeling after hypoxia injury.

Cisapride has been shown to have electrophysiological effects on the heart. The aim of this study was to investigate whether cisapride has effects on cardiac hypertrophy. Rat and cellular models of cardiac hypertrophy were used in this study. Cell surface area (CSA), mRNA and protein expression were used to evaluate cardiac hypertrophy. Cardiac function was measured by echocardiography. Cisapride attenuated ISO-induced increase in CSA in a dose-dependent manner in cultured neonatal rat cardiomyocytes. A significant anti-hypertrophic effect was achieved by cisapride 0.01μM (P0.05). Cisapride repressed the increased mRNA levels of ANP, BNP, β-MHC in ISO-treated cells (P0.05). However, mallotoxin or GR113808 did not influence anti-hypertrophic effects of cisapride. In addition, cisapride inhibited the increase of intracellular Ca(2+) ([Ca(2+)]i) and the upregulation of protein levels of calcineurin and NFATc-3 (P0.05) as well as prevented the downregulation of p-NFATc-3 (P0.01) induced by ISO. Consistently, cisapride (0.5mg/kg/day) produced inhibitory effects on cardiac hypertrophy, including the suppression of ANP, BNP, β-MHC, calcineurin, and NFATc-3; elevation of p-NFATc-3; reduction of cross-sectional area of cardiomyocytes in rat heart; and restoration of cardiac dysfunction by improving left ventricular diastolic and systolic performance. Importantly, cisapride 0.5 and 5.0mg/kg/day did not cause prolongation of QT and QTc intervals in rats. In conclusion, cisapride possesses a prominent anti-hypertrophic property which is likely to be conferred by its ability to downregulate Ca(2+)/calcineurin/NFAT and the present data provide new insight into this drug action.

Beta-adrenoceptor (β-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of β(1) -AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. β(1) -AR expression was significantly up-regulated and let-7a, c, d, e and i were markedly down-regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let-7e suppressed β(1) -AR expression at the protein level, without affecting β(1) -AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced β(1) -AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited β(1) -AR expression in rats, whereas len-AMO-let-7e up-regulated β(1) -AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of β(1) -AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on β(1) -AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of β(1) -AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective β-AR blocker and metoprolol, a selective β(1) -AR blocker. Down-regulation of let-7e contributes to the adverse increase in β(1) -AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse β(1) -AR up-regulation and treating AMI-induced arrhythmia.

Oxidative stress has been linked to endothelial dysfunction in atherosclerosis and hypertension. The present study was designed to investigate the effect of hydrogen peroxide (H2O2) on angiotensin-converting enzyme (ACE), a key regulator of the renin-angiotensin system, and the mechanisms underlying ACE regulation in human umbilical vein endothelial cells (HUVECs). We used Tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, enzyme-linked immunosorbent assay (ELISA) for cAMP measurement, real-time PCR for mRNA detection, and Western blot for protein analysis in the study. Our results demonstrated that H2O2 (50-1000 μM) decreased HUVECs viability by inducing apoptosis. Notably, H2O2 upregulated ACE expression in a concentration-dependent manner. H2O2 100 μM significantly enhanced cyclic adenosine monophosphate (cAMP) expression by 1.48-fold (P0.05). Additionally, forskolin 10 μM, a cAMP agonist, was also found to enhance ACE expression by 1.78-fold (P0.05); in contrast, H-89 10 μM, a protein kinase A (PKA) inhibitor, abolished H2O2-induced ACE expression and prevented the enhancing effect of forskolin-induced ACE expression. Similar effects on ACE mRNA were also observed. cAMP-response element-specific decoy oligodeoxynucleotides (CRE-dODN) containing binding sites for cAMP-response element-binding protein (CREB) inhibited ACE expression at both the mRNA and protein levels. Negative control CRE-dODN had no effect on ACE expression. We conclude that H2O2 upregulates the expression of ACE through the activation of cAMP/PKA/CREB signal pathway in HUVECs, indicating a role of oxidative stress in the pathophysiology of hypertension.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF-1α in mild hypoxia-induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O(2)) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF-1α mRNA and accumulation of HIF-1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca(2+)-calcineurin signals were also enhanced in a time-dependent manner under MH condition. MH-induced cardiomyocytes hypertrophy, TRPC up-regulation and enhanced Ca(2+)-calcineurin signals were inhibited by an HIF-1α specific blocker, SC205346 (30 μM), whereas promoted by HIF-1α overexpression. Electrophysiological voltage-clamp demonstrated that DAG analogue, OAG (30 μM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF-1α compared to negative control. These results implicate that HIF-1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up-regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca(2+)-calcineurin signals.

Background MicroRNAs (miRNAs) are noncoding RNAs of 18-25 nucleotides that post-transcriptionally regulate gene expression and are involved in a wide range of physiological and pathological conditions. The β-adrenergic signaling pathway plays a fundamental role in regulation of heart function. The present study was designed to investigate the expression profile of miRNAs and functional implications under conditions of β-adrenoceptor activation or inhibition in rat heart. Material/methods Hemodynamic parameters were measured to assess heart function in Wistar rats treated with isoproterenol (ISO) or propranolol (PRO). miRNA expression was analyzed by miRNA Microarray and confirmed by real-time quantitative reverse transcription PCR (real-time qRT-PCR). Results Isoproterenol (ISO, a β-adrenoceptor activator) and propranolol (PRO, a β-adrenoceptor inhibitor) induced differential miRNA expression profiles. Out of 349 miRNAs measured, 43 were upregulated and nine downregulated in the ISO group, while five miRNAs were upregulated and 28 downregulated in PRO group. Among these altered miRNAs in both PRO and ISO groups, 11 were cardiac abundant and 11 showed opposite profiles between the PRO and ISO groups. The recognized anti-hypertrophic miRNAs miR-1, miR-21 and miR-27b, and the pro-hypertrophic miRNAs miR-22, miR-24, miR-199a, miR-212 and miR-214, were upregulated in the ISO group. In the PRO group, pro-hypertrophic miRNA miR-30c was upregulated, whereas miR-212 was downregulated. Conclusions β-adrenoceptor intervention alters miRNA expression profile, and miRNAs may be involved in the β-adrenoceptor signaling pathway. Cardiomyocyte hypertrophy is a balanced process between pro-hypertrophic and anti-hypertrophic regulation and involves, at the very least, miRNA participation.

Background Previous studies suggested that platelet-activating factor (PAF) plays an important role in ischemic diseases. Apoptosis has been implicated in myocardial infarction-related cell death. The present study was designed to determine whether PAF could induce apoptosis in cardiac myocytes and the underlying mechanisms by which PAF causes apoptosis. Methods H9c2 cardiac myocytes were used to investigate the effect of PAF on intracellular calcium concentration, cell viability and cell apoptosis. Signaling pathway of caspase-3, cytochrome c and MAPK (ERK, JNK, p38) was determined during the PAF induced apoptosis. Results First, our results showed that treatment of H9c2 cardiomyocytes with PAF (0.2 to 20 µM) caused apoptosis in these cells and the apoptotic process was suppressed by either BN52021 (an antagonist of PAF receptor) or BAPTA/AM (an intracellular Ca 2+ chelator), suggesting an involvement of PAF and its receptor mediated calcium-dependent signaling. Second, we found that activity of p38-MAPK (mitogen-activated protein kinase) and caspase-3 was elevated in the cells treated with PAF, without altering activity of ERK and JNK, and that PAF-induced enhancement of caspase-3 activity was attenuated by application of either BAPTA/AM or SB203580 (p38 inhibitor). Furthermore, PAF-induced apoptosis and release of cytochrome c from mitochondria was blunted by SB203580, and PAF-induced enhancement of p38 activity was also attenuated by BAPTA/AM. Conclusion Our data implicate that a PAF and its receptor in triggering apoptosis occurs in cultured H9c2 cardiac myocytes via a calcium-dependent p38 MAPK activated cytochrome c /caspase-3 apoptosis signaling pathway.

Background and purpose: Tanshinone IIA is an active component of a traditional Chinese medicine based on Salvia miltiorrhiza, which reduces sudden cardiac death by suppressing ischaemic arrhythmias. However, the mechanisms underlying the anti-arrhythmic effects remain unclear.

Scutellarin is a flavonoid extracted from the traditional Chinese herb, Erigeron breviscapus Hand Mazz. In the present study, the vasorelaxant effects of scutellarin and the underlying mechanism were investigated in isolated rat aorta. Scutellarin (3, 10, 30, 100 microm) caused a dose-dependent relaxation in both endothelium-intact and endothelium-denuded rat aortic rings precontracted with noradrenaline bitartrate (IC(50) = 7.7 +/- 0.6 microm), but not with potassium chloride. Tetraethylammonium, glibenclamide, atropine, propranolol, indomethacin and N(G)-nitro-l-arginine methyl ester had no influence on the vasorelaxant effect of scutellarin, which further excluded the involvement of potassium channels, muscarinic receptor, nitric oxide pathway and prostaglandin in this effect. Pretreatment with scutellarin decreased the tonic phase, but not the phasic phase of the noradrenaline bitartrate induced tension increment. Scutellarin also alleviated Ca(2+)-induced vasoconstriction in Ca(2+)-depleted/noradrenaline bitartrate pretreated rings in the presence of voltage-dependent calcium channel blocker verapamil. The noradrenaline bitartrate evoked intracellular calcium increase was inhibited by scutellarin. Scutellarin had no effect on phorbol-12,13-diacetate induced contraction in a calcium-free bath solution. These results showed that scutellarin could relax thoracic artery rings in an endothelium-independent manner. The mechanism seems to be the inhibition of extracellular calcium influx independent of the voltage-dependent calcium channel.

Viscum coloratum has been used in the indigenous system of medicine for the treatment of various diseases such as myocardial ischemia and arrhythmia. Platelet-activating factor (PAF) is an important player in cardiovascular diseases. The aim of this study was to investigate the protective effects of Viscum coloratum flavonoids (VCF) against ischemic myocardial injuries in vivo and to further investigate its regulatory effect on PAF. Studies were performed in a rat model of myocardial infarction and in isolated myocytes. It was found that VCF relieved myocardial injuries during ischemia. PAF (10−11m) significantly increased the intracellular free Ca2+ concentration ([Ca2+]i) and VCF inhibited the changes induced by PAF in single cardiac myocytes. The results suggest that VCF can improve cardiac function and that VCF reduces ischemic myocardial injuries via blocking the signaling pathway of PAF. Therefore, PAF blockers may be candidate drugs for preventing cardiac injuries during ischemia/reperfusion, and subsequently improving cardiac function. Copyright © 2007 John Wiley & Sons, Ltd.

Arsenic trioxide (As(2)O(3)) has been found to be effective for relapsed or refractory acute promyelocytic leukaemia, but its clinical use is burdened by QT prolongation, Torsade de pointes tachycardias, and sudden cardiac death. The aim of the present study was to elucidate the ionic mechanisms of As(2)O(3)-induced abnormalities of cardiac electrophysiology and the therapeutic action of choline on As(2)O(3)-caused QT prolongation in guinea pig. Intravenous administration of As(2)O(3) prolonged the QT interval in a dose- and time-dependent manner in guinea pig hearts, and the QT prolongation could be modulated by choline. By using whole-cell patch clamp technique and confocal laser scanning microscopy, we found that As(2)O(3) significantly lengthened action potential duration measured at 50 and 90% of repolarization, enhanced L-type calcium currents (I(Ca-L)), inhibited delayed rectifier potassium currents (I(K)), and increased intracellular calcium concentration ([Ca(2+)](i)) in guinea pig ventricular myocytes. Choline corrected As(2)O(3)-mediated alterations of action potential duration, I(Ca-L) and [Ca(2+)](i), but had no effect on the I(K) inhibition. As(2)O(3) markedly disturbed the normal equilibrium of transmembrane currents (increasing I(Ca-L) and suppressing I(K)) in guinea pig cardiomyocyte, and induced prolongation of action potential duration, further degenerated into QT prolongation. Choline normalized QT interval abnormality and corrected lengthened action potential duration by inhibiting the elevated I(Ca-L) and [Ca(2+)](i) in ventricular myocytes during As(2)O(3) application.

Statins decrease heart disease risk, but their mechanisms are not completely understood. We examined the role of the TGF-β receptor III (TGFBR3) in the inhibition of cardiac fibrosis by simvastatin.Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in mice given simvastatin orally for 7 days. Cardiac fibrosis was measured by Masson staining and electron microscopy. Heart function was evaluated by echocardiography. Signalling through TGFBR3, ERK1/2, JNK and p38 pathways was measured using Western blotting. Collagen content and cell viability were measured in cultures of neonatal mouse cardiac fibroblasts (NMCFs). Interactions between TGFBR3 and the scaffolding protein, GAIP-interacting protein C-terminus (GIPC) were detected using co-immunoprecipitation (co-IP). In vivo, hearts were injected with lentivirus carrying shRNA for TGFBR3.Simvastatin prevented fibrosis following MI, improved heart ultrastructure and function, up-regulated TGFBR3 and decreased ERK1/2 and JNK phosphorylation. Simvastatin up-regulated TGFBR3 in NMCFs, whereas silencing TGFBR3 reversed inhibitory effects of simvastatin on cell proliferation and collagen production. Simvastatin inhibited ERK1/2 and JNK signalling while silencing TGFBR3 opposed this effect. Co-IP demonstrated TGFBR3 binding to GIPC. Overexpressing TGFBR3 inhibited ERK1/2 and JNK signalling which was abolished by knock-down of GIPC. In vivo, suppression of cardiac TGFBR3 abolished anti-fibrotic effects, improvement of cardiac function and changes in related proteins after simvastatin.TGFBR3 mediated the decreased cardiac fibrosis, collagen deposition and fibroblast activity, induced by simvastatin, following MI. These effects involved GIPC inhibition of the ERK1/2/JNK pathway.

Deregulated myocardial fibrosis is a major cause of cardiac dysfunction. The focus of this investigation was to elucidate the importance of miRNAs in the control of pathophysiological processes fol...

NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca2+]i (intracellular Ca2+ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca2+]i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy.

Arsenic trioxide (As2O3; ATO) is clinically effective in treating acute promyelocytic leukemia (APL); however, it frequently causes cardiotoxic effects. This study was designed to investigate whether ATO could induce apoptosis of cardiac fibroblasts (CFs) that play very important roles in maintaining the structure integrity and function of the heart. Cardiac fibroblasts from guinea pigs administered with ATO (1mg/kgbw) were used to test the pro-apoptotic role of ATO in vivo. The current study demonstrated that ATO induced morphological characteristics of apoptosis and Caspase-3 activation in CFs of guinea pigs along with a significant up-regulation in TGF-β1 protein expression, Bax/Bcl-2 ratio and ERK1/2 phosphorylation. In vitro MTT assay showed that ATO remarkably reduced the viability of cultured cardiac fibroblasts (NRCFs) from neonatal rat in a concentration- and time-dependent manner. Consistent with the notions in vivo, ATO significantly induced the apoptosis in NRCFs, dramatically up-regulated TGF-β1 protein level and Bax/Bcl-2 ratio in a time-dependent fashion and activated Caspase-3 and ERK1/2. Finally, pretreatment with LY364947, an inhibitor of TGF-β signaling could apparently reverse these changes. We therefore conclude that TGF-β is functionally linked to ERK1/2 and that TGF-β signaling is responsible for ATO-induced CFs apoptosis, which provides a novel mechanism of ATO related cardiac toxicology.

Aims Arsenic trioxide (ATO), an effective therapeutic agent for acute promyelocytic leukaemia, can cause sudden cardiac death due to long QT syndrome (LQTS). The present study was designed to determine whether ATO could induce cardiac fibrosis and explore whether cardiac fibroblasts (CFs) are involved in the development of LQTS by ATO. Methods and results ATO treatment of guinea pigs caused substantial interstitial myocardial fibrosis and LQTS, which was accompanied by an increase in transforming growth factor β1(TGF-β1) secretion and a decrease in ether-a-go-go -related gene (HERG) and inward rectifying potassium channel (IK1) subunit Kir2.1 protein levels. ATO promoted collagen production and TGF-β1 expression and secretion in cultured CFs. Whole-cell patch clamp and western blotting showed that treatment with TGF-β1 markedly reduced HERG and I K1 current densities and downregulated HERG and Kir2.1 protein expression in HEK293 cells stably transfected with the human recombinant HERG channel and in cardiomyocytes (CMs). These changes were completely reversed by treatment with the protein kinase A (PKA) antagonist, H89. CM and CF co-cultures showed that ATO significantly increased TGF-β1 levels in the culture medium, whereas markedly reduced HERG and Kir2.1 protein levels were observed in CMs compared with ATO-treated CMs not co-cultured with CFs. Finally, in vivo administration of LY364947, a pharmacological antagonist of TGF-β signalling, dramatically prevented interstitial fibrosis and LQTS and abolished aberrant expression of TGF-β1, HERG, and Kir2.1 in ATO-treated guinea pigs. Conclusion ATO-induced TGF-β1 secretion from CFs aggravates QT prolongation, suggesting that modulation of TGF-β signalling may provide a novel strategy for the treatment of drug-induced LQTS.

Summary Background MicroRNAs (miRNAs) are noncoding RNAs of 18–25 nucleotides that post-transcriptionally regulate gene expression and are involved in a wide range of physiological and pathological conditions. The β-adrenergic signaling pathway plays a fundamental role in regulation of heart function. The present study was designed to investigate the expression profile of miRNAs and functional implications under conditions of β-adrenoceptor activation or inhibition in rat heart. Material/Methods Hemodynamic parameters were measured to assess heart function in Wistar rats treated with isoproterenol (ISO) or propranolol (PRO). miRNA expression was analyzed by miRNA Microarray and confirmed by real-time quantitative reverse transcription PCR (real-time qRT-PCR). Results Isoproterenol (ISO, a β-adrenoceptor activator) and propranolol (PRO, a β-adrenoceptor inhibitor) induced differential miRNA expression profiles. Out of 349 miRNAs measured, 43 were upregulated and nine downregulated in the ISO group, while five miRNAs were upregulated and 28 downregulated in PRO group. Among these altered miRNAs in both PRO and ISO groups, 11 were cardiac abundant and 11 showed opposite profiles between the PRO and ISO groups. The recognized anti-hypertrophic miRNAs miR-1, miR-21 and miR-27b, and the pro-hypertrophic miRNAs miR-22, miR-24, miR-199a, miR-212 and miR-214, were upregulated in the ISO group. In the PRO group, pro-hypertrophic miRNA miR-30c was upregulated, whereas miR-212 was downregulated. Conclusions β-adrenoceptor intervention alters miRNA expression profile, and miRNAs may be involved in the β-adrenoceptor signaling pathway. Cardiomyocyte hypertrophy is a balanced process between pro-hypertrophic and anti-hypertrophic regulation and involves, at the very least, miRNA participation.

Cardiac fibrosis is characterized by aberrant proliferation of cardiac fibroblasts and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-21 (miR-21) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming growth factor beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-21 and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-21 were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-21 into cardiac fibroblasts, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-21. It suggests that up-regulation of miR-21 could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-21 and reduce collagen production in fibroblasts. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-21. Taken together, we found a novel reciprocal loop between miR-21 and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.

A lot of evidence indicates that cardiac fibroblasts are essential for maintaining the structure and function of heart. The present study examined whether TGFBR3 (transforming growth factor type III receptor, also known as betaglycan) could prevent hypoxia-induced injury in neonatal mice cardiac fibroblasts, if so, its possible molecular targets. MTT, electron microscopy and TUNEL assay were used to identify cell viability and apoptosis in neonatal mice cardiac fibroblasts. Results showed that hypoxia for 24 h markedly reduce cell viability by 49.8 ± 8.9%, largely via apoptosis. However, hypoxia-induced apoptosis in cardiac fibroblasts were almost completely prevented by overexpression of TGFBR3. In the present study, hypoxia also induced TGF-β1, p-Smad2/3 expression, TGFBR1-TGFBR2 complex formation and collagen production in cardiac fibroblasts, which were attenuated substantially by TGFBR3 overexpression. TGFBR3 also reversed Bax up-regulation, Bcl-2 down-regulation and Caspase-3 activation induced by hypoxia in cardiac fibroblasts. Hypoxia or TGF-β1 itself triggered an increase of [Ca(2+) ](i) in cardiac fibroblasts, which were both inhibited by TGFBR3 overexpression. Taken together, our results indicate that TGFBR3 may act as a protective factor in apoptotic process of cardiac fibroblasts by negative regulation of TGF-β signaling and represent a potential therapeutic target for heart remodeling after hypoxia injury.

Viscum coloratum has been used in the indigenous system of medicine for the treatment of various diseases such as myocardial ischemia and arrhythmia. Platelet-activating factor (PAF) is an important player in cardiovascular diseases. The aim of this study was to investigate the protective effects of Viscum coloratum flavonoids (VCF) against ischemic myocardial injuries in vivo and to further investigate its regulatory effect on PAF. Studies were performed in a rat model of myocardial infarction and in isolated myocytes. It was found that VCF relieved myocardial injuries during ischemia. PAF (10(-11) m) significantly increased the intracellular free Ca2+ concentration ([Ca2+]i) and VCF inhibited the changes induced by PAF in single cardiac myocytes. The results suggest that VCF can improve cardiac function and that VCF reduces ischemic myocardial injuries via blocking the signaling pathway of PAF. Therefore, PAF blockers may be candidate drugs for preventing cardiac injuries during ischemia/reperfusion, and subsequently improving cardiac function.

We used Fluo-3/AM to examine the effect of platelet-activating factor on the intracellular Ca 2+ ([Ca 2+ ] i ) levels in isolated myocytes of guinea pig ventricle. Myocytes were isolated with Langendorff perfusion technique and were challenged with platelet-activating factor. Addition of platelet-activating factor (I pM to 10 nM) significantly increased the [Ca 2+ ] i in the presence and absence of extracellular Ca 2+ . The notion that increases in intracellular Ca 2+ induced by platelet-activating factor is the result of stimulation of intracellular Ca 2+ pool rather than increasing Ca 2+ influx was further supported by the whole cell patch-clamp experiments in which the platelet-activating factor did not alter the activity of L-type of Ca 2+ channels (I Ca-L ). Treatment of myocytes with ryanodine failed to abolish the stimulatory effect of platelet-activating factor on [Ca 2+ ] i . In contrast, inhibition of IP 3 -sensitive Ca 2+ release pool with 2-aminoethoxydiphenyl borate (2-APB) blocked the effect of platelet-activating factor. We conclude that the platelet-activating factor-induced increase in intracellular Ca 2+ is mediated by stimulation of IP 3 receptor but not by stimulation of I Ca-L and ryanodine-sensitive receptor.

The microRNAs miR-1 and miR-133 are preferentially expressed in cardiac and skeletal muscles and have been shown to regulate differentiation and proliferation of these cells. We report here a novel aspect of cellular function of miR-1 and miR-133 regulation of cardiomyocyte apoptosis. miR-1 and miR-133 produced opposing effects on apoptosis, induced by oxidative stress in H9c2 rat ventricular cells, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. miR-1 level was significantly increased in response to oxidative stress. We identified single target sites for miR-1 only, in the 3′-untranslated regions of the HSP60 and HSP70 genes, and multiple putative target sites for miR-133 throughout the sequence of the caspase-9 gene. miR-1 reduced the levels of HSP60 and HSP70 proteins without changing their transcript levels, whereas miR-133 did not affect HSP60 and HSP70 expression at all. By contrast, miR-133 repressed caspase-9 expression at both the protein and mRNA levels. The post-transcriptional repression of HSP60 and HSP70 and caspase-9 was further confirmed by luciferase reporter experiments. Our results indicate that miR-1 and miR-133 are involved in regulating cell fate with increased miR-1 and/or decreased miR-133 levels favoring apoptosis and decreased miR-1 and/or miR-133 levels favoring survival. Post-transcriptional repression of HSP60 and HSP70 by miR-1 and of caspase-9 by miR-133 contributes significantly to their opposing actions.