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Both aldosterone and arginine vasopressin (AVP) are produced in the heart and may participate in cardiac fibrosis. However, their relationship remains unknown. This study aims to demonstrate the regulation and role of AVP in aldosterone synthesis in the heart. Rats were subjected to a sham operation or myocardial infarction (MI) by ligating the coronary artery. Cardiac function and fibrosis were assessed using echocardiography and immunohistochemical staining, respectively. In addition, the effects of AVP stimulation on cardiac microvascular endothelial cells (CMECs) were studied using ELISA, real-time PCR, and Western blotting. Compared with the rats having undergone a sham operation, the MI rats had an increased LVMI, type I collagen composition, and concentrations of aldosterone and AVP in the heart but decreased cardiac function. As the MI rats aged, the LVMI, type I collagen, aldosterone, and AVP increased, while the LVMI decreased. Furthermore, AVP time-dependently induced aldosterone secretion and CYP11B2 mRNA expression in CMECs. The p-CREB levels were significantly increased by AVP. Nevertheless, these effects were completely blocked by SR49059 or partially inhibited by KN93. This study demonstrated that AVP could induce the secretion of local cardiac aldosterone, which may involve CaMK and CREB phosphorylation and CYP11B2 upregulation through V1 receptor activation.

Background Experimental and clinical trials have demonstrated the efficiency of bone marrow-derived mesenchymal stromal/stem cells (bMSCs) in the treatment of myocardial infarction. However, after intravenous injection, the ineffective migration of engrafted bMSCs to the hearts remains an obstacle, which has an undesirable impact on the efficiency of cell-based therapy. Therefore, we attempted to identify a marker that could distinguish a subpopulation of bMSCs with a promising migratory capacity. Methods Here, CD51-negative and CD51-positive cells were isolated by flow cytometry from Ter119−CD45−CD31−bMSCs and cultured in specifically modified medium. The proliferation ability of the cells was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) staining or continuously monitored during culture, and the differentiation potential was assessed by culturing the cells in the appropriate conditioned media. Wound healing assays, transwell assays and quantitative polymerase chain reaction (qPCR) were used to measure the migratory ability. The mice were subjected to a sham operation or myocardial infarction (MI) by permanently occluding the coronary artery, and green fluorescent protein (GFP)-labelled cells were transplanted into the mice via intravenous infusion immediately after MI. Heart function was measured by echocardiography; infarct myocardium tissues were detected by triphenyl tetrazolium chloride (TTC) staining. Additionally, immunofluorescence staining was used to verify the characteristics of CD51+bMSCs and inflammatory responses in vivo. Statistical comparisons were performed using a two-tailed Student’s t test. Results In this study, the isolated CD51−bMSCs and CD51+bMSCs, especially the CD51+ cells, presented a favourable proliferative capacity and could differentiate into adipocytes, osteocytes and chondrocytes in vitro. After the cells were transplanted into the MI mice by intravenous injection, the therapeutic efficiency of CD51+bMSCs in improving left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) was better than that of CD51−bMSCs. Compared with CD51−bMSCs, CD51+bMSCs preferentially migrated to and were retained in the infarcted hearts at 48 h and 8 days after intravenous injection. Accordingly, the migratory capacity of CD51+bMSCs exceeded that of CD51−bMSCs in vitro, and the former cells expressed higher levels of chemokine receptors or ligands. Interestingly, the retained CD51+bMSCs retained in the myocardium possessed proliferative potential but only differentiated into endothelial cells, smooth muscle cells, fibroblasts or cardiomyocytes. Transplantation of CD51+bMSCs partially attenuated the inflammatory response in the hearts after MI, while the potential for inflammatory suppression was low in CD51−bMSC-treated mice. Conclusions These findings indicated that the CD51-distinguished subpopulation of bMSCs facilitated proliferation and migration both in vitro and in vivo, which provided a novel cell-based strategy to treat acute MI in mice by intravenous injection.

A high-fat diet is a major risk factor for coronary heart diseases. Matrix metalloprotease (MMP) expression is changed in many cardiovascular diseases. Selenium, which is an important trace element in animals, has a close relationship with cardiovascular diseases. The TGFβ1/Smad signalling pathway is ubiquitous in diverse tissues and cells, and it is also associated with the occurrence and development of cardiovascular diseases. Therefore, in this study, we aimed to determine selenium's effect on lipid metabolism, atherosclerotic plaque formation, and MMP2 expression, as well as the underlying functional mechanism.In vivo tests: 24 male New Zealand white rabbits were randomly divided into 4 groups: regular diet, high-fat diet, high-fat diet+selenium and regular diet+selenium groups. The high-fat diet induced the lipid disturbances of rabbits at week 12. Selenium supplementation lowered total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels (p0.01). Selenium supplementation also suppressed MMP2 over-expression in thoracic aortas. In vitro tests: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of selenium or ox-LDL. Ox-LDL promoted MMP2 expression by increasing TGFβ1, pSmad2, pSmad3 and Smad3 expression (p0.01). Selenium attenuated MMP2 over-expression by regulating the TGFβ1/Smad signalling pathway.Selenium suppressed high-fat diet-induced MMP2 over-expression in vivo by improving lipid metabolism. In vitro, selenium attenuated MMP2 over-expression through the TGFβ1/Smad signalling pathway.

Our previous study demonstrated that angiotensin II (Ang II) upregulates the expression of Kv1.5, a promising target for atrial fibrillation (AF) therapy, by activating ROS-dependent P-Smad2/3 and P-ERK 1/2. A recent study showed that hydrogen sulfide (H2S) may modulate the effects of angiotensin II (Ang II) by inhibiting the NADPH oxidase 4 (Nox4)-ROS signaling in the heart. The present study aimed to determine whether H2S is involved in the regulation of atrial Kv1.5 via ROS-related mechanisms in AF. Cultured neonatal rat atrial myocytes and a beagle model of AF were used for this study. In the neonatal rat atrial myocytes, quantitative PCR and enzyme immunoassays revealed that the mRNA expression levels of angiotensinogen, angiotensin-converting enzyme, and Ang II type I receptor (AT1R) and the Ang II supernatant concentration were significantly increased by hydrogen peroxide (H2O2) incubation, and these H2O2-induced alterations were reversed by diphenyleneiodonium, apocynin and H2S supplementation. Flow cytometry and Western blotting revealed that blockade of H2S biosynthesis using dl-propargylglycine increased ROS production and the expression of Ang II and Kv1.5. Sodium hydrosulfide (an exogenous H2S donor) and Nox4 siRNA inhibited Ang II-induced ROS production and Ang II-induced expression of Kv1.5, P-Smad2/3, P-ERK 1/2. Sodium hydrosulfide suppressed the Ang II-induced upregulation of Nox4. In our beagle AF model, 24 h of rapid atrial pacing (RAP) increased the atrial Ang II concentration, ROS production and the protein expression of Nox4, Kv1.5, P-Smad2/3 and P-ERK 1/2. These RAP-induced changes were inhibited by H2S supplementation and losartan (an AT1R blocker) pretreatment. In conclusion, our study indicates that H2S downregulates Ang II-induced atrial Kv1.5 expression by attenuating Nox4-related ROS-triggered P-Smad2/3 and P-ERK 1/2 activation during AF. H2S supplementation would be beneficial for AF treatment via the suppression of atrial Kv1.5 expression.

Our previous investigation indicated that angiotensin II (Ang II) enhances the expression of Kv1.5, a promising target for the treatment of atrial fibrillation (AF), by activating reactive oxygen species (ROS)-dependent phosphorylation of Smad 2/3 (forming P-Smad 2/3) and ERK 1/2 (forming P-ERK 1/2). A recent study indicated that aldosterone (Aldo) upregulates atrial Kv1.5 protein in a rat AF model, but the mechanism remains unknown. The present study aimed to clarify the mechanism underlying Aldo-induced Kv1.5 expression and to test whether spironolactone may modulate atrial Kv1.5. Our Western blot analysis indicated that the Aldo/mineralocorticoid receptor (MR) interacts with Ang II/AT1R in upregulating Kv1.5 expression in cultured neonatal atrial myocytes (NRAMs). Blockade of MR with spironolactone and of AT1R with losartan significantly suppressed Kv1.5 expression induction by combined Aldo and Ang II treatment. Aldo increased the protein expression of Nox1, Nox2 and Nox4, but this effect was abolished by spironolactone pretreatment. The Aldo-induced upregulation of Kv1.5 was also reversed by the Src protein tyrosine kinase family inhibitor PP2, the Nox2 inhibitor gp91ds-tat and the Nox1/Nox4 inhibitor GKT137831 but not by the Rac GTPase inhibitor NSC23766. Flow cytometry showed that the Aldo-induced ROS production was inhibited by spironolactone, PP2, gp91ds-tat and GKT137831. Spironolactone suppressed the Aldo-induced protein expression phosphorylated Src (P-Src), P-Smad 2/3 and P-ERK 1/2. In conclusion, we have demonstrated that spironolactone suppresses Aldo-induced Kv1.5 expression by attenuating MR-Nox1/2/4-mediated ROS generation in NRAMs.

Additional file 1. Supplementary methods and figures. (DOCX 1275 kb)

SummaryWhat is known and objective Erlotinib is one of the epidermal growth factor receptor (EGFR) inhibitors and is widely used as a targeted therapy for advanced non-small-cell lung cancer (NSCLC). There are a few reports regarding ocular adverse effects of erlotinib. Herein, we report a case of rapid onset of ocular toxicity associated with overdosing of erlotinib. Case description A 72-year-old male with metastatic NSCLC developed conjunctivitis after accidentally taking erlotinib at a dosage of 300 mg/day for 4 days. Before that, the patient had been taking erlotinib at the prescribed dose of 150 mg/day for 17 days. Erlotinib was discontinued for 7 days, and the conjunctivitis was successfully treated symptomatically. The adverse effect did not recur when he resumed taking erlotinib 150 mg/day, suggesting the ocular change was related to the overdosing of erlotinib. What is new and conclusion Awareness and close monitoring of this adverse effect are helpful for doctors and pharmacists to identify inadvertent drug overdose. Patients should be provided comprehensive education before receiving targeted therapy.

Objective The aim of this study is to investigate local cardiac and circulating AVP secretion during heart failure and to determine whether AVP mediates ventricular remodeling. Methods We assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failure rats 3 weeks (n = 10), 4 weeks (n = 10), 6 weeks (n = 10), 9 weeks (n = 15) after the proximal left anterior descending coronary artery (LAD) ligation. Ten sham-operated rats were used as the control group. In vitro, cardiac microvascular endothelial cells (CMECs) were initiated from isolated Wistar rat hearts and subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulation on CMECs and cardiac fibroblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blotting and real-time PCR. Results With cardiac dysfunction, plasma and local cardiac AVP, aldosterone levels increased over time, peaking at 9 weeks post-MI. AVP levels were negatively correlated with serum Na + and LVEF but positively correlated with LVEDD and myocardial hydroxyproline. In CMECs treated with Ang II, AVP mRNA and protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the expression of endothelin-1 and connective tissue growth factor. Conclusion CMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. An intrinsic cardiac AVP system exists. Local cardiac and circulating AVP secretion were enhanced by deteriorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important mediator of myocardial fibrosis in late-stage heart failure.

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin-angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β1)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β1, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT1) receptor antagonist losartan, an anti-TGF-β1 antibody and the ERK 1/2 inhibitor PD98059 but not by the AT2 receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT1 receptor/TGF-β1/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β1 antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT1 receptor/TGF-β1/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.

Cardiac structures and functions change with advanced age, but the underlying mechanisms are not well understood. Autophagy and apoptosis play important roles in the process of cardiac remodeling. This study was designed to explore changes in cell autophagy and apoptosis during age-related left ventricular remodeling and to determine whether the mitogen-activated protein kinase (MAPK) pathway is an underlying mechanism. Eight 5-month-old (adult group) and eight 24-month-old male C57bl/6 mice (aged group) were studied. The heart mass index, left ventricular mass index and hydroxyproline content of both groups were compared. Western Blotting was used to quantitate the protein expression of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, caspase-3, B-cell leukemia-2 (Bcl-2) and MAPKs in the left ventricles of adult and aged mice. Our results showed that the heart mass index, left ventricular mass index and hydroxyproline content in the left ventricles of the aged mice were increased significantly compared with the adult mice, indicating that left ventricular remodeling occurs with aging. The expression of LC3 and Beclin-1 in the left ventricles of aged mice were decreased significantly compared to adult mice. Meanwhile, the level of myocardial caspase-3 in adult mice remained the same in aged mice, and the level of myocardial Bcl-2 increased significantly in aged mice. There were no differences in the expression level of myocardial extracellular signal-regulated kinase 1/2 (ERK1/2), activated/phospho-ERK1/2, c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 between aged and adult mice. However, the expression of myocardial activated/phospho-JNK1/2 increased significantly in aged mice, while activated/phospho-p38 decreased significantly. These findings indicate that autophagy decreases without a concurrent change in apoptosis during age-related left ventricular remodeling in mice. The MAPK pathway may be involved in the regulation of age-related left ventricular remodeling by modulating autophagy.

Objective: To explore the mechanisms of Kv2.1 on the secretion of ADH in rats with heart failure. Methods: In the animal study, 70 healthy male SD rats were selected. Ligation of coronary heart failure model surgery was performed in 60 rats and sham surgery was performed in the other 10 rats. Q-PCR was used to detect the mRNA expression of Kv2.1 in hypothalamus and heart. The protein expression of Kv2.1 and ADH was detected by western blot. In the cell culture study, hypothalamic neurons were cultured and divided into 7 groups. The mRNA expression of Kv2.1 and ADH was detected by Q-PCR. The protein expression of Kv2.1, CamKII, phosphorylation SynapsinI, dephosphorylation SynapsinI and ADH was detected by western blot. Results: Compared with the control group of heart failure, LVEDD, LVESD, LVEDV and LVESV were significantly decreased (P < 0.01), and LVEF and LVFS were significantly increased (P < 0.01) in the Kv2.1 agonist group; in the Kv2.1 inhibitor group, LVEDD, LVESD, LVEDV and LVESV were significantly increased (P < 0.01), and LVEF and LVFS were significantly decreased (P < 0.01). In cell culture study, after the different concentrations of Kv2.1 inhibitor gradient down the expression of Kv2.1, intracellular Ca2+ concentration gradient increased (P < 0.01), CamKII and phosphorylation of SynapsinI protein expression gradient increased (P < 0.01), dephosphorylation of SynapsinI protein expression gradient decreased (P < 0.01), and the ADH mRNA and protein expression of gradient increased (P < 0.01). Conclusions: Kv2.1 agonist can prevent the calcium overload by reducing the intracellular Ca2+ concentration, so that the phosphorylation of SynapsinI reduces and exocytosis in hypothalamic neurons is inhibited, which ease the secretion of ADH.

Heart failure is a major cause of morbidity and mortality worldwide. Diuretics are regarded as the first-line treatment for patients with heart failure because they provide symptomatic relief. However, the specific benefits of diuretics and their effects on heart failure survival remain unclear. This study was designed to investigate the potential of hydrochlorothiazide to improve cardiac remodeling compared with spironolactone. Heart failure was produced by ligation of the left anterior descending coronary artery in male Sprague-Dawley rats. Two weeks after coronary artery ligation, 55 rats were randomly divided into four groups: sham-operated group (n=10), control group (n=15), hydrochlorothiazide group (12.5 mg/kg/day, n=15) and spironolactone group (20 mg/kg/day, n=15). Cardiac function was assessed by echocardiography and Millar catheter after treatment with drugs for 8 weeks. Compared with the control group, ejection fraction and left ventricular end-systolic pressure were significantly improved in the hydrochlorothiazide and spironolactone treatment groups (P0.05). In addition, hydrochlorothiazide and spironolactone reduced collagen volume fraction and proinflammatory cytokine levels. Moreover, gene and protein expression of TGF-β1, Smad2, Smad3 and Smad7 (P0.05) were also reduced. Nevertheless, no significant differences were observed between the hydrochlorothiazide and spironolactone groups. These results suggest that hydrochlorothiazide improves cardiac remodeling as effectively as spironolactone by reducing proinflammatory cytokine levels and inhibiting the TGF-β signaling pathway in post-myocardial infarction congestive heart failure. Moreover, the effects of the drugs on the TGF-β signaling pathway are likely to result from inhibited TGF-β and R-Smads expression rather than increased Inhibitory-Smad7 expression.

Diastolic heart failure (DHF) is a clinical syndrome characterized by the symptoms and signs of heart failure, preserved ejection fraction (EF) and abnormal diastolic function [1]. Hypertension is the most common risk factor and the principal precursor for DHF [2]. Hypertension-induced left ventricular (LV) hypertrophy that includes myocardial hypertrophy and abnormal accumulation of fibrillar collagen causes a decreased compliance and LV diastolic dysfunction. It is estimated that 20–60% of patients with heart failure have preserved EF [3,4]. Unfortunately, in contrast to the situation of heart failure with low EF, the mortality and morbidity of DHF have not been significantly decreased in the past decades [5,6]. Magnesium is the second most abundant intracellular cation. As a cofactor formany enzymes,magnesiumplays a role in various biological functions including energy metabolism and muscle contraction [7]. It is known that activation of the rennin–angiotensin–aldosterone system and the use of diuretics in patients with essential hypertension or cardiac failure may deplete serum potassium and affect intracellular magnesium homeostasis [8,9]. We have shown the importance of maintaining magnesium homeostasis in reducing arrhythmia and myocardial hypertrophy in patients with heart failure or hypertension [10,11]. However, the role of magnesium homeostasis in diastolic heart dysfunction is not known yet. Thus, we designed this study to address this issue. Since myocardium of patients is usually not easily accessible, we measured intralymphocytic magnesium content to reflect the magnesium content in myocardial cells. This was a single center prospective study that conformed to the ethical principles outlined in the Declaration of Helsinki. The study protocol was approved by the ethics committee of the First Affiliated Hospital of the SunYat-SenUniversity (Guangzhou, China). Consecutive 206 essential hypertension patients with diastolic heart dysfunction documented by Doppler echocardiography from November 2007 to October 2009 were enrolled. Patients with diastolic heart dysfunction and clinical signs and symptoms of heart failure as described in the European Society of Cardiology Guidelines published in 2008 [12] were attributed to DHF group (group A). Those free of signs and symptoms of

Rationale: Tachycardia-induced atrial fibrosis is a hallmark of structural remodeling of atrial fibrillation (AF). The molecular mechanisms underlying the AF-induced atrial fibrosis remain unclear. Objective: To determine the role of angiotensin II (Ang II)/Ang II type 1 (AT 1 ) receptor–coupled transforming growth factor (TGF)-β 1 /Smad signaling pathway in the AF-induced atrial fibrosis. Methods and Results: Rapid atrial pacing (1000 ppm) was applied to the left atrium of rabbit heart to induce atrial fibrillation and fibrosis. Quantitative PCR and Western blot analysis revealed that rapid atrial pacing caused a marked increase in the expression of Ang II, TGF-β 1 , phosphorylated Smad2/3 (P-Smad2/3), Arkadia, and hydroxyproline synthesis. However, the expression of Smad7, a key endogenous antagonist of the TGF-β 1 /Smad-mediated fibrosis, was significantly decreased. These changes were dose-dependently reversed by AT 1 receptor antagonist losartan, implicating the involvement of AF-induced release of Ang II and activation of AT 1 receptor–specific pathway. In the adult rabbit cardiac fibroblasts, Ang II increased the expression of TGF-β 1 , P-Smad2/3, Smad4, Arkadia, and collagen I synthesis and significantly reduced Smad7 expression. These effects of Ang II were reversed by losartan but not by the AT 2 antagonist (PD123319). In addition, extracellular signal-regulated kinase inhibitor and anti–TGF-β 1 antibody also blocked the Ang II–induced downregulation of Smad7. Silencing of Smad7 gene by small interfering RNA abolished the antagonism of losartan on the fibrogenic effects of Ang II on cardiac fibroblasts, whereas overexpression of Smad7 blocked Ang II–induced increase in collagen I synthesis. Conclusions: Ang II/AT 1 receptor–specific activation of Arkadia-mediated poly-ubiquitination and degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β 1 /Smad signaling and serves as a key mechanism for AF-induced atrial fibrosis.

On this article the authors requested to add another affiliation which is Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou 510,080, China for Juhong Zhang, Xiuren Gao and Zhibin Huang. The new affiliation is now added in this article. The remainder of the article remains unchanged.

Objective: It was our aim to study the outcome of autonomic nervous function (ANF), heart rate variability (HRV) and arrhythmias in patients with acute viral myocarditis in a 6-month follow-up period after the diagnosis. Methods: ANF was measured in patients with acute viral myocarditis 1, 3 and 6 months after the disease was diagnosed. HRV and arrhythmias were monitored by 24-hour electrocardiogram during the follow-up examination. Results: Ninety-six patients with acute viral myocarditis were enrolled in this study; 58% showed abnormal ANF tests, and the incidence was reduced to 17% after 6 months (p < 0.01). Time and frequency domain analyses of HRV were significantly reduced in the early stage of the disease and recovered after 6 months (p < 0.05). The incidence of arrhythmias in patients with abnormal ANF was significantly higher than in patients with normal ANF (p < 0.05). The incidence of arrhythmias in patients with abnormal ANF at 6 months was significantly lower than that at the first month (p < 0.05). Conclusions: There is a significant impairment of ANF and a higher incidence of arrhythmias in the early stage of acute myocarditis. ANF and HRV abnormalities are significantly improved, and the incidence of arrhythmia is significantly reduced after 6 months in patients with acute myocarditis.

Objectives To study the role of the Rho/ROCK pathway in Ang II and TGF-β1-induced atrial remodeling. Methods and Results A canine atrial fibrillation (AF) model was established by rapid atrial pacing (RAP) of the left atrium. The roles of TGF-β1, the RhoA/ROCK signaling pathway and connective tissue growth factor (CTGF) in atrial remodeling were studied via both in vitro and in vivo experiments. Each of the dogs that received RAP developed persistent AF within 4 weeks. The mRNA expression levels of TGF-β1 (1.32±0.38), Collagen-I(1.33±0.91), CTGF(5.83±3.71), RhoA(1.23±0.57) and ROCK-1 (1.02±0.27) in the left atrium were significantly increased following 4 weeks of RAP. Angiotensin II (Ang II) induced the proliferation of atrial fibroblasts and up-regulated the expression of both CTGF and ROCK-1 in a dose-dependent manner. Simvastatin and Y27632 reversed Ang II-induced CFs proliferation, as well as ROCK-1(0.89±0.05 and 1.27±0.03, respectively) and CTGF (0.87±0.04 and 0.91±0.02, respectively) expression. The expression mRNA of ROCK-1(1.74±0.13) and CTGF (2.28±0.11) can upregulated by TGF-β1, and down-regulated by Simvastatin (1.22±0.03 vs 2.27±0.11), Y27632 (1.01±0.04 vs 1.64±0.03), Los (1.04±0.11 vs 1.26±0.05), respectively. Losartan and Simvastatin attenuated the effects of TGF-β1, inhibited RhoA activity as opposed to RhoA protein expression. Y27632 had no effect on either the expression or the activity of RhoA. Conclusions The increased expression of profibrotic factors (CTGF, ROCK1 and Smad2/3) played an important role in our RAP-induced AF model. Increased atrial profibrotic factors involve the activation of either the TGF-β1/RhoA/ROCK-1 or the TGF-β1/Smad2/3 signaling pathway.

Lipid disturbance induced by high-fat diet is a worldwide problem, and it can induce inflammation and oxidative stress in vivo. Zinc is considered as an antioxidant, anti-inflammatory agent. Since matrix metalloprotease 2 (MMP2) and matrix metalloprotease 9 (MMP9)'s expressions are changed under many pathological conditions, we would like to know how zinc affects lipid metabolism and MMP2, MMP9's expressions in the lipid disturbance rabbits. Twenty-four male New Zealand white rabbits were randomly divided into four groups. Each group had six rabbits, and they were fed with regular diet, high-fat diet, high-fat diet+zinc, and regular diet+zinc separately for 12 weeks. High-fat diet induced lipid disturbance significantly which raised the level of aspartate aminotransferase (p0.01) and alanine transaminase (p0.05) in the high-fat diet group, but zinc supplement reversed this phenomenon (p0.05). Zinc did not reduce total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (p0.05), but it lowered triglyceride (TG) and raised high-density lipoprotein cholesterol (HDL-C) (p0.01). Zinc also reduced high-sensitivity C-reactive protein (hs-CRP) (p0.01) and interleukin-6 (IL-6)'s expressions (p0.05). Zinc reduced the epicardial adipose tissue and alleviated the hepatic steatosis. Zinc suppressed MMP2 and MMP9's expressions in vivo, but it did not alleviate the aorta fatty streak's severity in the lipid disturbance rabbits. Zinc protected the liver, reduced TG, hs-CRP, and IL-6 and raised HDL-C in the lipid disturbance rabbits. Zinc suppressed MMP2 and MMP9's expressions in vivo, but it did not alleviate the severity of aorta fatty streak induced by the high-fat diet.

High glucose‑induced endothelial cell apoptosis is considered to be the initiator of diabetes‑associated vascular complications. Experiments in vivo and in vitro have demonstrated that high glucose levels contribute to the apoptosis of endothelial cells by mediating cellular dysfunction and metabolic disorder via the production of various cytokines. As the most important endogenous vascular regulators, the balance between pro‑proliferative effector vascular endothelial growth factor (VEGF) and anti‑proliferative effector tumor necrosis factor‑like cytokine 1A (TL1A) is important in the modulation of endothelial cell survival and proliferation, and neovascularization. The present study aimed to explore whether the imbalance between VEGF and TL1A affected the apoptosis of human umbilical vein endothelial cells (HUVECs) exposed to high glucose conditions and then further investigated the potential mechanism. The results showed that the downregulation of VEGF in combination with the upregulation of TL1A in response to high glucose levels led to enhanced HUVEC apoptosis. Further experiments revealed that silencing high glucose‑induced TL1A expression using TL1A small interfering (si)RNA or the overexpression of VEGF by transfection with VEGF DNA resulted in a reduced HUVEC apoptosis rate compared with the controls. The effects occurred by attenuating and activating the phosphoinositide 3‑kinase/Akt/endothelial nitric oxide synthase pathway, respectively. In addition, VEGF and TL1A inhibited each other in hyperglycemia. In conclusion, these findings provide theoretical support for the further investigation of novel therapeutic strategies designed to maintain the balance between VEGF and TL1A and, thus, to prevent the onset and progression of endothelial cell apoptosis in response to high glucose stimuli.

Aims Our previous study indicates that hydrochlorothiazide inhibits transforming growth factor (TGF)-β/Smad signaling pathway, improves cardiac function and reduces fibrosis. We determined whether these effects were common among the diuretics and whether angiotensin II receptor type 1 (AT1) signaling pathway played a role in these effects. Methods Heart failure was produced by ligating the left anterior descending coronary artery in adult male Sprague-Dawley rats. Two weeks after the ligation, 70 rats were randomly divided into five groups: sham-operated group, control group, valsartan group (80 mg/kg/day), hydrochlorothiazide group (12.5 mg/kg/day) and furosemide group (20 mg/kg/day). In addition, neonatal rat ventricular fibroblasts were treated with angiotensin II. Results After eight-week drug treatment, hydrochlorothiazide group and valsartan group but not furosemide group had improved cardiac function (ejection fraction was 49.4 ± 2.1%, 49.5 ± 1.8% and 39.9 ± 1.9%, respectively, compared with 40.1 ± 2.2% in control group), reduced cardiac interstitial fibrosis and collagen volume fraction (9.7 ± 1.2%, 10.0 ± 1.3% and 14.1 ± 0.8%, respectively, compared with 15.9 ± 1.1% in control group), and decreased expression of AT1, TGF-β and Smad2 in the cardiac tissues. In addition, hydrochlorothiazide reduced plasma angiotensin II and aldosterone levels. Furthermore, hydrochlorothiazide inhibited angiotensin II-induced TGF-β1 and Smad2 protein expression in the neonatal rat ventricular fibroblasts. Conclusions Our study indicates that the cardiac function and remodeling improvement after ischemic heart failure may not be common among the diuretics. Hydrochlorothiazide may reduce the left ventricular wall stress and angiotensin II signaling pathway to provide these beneficial effects. This article is protected by copyright. All rights reserved.

Background— Although increased coronary microvascular resistance (CMR), resulting in coronary microvascular dysfunction, is speculated to be responsible for myocardial ischemia in patients with cardiac syndrome X (CSX), it has never been directly demonstrated, and the correlation between CMR and severity of myocardial ischemia has not been elucidated in this setting. This study aimed to ascertain the increased CMR directly and to explore the relationship between CMR and severity of ischemia in patients with CSX. Methods and Results— We studied 18 patients with CSX and 18 age- and sex-matched control subjects. Thermodilution-derived coronary flow reserve and index of microvascular resistance were measured using a pressure–temperature sensor-tipped coronary wire. Exercise treadmill test was performed by the Bruce protocol for calculating Duke treadmill score. Coronary flow reserve was significantly lower (2.37±0.81 versus 3.68±0.72; P P r =0.539; P =0.021) and negatively to index of microvascular resistance ( r =−0.742; P Conclusions— Using an intracoronary thermodilution method, we for the first time directly demonstrated an increased microvascular resistance in patients with CSX. Furthermore, severity of ischemia was found to be intimately associated with CMR in this setting.

This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72 h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.

Malignant gliomas are the most devastating and aggressive brain tumors affecting the central nervous system. The insidious growth and infiltration are the most prominent characteristics of malignant gliomas, which render the current therapies for malignant gliomas including surgery, radiation and chemotherapy unsuccessful. Inhibition of infiltration as well as proliferation in combination with surgery might be more effective in the treatment of malignant gliomas. In the current study, we demonstrate the alphaxalone (3-hydroxypregnane-11,20-dione) could effectively inhibit the proliferation of C6 glioma cells in a concentration dependent manner. Moreover, this compound could also suppress the migration and invasion of C6 glioma cells at a concentration without causing significant cytotoxicity. Except the in vitro anti-glioma activity, alphaxalone effectively delayed the growth of rat C6 malignant glioma xenografts in vivo. Together, these findings suggest alphaxalone might be a promising candidate for the treatment of malignant gliomas and may also provide helpful clues for anti-glioma drugs development in future.

INTRODUCTION: The function of the repolarization reserve in the prevention of ventricular arrhythmias during cardiac ischemia/reperfusion and the impact of ischemia on slowly activated delayed rectifier potassium current (I(Ks)) channel subunit expression are not well understood. METHODS AND RESULTS: The responses of monophasic action potential duration (MAPD) prolongation and triangulation were investigated following an L-768,673-induced blockade of I(Ks) with or without ischemia/reperfusion in a rabbit model of left circumflex coronary artery occlusion/reperfusion. Ischemia/reperfusion and I(Ks) blockade were found to significantly induce MAPD90 prolongation and increase triangulation at the epicardial zone at 45 min, 60 min, and 75 min after reperfusion, accompanied with an increase in premature ventricular beats (PVBs) during the same period. Additionally, I(Ks) channel subunit expression was examined following transient ischemia or permanent infarction and changes in monophasic action potential (MAP) waveforms challenged by β-adrenergic stimulation were evaluated using a rabbit model of transient or chronic cardiac ischemia. The epicardial MAP in the peri-infarct zone of hearts subjected to infarction for 2 days exhibited increased triangulation under adrenergic stimulation. KCNQ1 protein, the α subunit of the I(Ks) channel, was downregulated in the same group. Both findings were consistent with an increased incidence of PVBs. CONCLUSION: Blockade of I(Ks) caused MAP triangulation, which precipitated ventricular arrhythmias. Chronic ischemia increased the incidence of ventricular arrhythmias under adrenergic stimulation and was associated with increased MAP triangulation of the peri-infarct zone. Downregulation of KCNQ1 protein may be the underlying cause of these changes.

Atrial fibrillation (AF) is associated with impaired coronary flow and diminished myocardial perfusion. In the present study we aimed to evaluate coronary blood flow by means of Thrombolysis in Myocardial Infarction (TIMI) frame count (TFC) in patients with AF in the absence of obstructive coronary artery disease (CAD).This prospective study initially enrolled 166 patients with AF and 332 age- and gender-matched control subjects without AF. After diagnostic coronary angiography, TFC was assessed in the participants without obstructive CAD, with 146 in the AF group and 150 in the control group.The TFC for three major coronary arteries and the mean TFC were found to be significantly higher in AF patients compared to control subjects (34.1 ± 10.4 vs. 25.0 ± 10.4, 31.8 ± 9.7 vs. 23.7 ± 9.1, and 32.3 ± 9.5 vs. 24.1 ± 8.4 for each artery and 32.8 ± 9.2 vs. 24.3 ± 8.9 for mean TFC, p0.001 for all comparisons). The mean TFC was 28.8 ± 7.9 in patients with paroxysmal AF, 33.7 ± 8.7 in those with persistent AF, and 39.0 ± 8.8 in those with long-standing or permanent AF (p0.01 for all comparisons). After multivariate analysis, we found that the presence of AF remains to be independently associated with mean TFC. In AF group, baseline heart rate, left ventricular ejection fraction, AF duration and left atrium diameter were found to be independently associated with mean TFC.Patients with atrial fibrillation in the absence of obstructive coronary artery disease have significantly higher TIMI frame counts for all three coronary vessels, indicating impaired coronary blood flow, compared to the control subjects without atrial fibrillation.

ST-segment elevation is the major clinical criterion for committing patients with chest pain to have emergent coronary revascularizations; however, the mechanism responsible for ST-segment elevation is unknown. In a guinea pig model of ST-segment elevation acute myocardial infarction (AMI), local application of hirudin, a thrombin antagonist, significantly decreased AMI-induced ST-segment elevation in a dose-dependent manner. Hirudin-induced (5 antithrombin units [ATU]) decrease in ST elevation was reversed by 250 nmol/L thrombin receptor activator peptide (TRAP). TRAP (250 nmol/L [100 microL]) significantly induced ST-segment elevation in hearts without AMI. The TRAP effect was blocked by 4 mg/kg glibenclamide and 4 mg/kg HMR1098 and partially blocked by 3 mg/kg 5HD. Pinacidil (0.45 mg/kg) simulated the effect of TRAP (250 nmol/L [100 microL]) on hearts without AMI. Moreover, single-channel recordings showed that TRAP induced ATP-sensitive K+ channel (KATP channel) activity, and this effect was blocked by HMR1098 but not 5HD. Finally, TRAP significantly shortened the monophasic action potential (MAP) at 90% repolarization (MAP90) and epicardial MAP (EpiMAP) duration. These effects of TRAP were completely reversed by HMR1098 and partially reversed by 5HD. Thrombin and its receptor activation enhanced ST-segment elevation in an AMI model by activating the sarcolemmal KATP channel.

Angiotensin II is a powerful mediator to induce cardiac remodeling and fibrosis. Transforming growth factor-beta1 (TGF-beta1) and extracellular signal-regulated kinase (ERK) have been implicated in the angiotensin II-induced cardiac fibrosis. However, the signaling pathways for this angiotensin II effect and the interaction between ERK and the TGF-beta1 signaling in this effect have not been well-illustrated. Cardiac fibroblasts were prepared from the ventricles of adult male Sprague-Dawley rats. They were treated with 1 microM angiotensin II in the presence or absence of losartan (angiotensin II AT(1) receptor antagonist), PD123319 (angiotensin II AT(2) receptor antagonist), an anti-TGF-beta1 antibody or PD98059 (ERK inhibitor). The cells were collected for Western blotting and reverse transcription-polymerase chain reaction. Angiotensin II caused a significant increase of the expression of TGF-beta(1), ERK1, phosphorylated-Smad2/3, Smad4 and collagen I. This increase was attenuated by losartan but was not affected by PD123319. An anti-TGF-beta(1) antibody and PD98059 diminished angiotensin II-induced Smad2/3 phosphorylation and the expression of Smad7 and collagen I. Our results suggest that angiotensin II induces collagen I expression through angiotensin II AT(1) receptor-TGF-beta(1)-Smads signaling pathway in cardiac fibroblasts. ERK, by regulating Smads signaling, also participated in the angiotensin II-induced collagen I expression.

Statins ameliorate myocardial fibrosis after myocardial infarction. We designed this study to determine whether fluvastatin reduced hypertension-induced myocardial hypertrophy and fibrosis and whether these fluvastatin effects involved transforming growth factor beta1 (TGF-beta1) and Smad 7, factors known to play a role in the myocardial hypertrophy and fibrosis. We randomized 14 week old spontaneously hypertensive rats (SHRs) to receiving vehicle or 5-20 mg/kg/day fluvastatin for 8 weeks. Wistar Kyoto (WKY) rats receiving vehicle or 10 mg/kg/day fluvastatin were also studied. SHRs had an increased blood pressure, left ventricular hypertrophy and fibrosis compared with WKY rats. SHRs also had an elevated TGF-beta1 expression and a decreased Smad 7 expression. These changes in SHRs were dose-dependently attenuated by fluvastatin. For example, the hydroxyproline content was 3.2+/-0.1, 4.0+/-0.1 and 3.5+/-0.1 microg/mg heart and the Smad 7 protein expression was 5.1+/-0.6, 1.0+/-0.1 and 4.1+/-0.7 arbitrary units for WKY rats, SHRs and SHRs receiving 20 mg/kg/day fluvastatin, respectively. The hydroxyproline content in the SHRs treated with or without fluvastatin was positively correlated with the left ventricular mass index, systolic blood pressure and the amount of TGF-beta1 proteins and negatively correlated with the Smad 7 expression level. The left ventricular mass index was positively correlated with the systolic blood pressure. Fluvastatin did not alter the blood pressure, left ventricular mass index and collagen content of WKY rats. These results suggest that fluvastatin reduces hypertension-induced myocardial hypertrophy and fibrosis. These effects may involve an increased expression of Smad 7 and a decreased expression of TFG-beta1. Our results call for clinical studies to evaluate these fluvastatin effects in hypertensive patients.

Tachycardia-induced atrial fibrosis is a hallmark of the structural remodeling of atrial fibrillation (AF). The mechanisms underlying tachycardia-induced atrial fibrosis remain unclear. In our previous study, we found that Smad7-downregulation promoted the development of atrial fibrosis in AF. Fibroblasts are enriched in microRNA-21 (miR-21), which contributes to the development of fibrosis and heart failure in the cardiovascular system. Our study was designed to test the hypothesis that miR-21 reinforces the TGF-β1/Smad signaling pathway in AF-induced atrial fibrosis by down-regulating Smad7. Rapid atrial pacing (RAP, 1000 ppm) was applied to the left atrium of the rabbit heart to induce atrial fibrillation and fibrosis. qRT-PCR and northern blot analysis revealed that RAP caused a marked increase in the expression of miR-21. Transfection with a miR-21 inhibitor significantly increased the expression of Smad7, while the expression of collagen I/III significantly decreased. These changes were implicated in the AF-induced release of miR-21 and down-regulation of Smad7. Adult rat cardiac fibroblasts treated with TGF-β1 showed increased miR-21 expression and decreased Smad7 expression. Pretreatment with a TGF-β1 inhibitor reduced the TGF-β1-induced up-regulation of miR-21. Pretreatment with pre-miR-21 and a miR-21 inhibitor significantly decreased and increased Smad7 expression, respectively. This result was negatively correlated with the expression of collagen I/III in fibroblasts. Moreover, the results of a luciferase activity assay suggest that Smad7 is a validated miR-21 target in CFs. Our results provide compelling evidence that the miR-21 specific degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β1/Smad signaling and serves as a new insight of the mechanism of atrial fibrosis in atrial fibrillation. Electronic supplementary material The online version of this article (doi:10.1007/s00380-016-0808-z) contains supplementary material, which is available to authorized users.