Chinese society of cardiology expert consensus statement on the diagnosis and treatment of adult fulminant myocarditis.

Fulminant myocarditis is primarily caused by infection with any number of a variety of viruses. It arises quickly, progresses rapidly, and may lead to severe heart failure or circulatory failure presenting as rapid-onset hypotension and cardiogenic shock, with mortality rates as high as 50%-70%. Most importantly, there are no treatment options, guidelines or an expert consensus statement. Here, we provide the first expert consensus, the Chinese Society of Cardiology Expert Consensus Statement on the Diagnosis and Treatment of Fulminant Myocarditis, based on data from our recent clinical trial (NCT03268642). In this statement, we describe the clinical features and diagnostic criteria of fulminant myocarditis, and importantly, for the first time, we describe a new treatment regimen termed life support-based comprehensive treatment regimen. The core content of this treatment regimen includes (i) mechanical life support (applications of mechanical respirators and circulatory support systems, including intraaortic balloon pump and extracorporeal membrane oxygenation, (ii) immunological modulation by using sufficient doses of glucocorticoid, immunoglobulin and (iii) antiviral reagents using neuraminidase inhibitor. The proper application of this treatment regimen may and has helped to save the lives of many patients with fulminant myocarditis.

Pioglitazone modulates the proliferation and apoptosis of vascular smooth muscle cells via peroxisome proliferators-activated receptor-gamma.

BACKGROUND: PPARγ is a member of the nuclear hormone receptor superfamily. It has been considered as a mediator regulating metabolism, anti-inflammation, and pro-proliferation in the Vascular Smooth Muscle Cells (VSMCs). Thiazolidinediones (TZDs), synthetic ligands of PPARγ, have anti-proliferative and pro-apoptotic effects on VSMCs, which prevent the formation and progression of atherosclerosis and restenosis following percutaneous coronary intervention (PCI). However, the underlying mechanism remains elusive. This present study therefore aimed to investigate the signaling pathway by which pioglitazone, one of TZDs, inhibits proliferation and induces apoptosis of VSMCs. METHODS: The effects of pioglitazone on VSMC proliferation and apoptosis were studied. Cell proliferation was determined using BrdU incorporation assay. Cell apoptosis was monitored with Hoechst and Annexin V staining. The expression of caspases and cyclins was determined using real-time PCR and Western blot. RESULTS: Pioglitazone treatment and PPARγ overexpression inhibited proliferation and induced apoptosis of VSMCs, whereas blocking by antagonist or silencing by siRNA of PPARγ significantly attenuated pioglitazone's effect. Furthermore, pioglitazone treatment or PPARγ overexpression increased caspase 3 and caspase 9 expression, and decreased the expression of cyclin B1 and cyclin D1 in VSMCs. CONCLUSIONS: Pioglitazone inhibits VSMCs proliferation and promotes apoptosis of VSMCs through a PPARγ signaling pathway. Up-regulation of caspase 3 and down-regulation of cyclins mediates pioglitazone's anti-proliferative and pro-apoptotic effects. Our results imply that pioglitazone prevents the VSMCs proliferation via modulation of caspase and cyclin signaling pathways in a PPARγ-dependent manner.

Effect of oxidative stress on ventricular arrhythmia in rabbits with adriamycin-induced cardiomyopathy.

The purpose of the present study was to examine the effects of oxidative stress on ventricular arrhythmias in rabbits with adriamycin-induced cardiomyopathy and the relationship between oxidative stress and ventricular arrhythmia. Forty Japanese white rabbits were randomly divided into four groups (n=10 in each): control group, metoprolol (a selective ß1 receptor blocker) group, carvedilol (a nonselective ß blocker/α-1 blocker) group and adriamycin group. Models of adriamycin-induced cardiomyopathy were established by intravenously injecting adriamycin hydrochloride (1 mg/kg) to rabbits via the auri-edge vein twice a week for 8 weeks in the adriamycin, metoprolol and carvedilol groups. Rabbits in the control group were given equal volume of saline through the auri-edge vein. Rabbits in the metoprolol and carvedilol groups were then intragastrically administrated metoprolol (5 mg/kg/d) and carvedilol (5 mg/kg/d) respectively for 2 months, while those in the adriamycin and control groups were treated with equal volume of saline in the same manner as in the metroprolol and carvedilol groups. Left ventricular end diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) were measured by echocardiography. Plasma levels of N-terminal pro B-type natriuretic peptide (NT-proBNP), malondialdehyde (MAD) and superoxide dismutase (SOD) were detected. The left ventricular wedge preparations were perfused with Tyrode's solution. The transmural electrocardiogram, transmural action potentials from epicardium (Epi) and endocardium (Endo), transmural repolarization dispersion (TDR) were recorded, and the incidences of triggered activity and ventricular arrhythmias were obtained at rapid cycle lengths. The results showed that TDR and the serum MDA and NT-proBNP levels were increased, and LVEF and the serum SOD level decreased in the adriamycin group compared with the control group. The incidences of triggered activity and ventricular arrhythmia were significantly higher in the adriamycin group than those in the control group (P<0.05). In the carvedilol group as compared with the adriamycin group, the serum SOD level and the LVEF were substantially increased; the TDR, and the serum MDA and NT-proBNP levels were significantly decreased; the incidences of triggered activity and ventricular arrhythmia were obviously reduced (P<0.05). There were no significant differences in the levels of MDA and SOD, LVEF, TDR and the incidences of triggered activity and ventricular arrhythmia between the adriamycin group and the metoprolol group. It was concluded that carvedilol may inhibit triggered activity and ventricular arrhythmias in rabbit with adriamycin-induced cardiomyopathy, which is related to the decrease in oxygen free radials.

PI3K-like kinases restrain Pim gene expression in endothelial cells.

Pim kinases contribute to tumor formation and development of lymphoma, which shows enhanced DNA replication, DNA recombination and repair. Endothelial cells^(ECs) express all the three members of Pim kinase gene family. We hypothesized that DNA repair gene would regulate Pim expression in ECs. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium. The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining. The siRNA fragments were synthesized and transfected by using Lipofectamine LTX. The total cellular RNA was extracted from the cells by using Trizol reagent. cDNAs were quantified by semi-quantity PCR. The effects of LY294002 and wortmannin on RNA stability in ECs were also examined. Our data showed that LY294002 and wortmannin, phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors, increased Pim mRNA expression in ECs without altering the mRNA stability. RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1, respectively. Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs. But etoposide, a nucleoside analogue, which could activate DNA-PKcs and ATM, increased Pim expression in ECs. Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

PI3K-like Kinases Restrain Pim Gene Expression in Endothelial Cells / 华中科技大学学报（医学）（英德文版）

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim expression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also examined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stability.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.

Influence of silencing soluble epoxide hydrolase with RNA interference on cardiomyocytes apoptosis induced by doxorubicin.

In order to investigate the influence of silencing soluble epoxide hydrolase (sEH) with double-stranded small interfering RNA (siRNA) on cardiomyocytes apoptosis induced by doxorubicin (DOX), two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents. The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively, and the plasmids that silenced sEH most significantly were selected, and renamed EH-R. The plasmids carrying a nonspecific siRNA coding sequence (PCN) served as the negative control. Cardiomyocytes were divided into four groups: control group, DOX group, PCN+DOX group, and EH-R+DOX group. Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 µmol/L. Apoptosis rate of cardiomyocytes was determined by flow cytometery. The protein expression levels of Bcl-2 and Bax were detected by Western blotting. The results showed that the expression of sEH was down-regulated by EH-R plasmid. The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups (P<0.01). As compared with the control group, the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased, the expression levels of Bax increased, and those of Bcl-2 decreased (P<0.01). However, the expression levels of Bax were decreased, those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obviously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group (P<0.01 for each). It was concluded that the recombinant plasmids could be successfully constructed, and transfected into the primary cultured cardiomyocytes. They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

Influence of Silencing Soluble Epoxide Hydrolase with RNA Interference on Cardiomyocytes Apoptosis Induced By Doxorubicin / 华中科技大学学报（医学）（英德文版）

In order to investigate the influence of silencing soluble epoxide hydrolase (sEH) with double-stranded small interfering RNA (siRNA) on cardiomyocytes apoptosis induced by doxorubicin (DOX),two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence (PCN) served as the negative control.Cardiomyocytes were divided into four groups:control group,DOX group,PCN+DOX group,and EH-R+DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of l μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups (P＜0.01).As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased (P＜0.01).However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obviously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group (P＜0.01 for each).It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

PPARgamma gene C161T substitution alters lipid profile in Chinese patients with coronary artery disease and type 2 diabetes mellitus.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor, which regulates gene expression of the key proteins involved in lipid metabolism, vascular inflammation, and proliferation. PPARgamma may contribute to attenuating atherogenesis and postangioplasty restenosis. PPARgamma C161-->T substitution is associated with a reduced risk of coronary artery disease (CAD). Whether or not the gene substitution alters the risk of CAD in type 2 diabetes mellitus (T2DM) patients remains unclear. METHODS: A total of 556 unrelated subjects from a Chinese Han population, including 89 healthy subjects, 78 CAD patients, 86 T2DM patients, and 303 CAD combined with T2DM patients, were recruited to enroll in this study. PPARgammaC161-->T gene polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphisms. Plasma levels of lipoproteins, apolipoproteins, glucose, and insulin were measured by ELISA or radioimmunoassay (RIA). The coronary artery lesions were evaluated by coronary angiography. RESULTS: The frequency of the 161T allele in CAD, T2DM, and CAD combined with T2DM patients was similar to that observed in the healthy control group. However, in CAD combined with T2DM patients, the group with angiographically documented moderate stenoses had a higher frequency of the 161T allele in comparison to the group with severe stenoses (P < 0.05). Moreover, in CAD with T2DM patients, the triglyceride levels and apoB in CC homozygote carriers were significantly higher than those in "T" allele carriers. CONCLUSIONS: PPARgammaC161-->T genotypes weren't significantly associated with the risk of CAD, but were markedly correlated with severity of disease vessels in patients with CAD and T2DM. Furthermore, PPARgammaC161-->T substitution was associated with an altered adipose, but not glucose metabolism. These results indicate that the PPARgamma C161-->T polymorphism may reduce the risk of severe atherogenesis by modulation of adipose metabolism, especially triglycerides and apoB, in Chinese patients with CAD and T2DM.

[Kv1.3 potassium channel expression changes after CD4(+) and subsets CD28(null)/CD28(+)T cells activation in peripheral blood of patients with acute coronary syndrome].

OBJECTIVE: To study the Kv1.3 channel expression changes after CD4(+) and subsets CD28(null)/CD28(+)T cells activation in peripheral blood of patients with acute coronary syndrome (ACS). METHODS: CD4(+)T cell in 27 ACS patients and CD4(+)CD28(null)/CD4(+)CD28(+)T cells in 12 out of these 27 ACS patients were isolated from peripheral blood with magnetic cell sorting. The whole-cell Kv1.3 currents for three T cells were recorded with patch-clamp technique before and 72 hours after activation by purified anti-human CD3 Interferon gamma, tumor necrosis factor alpha (TNF-alpha), granzyme B mRNA expression were determined by reverse transcription-PCR before and 72 hours after activation by purified anti-human CD3 in the presence or absence of recombinant Margatoxin (rMgTX, 0.1, 1, 10 nmol/L), a specific Kv1.3 channel blocker. RESULTS: Peak Kv1.3 channel currents of CD4(+), CD4(+)CD28(null), CD4(+)CD28(+)T cells were significantly increased and the mean Kv1.3 channel numbers per cell of these cells were increased by about 90%, 60%, 80% (402 +/- 88 vs. 752 +/- 275, 553 +/- 328 vs. 874 +/- 400, 392 +/- 133 vs. 716 +/- 251, all P < 0.05) after activation compared to baseline values. Baseline CD4(+)CD28(null)T cell numbers were about 40% more than those of CD4(+)CD28(+)T cell (P < 0.05) and were similar after activation (P = 0.102). The mRNA expression of interferon gamma, TNF-alpha and granzyme B were dose-dependently down-regulated by rMgTX. CONCLUSIONS: Kv1.3 channels of peripheral CD4(+)T cell and CD28(null)/CD28(+)T cells from ACS patients significantly increased after activation and Kv1.3-specific channel blocker rMgTX could effectively abolish this effect suggesting a potential role of Kv1.3 channel blocker on plaque stabilization in ACS patients.

[Erythropoietin inhibits angiotensin II induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway].

OBJECTIVE: To explore the effect of erythropoietin (EPO) on angiotensin II (AngII) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. METHODS: Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by AngII in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. RESULTS: EPO (20 U/ml) significantly inhibited AngII induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P < 0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P < 0.05), increased the NO production (P < 0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P < 0.05). CONCLUSION: EPO attenuates AngII induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin-II in vitro.

OBJECTIVE: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin-II (Ang-II)-induced hypertrophy in cultured neonatal rat cardiomyocytes. MATERIAL AND METHODS: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang-II-induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor L-NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor-beta1 (TGF-beta1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. RESULTS: The results indicate that EPO significantly attenuates Ang-II-induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO-treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or L-NAME partially abolished the anti-hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down-regulated the protein expression of TGF-beta1. CONCLUSION: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K-Akt-eNOS-NO pathway and the down-regulation of TGF-beta1.

[Psychological stress status in patients with acute coronary syndrome and stable angina pectoris].

OBJECTIVE: To observe the psychological stress status in patients with acute coronary syndrome (ACS) and stable angina pectoris (SA). METHODS: The intensity of social psychological stress and the serum levels of IL-6, CRP and ICAM-1 were determined in patients with ACS (n = 67) and SA (n = 33). RESULTS: (1) The percentage of patients with psychological stress was significantly higher in ACS than that in SA group (78.8% vs. 21.2%, P < 0.01). (2) The serum levels of CRP [(14.82 +/- 5.07) g/L vs. (8.78 +/- 4.34) g/L], IL-6 [(101.7 +/- 22.2) ng/L vs. (71.1 +/- 23.5) ng/L] and sICAM-1 [(1.41 +/- 0.47) mg/L vs. (0.82 +/- 0.37) mg/L] were significantly higher in psychological stress group than those in non-psychological stress group (all P < 0.05). Serum CRP [(18.91 +/- 3.12) g/L vs. (6.20 +/- 2.46) g/L], IL-6 [(114.6 +/- 15.2) ng/L vs. (56.4 +/- 15.8) ng/L] and sICAM-1 [(1.67 +/- 0.39) mg/L vs. (0.63 +/- 0.28) mg/L] levels in ACS group were significantly higher than those in SA group (all P < 0.01). CONCLUSION: Higher psychological stress was associated with higher risk of ACS and increased serum inflammatory cytokines.

[Erythropoietin suppresses the expressions of TGF-beta1 and collagen in rat cardiac fibroblasts induced by angiotensin II].

OBJECTIVE: Recent studies have shown cardiac protection effects of erythropoietin (EPO). The present experiment was designed to investigate the effects of EPO on TGF-beta1, nitric oxide synthase (NOS), collagen contents induced by angiotensin II (Ang II) in rat cardiac fibroblasts (CFs) and explore the roles of PI3-K/Akt signaling pathway on related effects. METHODS: Neonatal rat CFs was isolated by collagenase and trypsinase digestion methods. PBS, EPO, Ang II in the absence or presence of LY294002, an inhibitor of PI3-K, or L-NAME, an inhibitor of NOS, were added to CFs and cultured for 24 hours. The concentration of collagen I and collagen III in culture medium were quantitated by ELISA. The levels of nitric oxide (NO) and the activities of NOS as well as NOS isoforms were measured by chemical enzymic method. Western blot was applied to detecting the protein expressions of Akt, p-Akt, eNOS, iNOS, and TGF-beta1. RESULTS: The concentrations of collagen I and collagen III in CFs culture medium were significantly increased while the level of NO was significantly decreased by Ang II and these changes were significantly suppressed by EPO in a dose dependent manner. The effects of EPO on eNOS and NO could be blocked by LY294002. L-NAME could block EPO's effect on NO but not on the eNOS expression. The suppression effects on expressions of TGF-beta1 and collagen by Ang II in CFs were blocked by both LY294002 and L-NAME. CONCLUSION: EPO suppresses the upregulated expressions of TGF-beta1 and increased production of collagen induced by Ang II through activating the PI3-K/Akt signaling pathway in neonatal rat CFs.

[Effects of adrenomedullin on angiotensin II-induced collagen synthesis in vascular adventitial fibroblasts].

OBJECTIVE: To explore the effects of adrenomedullin (ADM) on Angiotensin II (AngII)-induced collagen synthesis in cultured rat vascular adventitial fibroblasts. METHODS: Rat vascular adventitial fibroblasts were cultured in vitro. ADM produced and secreted from adventitia in the presence of AngII was detected by radioimmunoassay, type I, III collagen contents in adventitia fibroblasts were measured by ELISA and the expressions of TGFbeta1 and MMP-2 were determined by RT-PCR and Western blotting. RESULTS: AngII significantly induced ADM secretion in adventitial fibroblasts in a dose-dependent manner. These effects could be reduced by 45%, 3% and 46%, through pre-treatment with Losartan, PD123319 or both, respectively for 30 min in culture medium. The AngII-induced type I, III collagen secretion in adventitial fibroblasts was significantly reduced by AMD in a dose-dependent manner, (P < 0.01) while ADM agonist ADM(22 - 52) significantly potentiated the effect of AngII; ADM also reduced AngII-induced expression of TGFbeta1 at mRNA and protein levels in a dose-dependent manner. AngII reduced the expression of MMP-2 at mRNA and protein levels in adventitial fibroblasts and these effects could be reversed by AMD (10(-8) mol/L). CONCLUSION: AngII stimulated ADM secretion in adventitia fibroblasts, ADM in turn can inhibit AngII-induced type I, III collagen synthesis in adventitial fibroblasts probably by downregulating the TGFbeta1 expression and upregulating MMP-2 expression. ADM therefore served as an antifibrotic factor in vascular remodeling process.

Effect of calcium-channel antagonist on repolarization heterogeneity of ventricular myocardium in an in vitro rabbit model of long QT syndrome.

Intracellular Ca(2+) and Ca(2+)-dependent signaling molecule play an essential role in the genesis of long-QT (LQT) syndrome-related ventricular arrhythmias. The effect of calcium-channel antagonist verapamil on repolarization heterogeneity of ventricular myocardium was assessed in an in vitro rabbit model of LQT syndrome. By using the monophasic action potential (MAP) recording technique, MAPs of epicardium, mid-myocardium and endocardium were simultaneously recorded by specially designed plunge-needle electrodes across the left ventricular free wall in rabbit hearts perfused by Langendorff method with standard Tyrode's solution. Bradycardia was induced by complete ablation of atrioventricular node. A catheter was introduced into the right ventricle to pace at the cycle lengths (CLs) of 1500, 1000, and 500 ms, successively. Quinidine (2 micromol/L) prolonged QT interval and ventricular MAP duration (MAPD), and increased transmural dispersion of repolarization (TDR) in a reverse rate-dependent fashion in isolated rabbit heart. No polymorphic ventricular tachycardias were induced under this condition. The effective free therapeutic plasma concentrations of verapamil (0.01-0.05 micromol/L) used in this experiment had no effect on quinidine-induced changes of QT interval, MAPD and TDR. This study demonstrated that, in this model of LQT syndrome, blockade of calcium-channel with verapamil had no effect on quinidine-induced changes of repolatiation heterogeneity of ventricular myocardium.

[The effects of calmodulin kinase II inhibitor on ventricular arrhythmias in rabbits with cardiac hypertrophy].

OBJECTIVE: To investigate the effect of KN-93, a calmodulin kinase II inhibitor, on ventricular arrhythmias in rabbits with cardiac hypertrophy. METHODS: Female New Zealand white rabbits were randomly divided into four groups (n = 10 each): Sham; LVH; LVH + KN-92 and LVH + KN-93 group. LVH was induced by partially constricting the abdominal aorta. In Sham group, the abdominal aorta was exposed without constriction. Eight weeks later, the arterially perfused left ventricular wedge preparations were made and transmembrane action potentials (TAP) from epicardium and endocardium and transmural ECG were simultaneously recorded. Incidence of early after depolarization (EAD) and torsade de pointes (Tdp), QT interval, action potential duration (APD) and transmural depolarization dispersion (TDR) at different cycle lengths were observed under slow stimulation (2000 - 4000 ms), hypokalemic (2 mmol/L) and hypomagnesaemic (0.25 mmol/L) Tyrode's solution perfusion. RESULTS: Left ventricular hypertrophy was detected in LVH group by echocardiography and not affected by KN-92 and KN-93. Perfused with hypokalemic, hypomagnesaemic Tyrode's solution and under slow stimulation (2000 - 4000 ms), the incidences of EAD and Tdp in Sham group, LVH group, LVH + KN-92 group (0.5 micromol/L) and LVH + KN-93 group (0.5 micromol/L) were 0/10, 10/10, 9/10, 5/10 and 0/10, 5/10, 4/10, 1/10, respectively. With 1 micromol/L KN-92 and KN-93, the incidences of EAD and Tdp in LVH + KN-92 and LVH + KN-93 group were 9/10, 3/10 and 4/10, 1/10 respectively. The QT interval, APD and TDR were not affected by KN-93. CONCLUSION: The calmodulin kinase II inhibitor KN-93 can effectively suppress ventricular arrhythmias in rabbits with cardiac hypertrophy by decreasing EAD.

[Effects of RNA interference targeting angiotensin 1a receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C hypertension].

OBJECTIVE: To investigate the effects of RNA interference (RNAi) targeting angiotensin 1a (AT1a) receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C (2-kidney, 1-clip) hypertension. METHODS: Two kinds of RNAi plasmids, pAT1a-shRNA1 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 928 - 946 and pAT1a-shRNA2 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 978 - 996, and a blank plasmid pCon carrying a nonspecific shRNA-coding sequence were constructed. Thirty Sprague-Dawley rats underwent clipping of the left renal artery so as to establish two-kidney, one-clip (2K1C) hypertension models and then were randomly divided into 5 equal groups: pAT1a-shRNA1 group (injected with pAT1a-shRNA1 4 mg/kg only one time), pAT1a-shRNA2 group (injected with pAT1a-shRNA2 4 mg/kg only one time), pCon group (injected with pCon 4 mg/kg only one time), valsartan group (perfused into the stomach with valsartan, a AT1 receptor inhibitor 30 mg.kg(-1).d(-1), for 3 weeks), and control blank group (without any treatment). Three weeks later, the systolic pressure of the caudal artery was measured, catheterization through carotid artery was conducted to measure the systolic blood pressure (SBP) and diastolic blood pressure (DBP), and the left ventricular pressure curve was drawn. Then the rats were killed; the weight of the heart was measured, the ratio of left ventricle weight to body weight (LV/BW) was calculated, and pathological examination of the heart and thoracic aorta was performed. Western blotting was used to detect the protein expression of AT21 in the ventricle and aorta. Six age-matched healthy rats were used as normal controls. RESULTS: There was no significant difference in the caudal artery pressure among the 5 groups (all P > 0.05) before intervention. Three weeks later the caudal artery pressures of the blank control group and pCon group continued to significantly increase by about 25 mm Hg compared to the values before the intervention (both P < 0.001) and without significant difference between these 2 groups; however, the caudal artery pressures of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 15.1 mm Hg +/- 5.4 mm Hg, 16.4 mm Hg +/- 8.4 mm Hg, and 30.6 mm Hg +/- 18.2 mm Hg lower than those before the intervention respectively (all P < 0.01); and were also significantly lower than those of the blank groups (P < 0.01 or P < 0.05). There was no significant differences in the +/- dp/dt value and indicators of renal function among these groups. The carotid artery pressure of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 194 mm Hg +/- 5 mm Hg, 200 mm Hg +/- 5 mm Hg, and 164 mm Hg +/- 5 mm Hg, all significantly lower than those of the blank and pCon groups (234 mm Hg +/- 10 mm Hg and 232 mm Hg +/- 7 mm Hg respectively, all P < 0.01). The LV/BW of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 2.27 +/- 0.37, 2.31 +/- 0.26, and 2.26 +/- 0.39, all significantly lower than that of the blank and pCon groups (3.24 +/- 0.38 and 2.94 +/- 0.06, respectively, all P < 0.01), similar to that of the normal control group (P > 0.05). The myocardiocytes were significantly hypertrophic and the arterial tunica media was significantly thickened in the blank group and such changes were all improved to different degrees in the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups. The protein expression levels of AT1 receptor in the myocardium of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 53.3% and 47.8% respectively than that of the blank group, and the protein expression levels of AT1 receptor in the thoracic aorta of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 58.7% and 49.3% respectively than that of the blank group (all P < 0.01); however, there were no significant difference in the protein expression levels of AT1 receptor in the myocardium and thoracic aorta between the valsartan and blank groups (both P > 0.05). CONCLUSION: RNA interference targeting AT1a receptor inhibits the development of renovascular hypertension and the accompanying cardiac hypertrophy. The RNAi technology may become a new strategy of gene therapy for hypertension.

[Selective knockdown of Angiotensin II receptor subtype 1a in rat vascular smooth muscle cells by RNA interference].

OBJECTIVE: To selectively knockdown the expression of Angiotensin II receptor subtype 1a (AT1aR) in rat vascular smooth muscle cells (VSMCs) by RNA interference and the sequential effects on cellular viability and proliferation. METHODS: The primary cultured rat aortic VSMCs were transfected by plasmids pAT1a-shRNA1 and pAT1a-shRNA2, each carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence, or by a control plasmid pGenesil-Control (pCon) carrying a nonspecific shRNA-coding sequence. The mRNA and protein expressions of AT1a, AT2 were analyzed by semi-quantified RT-PCR and Western blot, respectively and normalized to the internal control gene beta-actin. Cellular viability and proliferation were determined with methylthiazoletetrazolium (MTT) assay. RESULTS: AT1a mRNA and protein were reduced by 82% and 69% by pAT1a-shRNA1, 77% and 56% by pAT1a-shRNA2, respectively while no change was found in pCon treated VSMCs. AT2 receptor level in VSMCs remains unchanged after various treatments. The A(490nm) values obtained by MTT measurements were similar among groups in the absence of Ang II but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 treated VSMCs in the presence of Ang II. CONCLUSION: RNA interference can selectively knockdown AT1a expression in cultured VSMCs and attenuate the Ang II induced cell proliferation. Future studies are warranted to explore the potential role of RNA interference on AT1 function and as a new gene therapy tool for cardiovascular diseases.

[Effects of endocannabinoids on cardiovascular system].

Endocannabinoids and its corresponding receptors exist in myocardium, vascular smooth muscle cells, endothelial cells, nerve cells within blood vessel walls and some of circulating cells in blood. Endocannabinoids have different roles such as modulating blood pressure and vascular dilation in cardiovascular system in different models and organs of animal or human. It might mediate cardiac protection and modulating circulation in shock and myocardial infarction. Also, endocannabinoids might play a key role in cardiac preconditioning. The study of endocannabinoids in cardiovascular system was just start. We introduced the progress in the source and distribution of endocannabinoids in cardiovascular system, and the action and mechanisms of endocannabinoids in blood vessels and hearts in the present review.

Endogenous nitric oxide mediates lipoteichoic acid induced preconditioning on reoxygenation injury of cultured human coronary artery endothelial cells.

AIM: To explore the effects of lipoteichoic acid (LTA) induced delayed preconditioning (PC) on hypoxia-reoxygenation (H/R) injury of cultured human coronary artery endothelial cells (HCAECs), and to investigate the potential role of endogenous nitric oxide (NO) participated in the protective mechanism. METHODS: HCAECs were incubated for 2 h in a hypoxic atmosphere and reoxygenated for 4 h in a normoxic atmosphere. The delayed PC was induced by pretreatment with LTA (30 or 300 microg x L(-1)) for 4 h before 24 h recovery. The extent of cellular injury after reoxygenation was assessed by the percentage of cellular injury with Trypan blue exclusion and by the amount of lactate dehydrogenase (LDH) in culture media. The NO level of the culture media was measured spectrophotometrically. Furthermore, HCAECs were exposed to 300 microg x L(-1) of LTA for 4 h, and to detect the expression of eNOS mRNA by RT-PCR method after cells were recovered from different points. RESULTS: LTA pretreatment significantly decreased the percentage of the killed cell and the concentration of LDH in media. Also, LTA pretreatment obviously raised the concentrations of NO in culture media. The protective effects of LTA were abrogated by pretreatment with N-monomethyl-L-arginine (L-NMMA). Moreover, the expression of eNOS mRNA was significantly upregulated after HCAECs exposure to LTA for 4 h following 2 h or 4 h recovery. CONCLUSION: LTA could induce the delayed protection against H/R induced endothelial injury and dysfunction of cultured HCAECs. NO produced by eNOS acts initially as a trigger and subsequently as a mediator of delayed PC.