Involvement of CaSR in hyperglycemia-induced macroangiopathy and related mechanism.

In order to clarify the potential role of calcium sensing receptor (CaSR), a typical G protein coupled receptor (GPCR), in hyperglacemia-induced macroangiopathy, experimental hyperglycemia models in vivo and in vitro were prepared. Firstly, SD rats were divided into control group (n=10) and diabetes group (n=10), and diabetic model was induced via high-fat diet feeding and streptozotocin (STZ, 30 mg/kg) injection. Hydroxyproline level, determined via Choramnie T oxidation method, in vessel wall in diabetic rats was 30% more than that in control group. The gene transcription and expression levels were detected by real-time PCR and Western blotting, respectively. Both of collagen I and III mRNA levels in diabetic aorta were nearly twice those in normal aorta. The cleaved caspase-3 and -9 were elevated 1.5 and 2.5 times respectively in diabetic vascular cells. As compared with controls, mRNA and protein levels of CaSR in aorta were increased by 3 and 1.5 times in diabetes group. The expression levels of Bax as well as pro-apoptotic kinases (phospho-p38 and phosphor-JNK) were also increased 2, 0.5 and 0.5 times respectively in diabetic rats. To further validate the involvement of CaSR in cell apoptosis and explore the potential mechanism, the endothelial cell line (human umbilical vascular endothelial cells, HUVECs) was stimulated with high concentration of glucose (33 mmol/L) to mimic hyperglycemia in vitro. Cell-based assays also showed that the CaSR level and key apoptotic proteins (cleaved caspase-3 and -9, Bax, phospho-p38 and phosphor-JNK) were elevated in response to stimulation, and inhibition of CaSR by using specific inhibitor (NPS-2143, 10 µmol/L) could protect cells against apoptosis. Our results demonstrated that CaSR might take important part in the development of diabetic macroangiopathy through promoting cell apoptosis induced by hyperglycemia.

N-acetylcysteine reduces oxidative stress, nuclear factor­κB activity and cardiomyocyte apoptosis in heart failure.

The roles of oxidative stress on nuclear factor (NF)­κB activity and cardiomyocyte apoptosis during heart failure were examined using the antioxidant N­acetylcysteine (NAC). Heart failure was established in Japanese white rabbits with intravenous injections of doxorubicin, with ten rabbits serving as a control group. Of the rabbits with heart failure, 12 were not treated (HF group) and 13 received NAC (NAC group). Cardiac function was assessed using echocardiography and hemodynamic analysis. Myocardial cell apoptosis, apoptosis­related protein expression, NF­κBp65 expression and activity, total anti­oxidative capacity (tAOC), 8­iso­prostaglandin F2α (8­iso­PGF2α) expression and glutathione (GSH) expression levels were determined. In the HF group, reduced tAOC, GSH levels and Bcl­2/Bax ratios as well as increased 8­iso­PGF2α levels and apoptosis were observed (all P<0.05), which were effects that were attenuated by the treatment with NAC. NF­κBp65 and iNOS levels were significantly higher and the P­IκB­α levels were significantly lower in the HF group; expression of all three proteins returned to pre­HF levels following treatment with NAC. Myocardial cell apoptosis was positively correlated with left ventricular end-diastolic pressure (LVEDP), NF­κBp65 expression and 8­iso­PGF2α levels, but negatively correlated with the maximal and minimal rates of increase in left ventricular pressure (+dp/dtmax and ­dp/dtmin, respectively) and the Bcl­2/Bax ratio (all P<0.001). The 8­iso­PGF2α levels were positively correlated with LVEDP and negatively correlated with +dp/dtmax and ­dp/dtmin (all P<0.001). The present study demonstrated that NAC increased the antioxidant capacity, decreased the NF­κB activation and reduced myocardial cell apoptosis in an in vivo heart failure model.

Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes.

AIM: To study the effects of Na(+) channel blocker flecainide and L-type Ca(2+) channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K(+) channel. METHODS: fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K(+) currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion. RESULTS: fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of -41.74 mV and a slow recovery from inactivation (τ=1.90 s at -90 mV). Flecainide and verapamil blocked the currents with IC(50) values of 512.29 ± 56.92 and 260.71 ± 18.50 µmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve. CONCLUSION: The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K(+) channel blockers to treat arrhythmia.

[Prognostic effects on percutaneous coronary intervention of different time windows in patients with acute myocardial infarction].

OBJECTIVE: To compare the prognostic effects of different time windows on initiating PCI (percutaneous coronary intervention) in AMI (acute myocardial infarction) patients. METHODS: Ninety-five AMI patients undergoing PCI were enrolled continuously from January 2007 to September 2008. According to the timing of direct PCI, the patients were divided into 3 groups: after AMI, < 6 h (Group A, n = 45), 6 - 12 h (Group B, n = 45) and 12 - 24 h (Group C, n = 35). Comparisons were made among these 3 groups in terms of the post-PCI incidence of heart failure and mortality. The left ventricular ejection fraction (LVEF) and left ventricular end-diastolic inside diameter (LVEDV) were measured by echocardiogram at Month 6. RESULTS: During the hospitalization and follow-up, Group A had a lower incidence of heart failure and mortality than Groups B and C. And Group B had a lower rate than Group C. The difference was significant (P < 0.05). Compared with LVEF(%) (52.25 ± 4.27) in Group B and LVEF (%) (47.75 ± 6.86) in Group C, LVEF(%) (57.51 ± 6.9) in Group A were significantly improved on ECG at Month 6 months. LVEDV (mm) (45.89 ± 4.23) in Group A were significantly lower than LVEDV (mm) (49.0 ± 3.1) in Group B and LVEDV (mm) (52.46 ± 4.9) in Group C. The differences were both statistically significant (P < 0.05). CONCLUSION: An early time windows of initiating PCI in AMI patients can significantly improve the left ventricular functions and reduce the incidence of heart failure, left ventricular remodeling and the mortality rate. All these measures could improve left ventricular functions and prognosis.

Genome-wide association identifies a susceptibility locus for coronary artery disease in the Chinese Han population.

Coronary artery disease (CAD) causes more than 700,000 deaths each year in China. Previous genome-wide association studies (GWAS) in populations of European ancestry identified several genetic loci for CAD, but no such study has yet been reported in the Chinese population. Here we report a three-stage GWAS in the Chinese Han population. We identified a new association between rs6903956 in a putative gene denoted as C6orf105 on chromosome 6p24.1 and CAD (P = 5.00 × 10⁻³, stage 2 validation; P = 3.00 × 10⁻³, P = 1.19 × 10⁻8 and P = 4.00 × 10⁻³ in three independent stage 3 replication populations; P = 4.87 × 10⁻¹², odds ratio = 1.51 in the combined population). The minor risk allele A of rs6903956 is associated with decreased C6orf105 mRNA expression. We report the first GWAS for CAD in the Chinese Han population and identify a SNP, rs6903956, in C6orf105 associated with susceptibility to CAD in this population.

Atorvastatin protects against angiotensin II-induced injury and dysfunction in human umbilical vein endothelial cells through bradykinin 2 receptors.

The morphological and functional integrity of the endothelial cell (EC) is compromised in many cardiovascular diseases such as atherosclerosis, hypertension, and diabetes. Angiotensin II (Ang II) plays important roles in the initiation and progression of these diseases. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) may have cholesterol-independent pleiotropic effects on preventing the EC injury and dysfunction that occurs in these diseases, and the protective effects may relate to bradykinin 2 receptors (B2Rs). Our study was designed to test the hypothesis that atorvastatin, via B2Rs, protects the viability and function of EC exposed to Ang II independent of hemodynamics. The experimental results showed that the cytotoxic effects of Ang II on human umbilical vein endothelial cells were significantly ameliorated by atorvastatin pretreatment (LDH tests, MTT assay, and propdium iodide (PI)/Annexin V-stating analysis), and atorvastatin treatment simultaneously enhanced expression of endothelial nitric oxide synthase and yielded of nitric oxide (NO) and cyclic guanosine monophosphate, but both effects were attenuated by the B2Rs antagonist HOE-140. This study proves the hypothesis and may be pertinent to the complex mechanism of action of statins explaining their long-term beneficial effects in maintaining the morphological and functional integrity of vascular ECs.

Compensatory function of bradykinin B1 receptor in the inhibitory effect of captopril on cardiomyocyte hypertrophy and cardiac fibroblast proliferation in neonatal rats.

BACKGROUND: Bradykinin (BK) acts mainly on two receptor subtypes: B(1) and B(2), and activation of B(2) receptor mediates the most well-known cardioprotective effects of angiotensin converting enzyme inhibitors (ACEi), however, the role that B(1) receptor plays in ACEi has not been fully defined. We examined the role of B(1) receptor in the inhibitory effect of ACE inhibitor captopril on rat cardiomyocyte hypertrophy and cardiac fibroblast proliferation induced by angiotensin II (Ang II) and explored its possible mechanism. METHODS: Neonatal cardiomyocytes and cardiac fibroblasts (CFs) were randomly treated with Ang II, captopril, B(2) receptor antagonist (HOE-140) and B(1) receptor antagonist (des-Arg(10), Leu(9)-kallidin) alone or in combination. Flow cytometry was used to evaluate cell cycle, size and protein content. Nitric oxide (NO) and intracellular cyclic guanosine monophosphate (cGMP) level were measured by colorimetry and radioimmunoassay. RESULTS: After the CFs and cardiomyocytes were incubated with 0.1 micromol/L Ang II for 48 hours, the percentage of CFs in the S stage, cardiomyocytes size and protein content significantly increased (both P < 0.01 vs control), and these increases were inhibited by 10 micromol/L captopril. However, NO and cGMP levels were significantly higher than that with Ang II alone (both P < 0.01). 1 micromol/L HOE-140 or 0.1 micromol/L des-Arg(10), Leu(9)-kallidin attenuated the effects of captopril, which was blunted further by blockade of both B(1) and B(2) receptors. CONCLUSIONS: Acting via B(2) receptor, BK contributes to the antihypertrophic and antiproliferative effects of captopril on cardiomyocytes and CFs. In the absence of B(2) receptor, B(1) receptor may act a compensatory mechanism for the B(2) receptor and contribute to the inhibition of cardiomyocyte hypertrophy and CFs proliferation by captopril. NO and cGMP play an important role in the effect of B(1) receptor.