Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca^(2+)-ATPase activity in rat cardiac pressure-overload hypertrophy

Aim： To observe effects of angiotensin （Ang） II receptor antagonist （ATI） irbesartan and angiotensin-converting enzyme （ACE） inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca^2＋-ATPase activity in the model of pressure-overload cardiac hypertrophy. Methods： Forty male adult Sprague Dawley rats were divided into 5 groups One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1： sham group, rats （n=8） were gavaged with normal saline 2 ml/（kg·d） （ig）; Group 2： control group, rats （n=8） were treated with normal saline 2 ml/（kg·d） （ig）; Group 3： rats （n=8） were given perindopril 2 mg/（kg·d） （ig）; Group 4： rats （n=8） were treated with irbesartan 20 mg/（kg·d） （ig）; Group 5： rats （n=8） were given irbesartan 20 mg/（kg·d） plus perindopril 2 mg/（kg·d） （ig）. Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca^2＋-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. Results： Left ventricular mass index （LVMI）, transverse diameter of myocardial cell （TDM）, calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca^2＋-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca^2＋-ATPase. Conclusion： These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca^2＋-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of ventricular hypertrophy.

Effect of Chaiqinchengqi decoction on sarco/endoplasmic reticulum Ca^(2+)-ATPase mRNA expression of pancreatic tissues in acute pancreatitis rats

AIM: To investigate the effect of Chaiqinchengqi decoction (CQCQD) on sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) mRNA expression of pancreatic tissues in acute pancreatitis (AP) rats. METHODS: Thirty Sprague-Dawley (SD) rats were randomized into control group, AP group and CQCQD group (n = 3 × 10). The rats in the CQCQD group were intragastrically administered with CQCQD (10 mL/kg every 2 h) after induction of AP by intraperitoneal injection of caerulein (50 μg/kg.h × 5) within 4 h. At 6 h after the induction of AP model, pancreatic tissues were collected for the pathological observation, mRNA extraction for determination of SERCA1 and SERCA2 mRNA expression or pancreatic acinar cell isolation for measurement of fluorescence intensity (FI) of intracellular calcium ion concentration [Ca2+]i. RESULTS: There was no expression of pancreatic SERCA1 mRNA in the control group and the AP group. The expression of pancreatic SERCA2 mRNA in the AP group was down-regulated (expression ratio = 0.536; P = 0.001) compared with the control group, while that in the CQCQD group was up-regulated (expression ratio= 2.00; P = 0.012) compared with AP group. The FI of intracellular [Ca2+] of pancreatic acinar cells in the AP group (138.2 ± 23.1) was higher than the C group (111.0 ± 18.4) and the CQCQD group (118.7 ± 15.2 ) (P < 0.05) and the pancreatic pathological score in the CQCQD group was lower than that in the AP group (5.7 ± 1.9 vs 9.2 ± 2.7, P < 0.05).CONCLUSION: CQCQD can up-regulate the expression of SERCA2 mRNA of pancreatic tissues, reduce intracellular calcium overload and relieve pancreatic tissue lesions.

Phospholamban Antisense RNA Improves SR Ca^(2+)-ATPase Activity and Left Ventricular Function in STZ-induced Diabetic Rats

Objective To study the effect of phospholamban antisense RNA （asPLB） on sarcoplasmic reticulum Ca2＋-ATPase activity and cardiac function in rats with diabetes mellitus （DM） mediated by recombinant adeno-associated virus （rAAV） vector. Methods Six weeks after the induction of DM by streptozotocin injected intraperitoneally, the rats were divided into three groups, namely： DM-rAAV-asPLB group, DM-saline group and DM group （control group）. The rats in the DM-rAAV-asPLB group were intramyocardially injected with rAAV-asPLB, the rats in the DM-saline group were injected with saline, and those in the control group did not receive any treatment. Six weeks after gene transfer, the expressions of PLB protein and PLB phosphorylation were detected by Western-blot, while the activity of sarcoplasmic reticulum （SR） Ca2＋-ATPase and left ventricular function were measured. Results The PLB protein expression level was significantly higher whereas the PLB phosphorylation, SR Ca2＋-ATPase activity and left ventricular function were significantly lower in the DM-saline group than in the control group. No significant difference was found in PLB protein expression level, PLB phosphorylation or SR Ca2＋-ATPase activity between the DM-rAAV-asPLB group and the control group. The left ventricular function in the DM-rAAV-asPLB group was poorer than in the control group and was better than in the DM-saline group. Conclusion rAAV-asPLB can down-regulate PLB protein expression and up-regulate phosphorylation and SR Ca2＋-ATPase activity, thus contributing to the improvement of in vivo ventricutar function. PLB left

Transmembrane Ca^(2+) gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum Ca^(2+)-ATPase

The sarcoplasmic reticulum (SR) Ca2+-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role ofphospholipids, especially phosphatidylcholine(PC), in the modulation of Ca2+-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows, (i) Incubated with phospholiplds, the enzyme activity of the delipidated Ca2+-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1 000μmol/L:50μmol/L, similar to the physiological condition), the inhibition of Ca2+-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50.50 molar ratio of PC:PE. (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of Ca2+-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of Ca2+-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.

Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis

Background Liraglutide is glucagon-like peptide-1 receptor agonist for treating patients with type 2 diabetes mellitus. Our previous studies have demonstrated that liraglutide protects cardiac function through improving endothelial function in patients with acute myocardial infarction undergoing percutaneous coronary intervention. The present study will investigate whether liraglntide can perform direct protective effects on cardiomyocytes against reperfusion injury. Methods In vitro experiments were performed using H9C2 cells and neonatal rat ventricular cadiomyocytes undergoing simulative hypoxia/reoxygenation （H/R） induction. Cardiomyocytes apoptosis was detected by fluorescence TUNEL. Mitochondrial membrane potential （AWm） and intracellular reactive oxygen species （ROS） was assessed by JC-1 and DHE, respectively. Fura-2/AM was used to measure intracellular Ca2＋ concentration and calcium transient. Immtmofluorescence staining was used to assess the expression level of sarcoplasmic reticulum Ca2＋-ATPase （SERCA2a）. In vivo experiments, myocardial apoptosis and expression of SERCA2a were detected by colorimetric TUNEL and by immunofluorescence staining, respectively. Results In vitro liraglutide inhibited cardiomyotes apoptosis against H/R. △mψ of cardiomyocytes was higher in liraglntide group than H/R group. H/R increased ROS production in H9C2 cells which was attenuated by liraglutide. Liraglutide significantly lowered Ca2＋ overload and improved calcium transient compared with H/R group, lmmunofluorescence staining results showed liraglutide promoted SERCA2a expression which was decreased in H/R group. In ischemia/reperfusion rat hearts, apoptosis was significantly attenuated and SERCA2a expression was increased by liraglutide compared with H/R group. Conclusions Liraglutide can directly protect cardiomyocytes against reperfusion injury which is possibly through modulation of intracellular calcium homeostasis.

The substitution of SERCA2 redox cysteine 674 promotes pulmonary vascular remodeling by activating IRE1α/XBP1s pathway

Pulmonary hypertension(PH) is a life-threatening disease characterized by pulmonary vascular remodeling, in which hyperproliferation of pulmonary artery smooth muscle cells(PASMCs)plays an important role. The cysteine 674(C674) in the sarcoplasmic/endoplasmic reticulum Ca^(2+)ATPase 2(SERCA2) is the critical redox regulatory cysteine to regulate SERCA2 activity. Heterozygous SERCA2 C674 S knock-in mice(SKI), where one copy of C674 was substituted by serine to represent partial C674 oxidative inactivation, developed significant pulmonary vascular remodeling resembling human PH, and their right ventricular systolic pressure modestly increased with age. In PASMCs, substitution of C674 activated inositol requiring enzyme 1 alpha(IRE1 a) and spliced X-box binding protein 1(XBP1 s) pathway, accelerated cell cycle and cell proliferation, which reversed by IRE1 a/XBP1 s pathway inhibitor 4μ8 C. In addition, suppressing the IRE1 a/XBP1 s pathway prevented pulmonary vascular remodeling caused by substitution of C674. Similar to SERCA2 a, SERCA2 b is also important to restrict the proliferation of PASMCs. Our study articulates the causal effect of C674 oxidative inactivation on the development of pulmonary vascular remodeling and PH, emphasizing the importance of C674 in restricting PASMC proliferation to maintain pulmonary vascular homeostasis. Moreover, the IRE1 a/XBP1 s pathway and SERCA2 might be potential targets for PH therapy.