17ß-Oestradiol facilitates M2 macrophage skewing and ameliorates arrhythmias in ovariectomized female infarcted rats.

Epidemiological studies have suggested a lower incidence of arrhythmia-induced sudden cardiac death in women than in men. 17ß-oestradiol (E2) has been reported to have a post-myocardial infarction antiarrhythmic effect, although the mechanisms have yet to be elucidated. We investigated whether E2-mediated antioxidation regulates macrophage polarization and affects cardiac sympathetic reinnervation in rats after MI. Ovariectomized Wistar rats were randomly assigned to placebo pellets, E2 treatment, or E2 treatment +3-morpholinosydnonimine (a peroxynitrite generator) and followed for 4 weeks. The infarct sizes were similar among the infarcted groups. At Day 3 after infarction, post-infarction was associated with increased superoxide levels, which were inhibited by administering E2. E2 significantly increased myocardial IL-10 levels and the percentage of regulatory M2 macrophages compared with the ovariectomized infarcted alone group as assessed by immunohistochemical staining, Western blot and RT-PCR. Nerve growth factor colocalized with both M1 and M2 macrophages at the magnitude significantly higher in M1 compared with M2. At Day 28 after infarction, E2 was associated with attenuated myocardial norepinephrine levels and sympathetic hyperinnervation. These effects of E2 were functionally translated in inhibiting fatal arrhythmias. The beneficial effect of E2 on macrophage polarization and sympathetic hyperinnervation was abolished by 3-morpholinosydnonimine. Our results indicated that E2 polarized macrophages into the M2 phenotype by inhibiting the superoxide pathway, leading to attenuated nerve growth factor-induced sympathetic hyperinnervation after myocardial infarction.

Exercise intensities modulate cognitive function in spontaneously hypertensive rats through oxidative mediated synaptic plasticity in hippocampus.

Oxidative damage in the brain may lead to cognitive impairments. There was considerable debate regarding the beneficial effects of physical exercise on cognitive functions because exercise protocols have varied widely across studies. We investigated whether different exercise intensities alter performance on cognitive tasks. The experiment was performed on spontaneously hypertensive rats (6 months at the established phase of hypertension) distributed into 3 groups: sedentary, low-intensity exercise and high-intensity exercise. Systolic blood pressure measurements confirmed hypertension in spontaneously hypertensive rats. In comparison to normotensive Wistar-Kyoto rats, sedentary spontaneously hypertensive rats had similar escape latencies and a similar preference for the correct quadrant in the probe trial. Compared to the sedentary group, the low-intensity exercise group had significantly better improvements in spatial memory assessed by Morris water maze. Low-intensity exercise was associated with attenuated reactive oxygen species, as measured by dihydroethidine fluorescence and nitrotyrosine staining in the dentate gyrus of the hippocampus. This was coupled with increased numbers of neurons and dendritic spines as well as a significant upregulation of synaptic density. In contrast, the beneficial effects of low-intensity exercise are abolished in high-intensity exercise as shown by increased free radical levels and an impairment in spatial memory. We concluded that exercise is an effective strategy to improve spatial memory in spontaneously hypertensive rats even at an established phase of hypertension. Low-intensity exercise exhibited better improvement on cognitive deficits than high-intensity exercise by attenuating free radical levels and improving downstream synaptic plasticity.

Taurine Alleviates Sympathetic Innervation by Inhibiting NLRP3 Inflammasome in Postinfarcted Rats.

ABSTRACT: The NLRP3 inflammasome is activated by myocardial infarction and then induces the activation of inflammatory caspase-1 activation and maturation of IL-1ß, a regulator of synthesis of the nerve growth factor (NGF). Here, we studied whether taurine, 2-aminoethanesulphonic acid, can attenuate cardiac sympathetic reinnervation by modulating NLRP3 inflammasome-mediated NGF in a rat model of myocardial infarction. Male Wistar rats were subjected to coronary ligation and then randomized to either saline or taurine for 3 days or 4 weeks. Postinfarction was associated with activation of NF-κB (p65) and NLRP3 inflammasome component and increased the protein and expression of IL-1ß. Macrophages at the border zone were shown to be positive for IL-1ß 3 days postinfarction. Compared with vehicle, infarcted rats treated with taurine significantly attenuated myocardial messenger RNA and protein levels of NF-κB, NLRP3 inflammasome, mature caspase-1, and IL-1ß. Immunofluorescent analysis, real-time quantitative reverse transcription polymerase chain reaction, and Western blotting of NGF showed that sympathetic hyperinnervation was blunted after administering taurine. Arrhythmia vulnerability in the taurine-treated infarcted rats was significantly improved than those in vehicle. Ex vivo studies showed that taurine infusion reduced myocardial IL-1ß level at the extent similar to either pyrrolidine dithiocarbamate or CP-456,773, inhibitors of NF-κB and NLRP3 inflammasome, implying the key axis of NF-κB/NLRP3 inflammasome in mediating taurine-related anti-inflammation. Furthermore, administration of anti-IL-1ß antibody reduced NGF levels. Taurine attenuated sympathetic innervation mainly by NLRP3 inflammasome/IL-1ß-dependent pathway, which downregulated expression of NGF in infarcted rats. These findings may provide a new insight into the anti-inflammation effect of taurine.

Effect of icosapent ethyl on susceptibility to ventricular arrhythmias in postinfarcted rat hearts: Role of GPR120-mediated connexin43 phosphorylation.

The ω-3 fatty acids exert as an antioxidant via the G protein-coupled receptor 120 (GPR120). Icosapent ethyl, a purified eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connexin43 expression and whether the GPR120 signalling is involved in the protection. Male Wistar rats after ligating coronary artery were assigned to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative-nitrosative stress. In concert, myocardial connexin43 levels revealed a significant decrease in vehicle-treated infarcted rats compared with sham. These changes of oxidative-nitrosative stress and connexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative-nitrosative stress and connexin43 phosphorylation were abolished by administering AH-7614, an inhibitor of GPR120. SIN-1 abolished the Cx43 phosphorylation of icosapent ethyl without affecting GPR120 levels. Taken together, chronic use of icosapent ethyl after infarction is associated with up-regulation of connexin43 phosphorylation through a GPR120-dependent antioxidant pathway and thus plays a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

Host pre-conditioning improves human adipose-derived stem cell transplantation in ageing rats after myocardial infarction: Role of NLRP3 inflammasome.

Functional decline of stem cell transplantation in ageing hosts is well documented. The mechanism for this is poorly understood, although it is known that advancing age does not provide an optimal milieu for exogenous stem cells to survive, engraft and differentiate. We showed that n-butylidenephthalide improved human adipose-derived stem cell (hADSC) engraftment via attenuating the production of reactive oxygen species (ROS). It remained unclear whether pre-treated hosts with n-butylidenephthalide can rejuvenate the ageing heart and improve hADSC engraftment by regulating the ROS/NLRP3 inflammasome-mediated cardiac fibrosis after myocardial infarction. One hour after coronary ligation, hADSCs were transplanted into the hearts of young and ageing Wistar rats that were pre-treated with or without n-butylidenephthalide for 3 days. At day 3 after infarction, myocardial infarction was associated with an increase in ROS levels and NLRP3 inflammasome activity with age. hADSC transplant effectively provided a significant decrease in ROS levels, NLRP3 inflammasome activity, IL-1ß levels and cardiac fibrosis in either young or old infarcted rats. However, the beneficial effects of hADSCs were greater in young compared with old rats in terms of NLRP3 inflammasome activity. The infarcted ageing rats pre-conditioned by n-butylidenephthalide improved engraftment and differentiation of hADSCs and additionally attenuated cardiac fibrosis compared with hADSCs alone. The anti-inflammation effects of n-butylidenephthalide were reversed by SIN-1. In conclusions, the increased NLRP3 inflammasome activity plays the pathogenesis of ageing-related functional hADSC decline in the ageing hosts. n-butylidenephthalide-pre-treated ageing hosts reversibly ameliorate the harsh microenvironments, improve stem cell engraftment and attenuate cardiac fibrosis after myocardial infarction.

Targeting the PI3K/STAT3 axis modulates age-related differences in macrophage phenotype in rats with myocardial infarction.

Ageing is associated with impaired repair mechanisms in cardiovascular diseases. Macrophages contribute to cardiac fibrosis after myocardial infarction (MI). The phosphatidyl-inositol-3-kinase (PI3K) pathway has been shown to play a role in cardiac remodelling after MI. It remained unclear whether n-butylidenephthalide, a major component of Angelica sinensis, can attenuate cardiac fibrosis by regulating the PI3K/signal transducer and activator of transcription 3 (STAT3)-mediated macrophage phenotypes in ageing rats after MI. Twenty-four hours after ligation of the left anterior descending artery, young (2-month-old) and ageing (18-month-old) male Wistar rats were treated with either vehicle or n-butylidenephthalide for 4 weeks. There were similar infarct sizes in both age groups. Compared with young rats, ageing rats exhibited significant increased cardiac fibrosis after MI, which can be attenuated after administering n-butylidenephthalide. MI was associated with decreased activities of PI3K and STAT3 in ageing rats compared with young rats. In both age groups, n-butylidenephthalide effectively provided a significant increase of STAT3 phosphorylation, STAT3 activity, STAT3 nuclear translocation, myocardial IL-10 levels and the percentage of M2c macrophage and a decrease of myofibroblast infiltration. The effects of n-butylidenephthalide on increased IL-10 levels were reversed by LY294002 or S3I-201. Furthermore, LY294002 abolished the STAT3 phosphorylation, whereas PI3K activity was not affected following the inhibition of STAT3. In conclusions, the host environment is responsible for ageing-related myofibroblast dysregulation in response to MI which can be improved by administering n-butylidenephthalide via macrophage differentiation towards M2 phenotype by targeting the PI3K/STAT3 axis.

Preconditioned adipose-derived stem cells ameliorate cardiac fibrosis by regulating macrophage polarization in infarcted rat hearts through the PI3K/STAT3 pathway.

Stem cells can modify macrophage phenotypes; however, the mechanisms remain unclear. We investigated whether n-butylidenephthalide (BP) primed adipose-derived stem cells (ADSCs) attenuated cardiac fibrosis via regulating macrophage phenotype by a PI3K/STAT3-dependent pathway in postinfarcted rats. Male Wistar rats after coronary ligation were allocated to receive either intramyocardial injection of vehicle, ADSCs (1 × 106 cells), BP-preconditioned ADSCs, (BP + lithium)-preconditioned ADSCs, (BP + LY294002)-preconditioned ADSCs, and (BP + S3I-201)-preconditioned ADSCs. ADSCs were primed for 16 h before implantation. BP-pretreated ADSCs increased the cell viability compared with naive ADSCs in the in vitro experiments. Infarct sizes were similar among the infarcted groups at the acute and chronic stages of infarction. At day 3 after infarction, post-infarction was associated with increased M1 macrophage infiltration, which was inhibited by administering naive ADSCs. Compared with naive ADSCs, BP-preconditioned ADSCs provided a significant increase of Akt and STAT3 phosphorylation, STAT3 activity, STAT3 nuclear translocation, myocardial IL-10 levels, and the percentage of M2 macrophage infiltration. The effects of BP on M2 polarization were reversed by LY294002 or S3I-201. Furthermore, the phosphorylation of both Akt and STAT3 was abolished by LY294002, whereas Akt phosphorylation was not affected following the inhibition of STAT3. The addition of lithium did not have additional effects compared with BP alone. After 4 weeks of implantation, ADSCs remained in the myocardium, and reduced fibrosis and improved cardiac function. BP-preconditioned ADSCs provided superior cardioprotection, greater ADSC engraftment, and antifibrotic effects compared with naive ADSCs. These results suggest that BP-pretreated ADSCs polarize macrophages into M2 cells more efficiently than naive ADSCs via the PI3K/STAT3 pathway.

Blockade of Cardiac Proton Pump Impairs Ventricular Remodeling Through a Superoxide-DDAH-Dependent Pathway in Infarcted Rats.

BACKGROUND: Proton pump inhibitors (PPIs) are frequently used to prevent or treat peptic ulcers. Recently, PPIs have been shown to increase the risk of myocardial infarction. The purpose of this study was to determine whether PPIs adversely affect ventricular remodeling in infarcted rats. METHODS: Male Wistar rats were randomly assigned to receive either vehicle, omeprazole, omeprazole + vitamin C, omeprazole + olmesartan, or famotidine treatment for 4 weeks starting 24 hours after inducing myocardial infarction by ligating coronary arteries. RESULTS: Compared with vehicle-treated infarcted rats, omeprazole-treated infarcted rats had significant changes with reduced myocardial vitamin C levels, increased oxidant production, and decreased dimethylarginine dimethylaminohydrolase 2 (DDAH2) activity, which in turn increased asymmetric dimethylarginine (ADMA) levels and impaired ventricular remodeling. With gastric protection similar to omeprazole, the H2 blocker famotidine had no effect on ventricular remodeling. In contrast to the in vivo results, the ex vivo study showed similar superoxide and DDAH2 protein levels between vehicle- and omeprazole-treated infarcted rats, suggesting involvement of gastric vitamin C uptake rather than myocardial vitamin C in mediating the impaired axis of vitamin C-superoxide-DDAH2 in the in vivo measurements. The administration of PPIs was associated with impaired DDAH2 expression and increased myocardial ADMA, which impaired ventricular remodeling after infarction. These effects were prevented by the coadministration of vitamin C or olmesartan. CONCLUSIONS: Our results indicate that the administration of PPIs was associated with impaired DDAH2 expression and increased myocardial ADMA by reducing gastric vitamin C uptake, which impaired ventricular remodeling after infarction.

Nicorandil regulates the macrophage skewing and ameliorates myofibroblasts by inhibition of RhoA/Rho-kinase signalling in infarcted rats.

We have demonstrated that ATP-sensitive potassium (KATP ) channel agonists attenuated fibrosis; however, the mechanism remained unclear. Since RhoA has been identified as a mediator of cardiac fibrosis, we sought to determine whether the anti-fibrotic effects of KATP channel agonists were mediated via regulating macrophage phenotype and fibroblast differentiation by a RhoA/RhoA-kinase-dependent pathway. Wistar male rats after induction of myocardial infarction were randomized to either vehicle, nicorandil, an antagonist of KATP channel glibenclamide, an antagonist of ROCK fasudil, or a combination of nicorandil and glibenclamide or fasudil and glibenclamide starting 24 hrs after infarction. There were similar infarct sizes among the infarcted groups. At day 3 after infarction, post-infarction was associated with increased RhoA/ROCK activation, which can be inhibited by administering nicorandil. Nicorandil significantly increased myocardial IL-10 levels and the percentage of regulatory M2 macrophages assessed by immunohistochemical staining, Western blot, and RT-PCR compared with vehicle. An IL-10 receptor antibody increased myofibroblast infiltration compared with nicorandil alone. At day 28 after infarction, nicorandil was associated with attenuated cardiac fibrosis. These effects of nicorandil were functionally translated in improved echocardiographically derived cardiac performance. Fasudil showed similarly increased expression of M2 macrophages as nicorandil. The beneficial effects of nicorandil on fibroblast differentiation were blocked by adding glibenclamide. However, glibenclamide cannot abolish the attenuated fibrosis of fasudil, implying that RhoA/RhoA-kinase is a downstream effector of KATP channel activation. Nicorandil polarized macrophages into M2 phenotype by inhibiting RhoA/RhoA-kinase pathway, which leads to attenuated myofibroblast-induced cardiac fibrosis after myocardial infarction.

Targeting the pathway of GSK-3ß/nerve growth factor to attenuate post-infarction arrhythmias by preconditioned adipose-derived stem cells.

Adipose-derived stem cell (ADSC) transplantation is a promising new therapy to improve cardiac function after myocardial infarction. However, its low efficacy of transdifferentiation hampers its usefulness. Glycogen synthase kinase-3ß (GSK-3ß) signal has been shown to play a role in preconditioning-induced cardioprotection. We assessed whether n-butylidenephthalide (BP) primed ADSCs can attenuate arrhythmias by a GSK-3ß-dependent pathway after myocardial infarction. Male Wistar rats after coronary ligation was randomly allocated to receive intramyocardial injection of vehicle, ADSCs, BP-preconditioned ADSCs, (BP+lithium)-preconditioned ADSCs, (BP+SB216763)-preconditioned ADSCs, and (BP+LY294002)-preconditioned ADSCs. ADSCs were primed for 16h before implantation. After 4weeks of implantation, ADSCs were retained in myocardium, reduced fibrosis and improved cardiac function. Sympathetic hyperinnervation was blunted after administering ADSCs, assessed by immunofluorescent analysis, and Western blotting and real-time quantitative RT-PCR of nerve growth factor. Arrhythmic scores during programmed stimulation in the ADSC-treated infarcted rats were significantly lower than vehicle. BP-preconditioned ADSCs had superior cardioprotection, greater ADSC engraftment and transdifferentiation, and antiarrhythmic effects compared with ADSCs alone. Simultaneously, BP increased the levels of phospho-Akt and down-regulated GSK-3ß activity. The effects of BP against sympathetic hyperinnervation were blocked by LY294002, a PI3K inhibitor. Addition of either lithium or SB216763 did not have additional effects compared with BP alone. Compared with ADSC alone, BP-primed ADSC implantation improved stem cell engraftment and attenuated sympathetic hyperinnervation and arrhythmias through a PI3K/Akt/GSK-3ß-dependent pathway, suggesting that a synergic action was achieved between BP pretreatment and ADSCs.

Effects of urate-lowering agents on arrhythmia vulnerability in post-infarcted rat hearts.

Hyperuricemia has been shown to be associated with ventricular arrhythmias. However, the mechanisms remained unknown. We assessed whether different urate-lowering agents can attenuate arrhythmias through lowering urate itself or inhibiting xanthenes oxidize (XO) activity in infarcted rats. Male Wistar rats after ligating coronary artery were randomized to either allopurinol, or febuxostat, chemically unrelated inhibitors of XO, benzbromarone or vehicle for 4 weeks. Post-infarction was associated with increased oxidant stress, as measured by myocardial superoxide, isoprostane, XO activity and dihydroethidine fluorescence staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham-operated rats. Sympathetic hyperinnervation was blunted after administering both XO inhibitors, assessed by immunofluorescent analysis, Western blotting and real-time quantitative RT-PCR. Besides, the XO inhibitors-attenuated nerve growth factor levels were reversed in the presence of peroxynitrite generator. Arrhythmic scores in the XO inhibitors-treated infarcted rats were significantly lower than that in vehicle. For similar levels of urate lowering, the uricosuric agent benzbromarone had no beneficial effects on oxidative stress, sympathetic hyperinnervation or arrhythmia vulnerability. Chronic use of XO inhibitors, but not uricosuric agent, down-regulated sympathetic innervation probably through a superoxide-dependent pathway and plays a role in the beneficial effect on arrhythmogenic response.

A Double-Blind, Randomized, Placebo-Controlled Study to Evaluate the Efficacy and Safety of STA-2 (Green Tea Polyphenols) in Patients with Chronic Stable Angina.

BACKGROUND: Green tea intake has been shown to improve endurance capacity in animal studies, but whether it has a similar effect on humans remains unclear. A randomized, double-blinded, parallel-controlled study was conducted to evaluate the short-term effect of STA-2, a pharmaceutical preparation of green tea polyphenols, in patients with effort-induced angina and documented positive exercise tolerance test. METHODS: A total of 79 patients recruited from three medical centers were randomly assigned to receive 2 STA-2 250 mg capsules, each containing 100 mg green tea polyphenols, three times daily, or placebo for six weeks after two consecutive symptom-limited treadmill exercise tests to ascertain the reproducibility of exercise tolerance. RESULTS: There was no difference in total exercise tolerance time from baseline to Week 6 between two groups (p = 0.639). There were also no observed improvements in subgroup analyses stratified by age, gender, and BMI categories. However, a significant reduction in low-density lipoprotein levels was shown in patients in the STA-2 group (-8.99 ± 19.18 mg/dL) versus the placebo group (0.57 ± 19.77 mg/dL), p = 0.037, with greater benefits in patients not taking antihyperlipidemic drugs (STA-2: -9.10 ± 19.96 mg/dL vs. placebo: 4.42 ± 15.08 mg/dL, p = 0.037). CONCLUSIONS: STA-2 treatment for 6 weeks did not increase exercise time as measured on a treadmill. However, this study also indicated that STA-2 treatment could have potential beneficial effects on LDL-cholesterol concentrations.

Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts.

We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats.

Dipeptidyl peptidase-4 inhibition attenuates arrhythmias via a protein kinase A-dependent pathway in infarcted hearts.

BACKGROUND: The effect of dipeptidyl peptidase-4 (DPP-4) inhibitors on arrhythmias remains unknown. The aim of this study was to investigate whether sitagliptin attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression, focusing on cyclic adenosine monophosphate (cAMP) downstream signaling such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). METHODS AND RESULTS: Male Wistar rats were randomized to either vehicle or sitagliptin for 4 weeks starting 24 h after ligating the coronary artery. Post-infarction was associated with increased oxidative stress. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated rats compared with sham. Compared with the vehicle, infarcted rats treated with sitagliptin had significantly increased cAMP levels, decreased DPP-4 activity, oxidative stress, NGF levels and immunofluorescence-stained sympathetic hyperinnervation. Arrhythmic scores were significantly lower in the sitagliptin-treated infarcted rats than in vehicle. Ex vivo studies showed that sitagliptin increased the phosphorylated cAMP response element-binding protein (CREB), which can be reversed by H-89 (a PKA inhibitor), not brefeldin A (an Epac inhibitor).Heme oxygenase-1(HO-1) expression was increased by a PKA agonist but not by an Epac agonist.HO-1expression was attenuated in KG-501 (a CREB inhibitor)-treated infarcted rats in the presence of a PKA agonist. CONCLUSIONS: Sitagliptin protects ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via upregulation ofHO-1expression in a cAMP/PKA/CREB-dependent antioxidant pathway in non-diabetic infarcted rats.

Both GPER and membrane oestrogen receptor-α activation protect ventricular remodelling in 17ß oestradiol-treated ovariectomized infarcted rats.

Clinical and experimental studies have established that gender is a factor in the development of ventricular hypertrophy. We investigated whether the attenuated hypertrophic effect of oestradiol was via activation of phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) through non-genomic action. Twenty-four hours after coronary ligation, female Wistar rats were randomized into control, subcutaneous oestradiol treatment or a G-protein coupled oestrogen receptor (GPER) agonist, G-1 and treated for 4 weeks starting from 2 weeks after bilateral ovariectomy. Ventricular hypertrophy assessed by cardiomyocyte size after infarction was similarly attenuated by oestradiol or G-1 in infarcted rats. The phosphorylation of Akt and eNOS was significantly decreased in infarcted rats and restored by oestradiol and G-1, implying the GPER pathway in this process. Oestradiol-induced Akt phosphorylation was not abrogated by G-15 (a GPER blocker). Akt activation was not inhibited by actinomycin D. When a membrane-impermeable oestrogen-albumin construct was applied, similar responses in terms of eNOS activation to those of oestradiol were achieved. Furthermore, PPT, an ERα receptor agonist, activated the phosphorylation of Akt and eNOS. Thus, membrane ERα receptor played a role in mediating the phosphorylation of Akt and eNOS. The specific PI3K inhibitor, LY290042, completely abolished Akt activation and eNOS phosphorylation in infarcted hearts treated with either oestradiol or oestradiol + G-15. These data support the conclusions that oestradiol improves ventricular remodelling by both GPER- and membrane-bound ERα-dependent mechanisms that converge into the PI3K/Akt/eNOS pathway, unveiling a novel mechanism by which oestradiol regulates pathological cardiomyocyte growth after infarction.

Differential effect of phosphodiesterase-3 inhibitors on sympathetic hyperinnervation in healed rat infarcts.

BACKGROUND: The effect of phosphodiesterase-3 (PDE-3) inhibitors on arrhythmia remains controversial, so the purpose of this study was to determine their differential effects on sympathetic hyperinnervation and the involved mechanisms in a rat model of myocardial infarction. METHODS AND RESULTS: After ligating the coronary artery, male Wistar rats were randomized to cilostazol or milrinone, chemically unrelated inhibitors of PDE-3, or vehicle for 4 weeks. The postinfarction period was associated with increased myocardial norepinephrine levels and oxidant release, as measured by myocardial superoxide level and dihydroethidine fluorescence staining. Infarcted rats in the milrinone- and cilostazol-treated groups had favorable ventricular remodeling with similar potency. Compared with milrinone, cilostazol significantly increased interstitial adenosine levels and reduced the production of myocardial cAMP and superoxide. Cilostazol significantly blunted sympathetic hyperinnervation, as assessed by immunofluorescent analysis of sympathetic innervation, and western blotting and real-time quantitative RT-PCR of nerve growth factor. Furthermore, the inhibitory effect of cilostazol on nerve growth factor was reversed by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1 receptor antagonist, and enhanced by tempol administration. In spite of similar arrhythmic vulnerability during programmed stimulation in both the vehicle-and cilostazol-treated groups, cilostazol did not have proarrhythmic effects compared with milrinone. CONCLUSIONS: Unlike milrinone, cilostazol has therapeutic neutrality in arrhythmias because of adenosine uptake inhibition, which antagonizes the PDE-3-induced increase of sympathetic reinnervation via mediation of an adenosine A1 receptor-mediated antioxidation.

K ATP channel agonists preserve connexin43 protein in infarcted rats by a protein kinase C-dependent pathway.

Downward remodelling of gap junctional proteins between myocytes may trigger ventricular arrhythmia after myocardial infarction. We have demonstrated that ATP-sensitive potassium (K(ATP)) channel agonists attenuated post-infarction arrhythmias. However, the involved mechanisms remain unclear. The purpose of this study was to determine whether K(ATP) channel agonists can attenuate arrhythmias through preserving protein kinase C (PKC)-(ε)dependent connexin43 level after myocardial infarction. Male Wistar rats after ligating coronary artery were randomized to either vehicle, nicorandil, pinacidil, glibenclamide or a combination of nicorandil and glibenclamide or pinacidil and glibenclamide for 4 weeks. To elucidate the role of PKC(ε) in the modulation of connexin43 level, carbachol and myristoylated PKC(ε) V1-2 peptide were also assessed. Myocardial connexin43 level was significantly decreased in vehicle-treated infarcted rats compared with sham. Attenuated connexin43 level was blunted after administering K(ATP) channel agonists, assessed by immunofluorescent analysis, Western blotting, and real-time quantitative reverse transcription-PCR of connexin43. Arrhythmic scores during programmed stimulation in the K(ATP) channel agonists-treated rats were significantly lower than those treated with vehicle. The beneficial effects of K(ATP) channel agonists were blocked by either glibenclamide or 5-hydroxydecanoate. Addition of the PKC activator, phorbol 12-myristate 13-acetate and the specific PKC(ε) agonist, carbachol, blocked the effects of nicorandil on connexin43 phosphorylation and dye permeability. The specific PKC(ε) antagonist, myristoylated PKC(ε) V1-2 peptide, did not have additional beneficial effects on connexin43 phosphorylation compared with rats treated with nicorandil alone. Chronic use of K(ATP) channel agonists after infarction, resulting in enhanced connexin43 level through a PKC(ε)-dependent pathway, may attenuate the arrhythmogenic response to programmed electrical stimulation.

Cardiac sympathetic hyperinnervation in deoxycorticosterone acetate-salt hypertensive rats.

Sympathetic activities are elevated in the central SNSs (sympathetic nervous systems) of hypertensive animals, but it is not known whether sympathetic innervation is also elevated in the heart. Sympathetic hyper-responsiveness in hypertension may result from oxidative stress. The aim of the present study was to investigate sympathetic hyperinnervation in DOCA (deoxycorticosterone acetate)-salt hypertensive rats with established hypertension. At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 8 weeks: vehicle, NAC (N-acetylcysteine) and triple therapy (hydralazine, hydrochlorothiazide and reserpine). DOCA-salt was associated with increased oxidant release. DOCA-salt produced concentric left ventricular hypertrophy and cardiomyocyte hypertrophy. Sympathetic hyperinnervation was observed in DOCA-salt rats, as assessed by myocardial noradrenaline levels, immunofluorescent analysis of tyrosine hydroxylase, growth-associated factor 43 and neurofilament and Western blotting and real-time quantitative RT-PCR (reverse transcription-PCR) of NGF (nerve growth factor). Arrhythmic scores during programmed stimulation in DOCA-salt rats were significantly higher than those in the control rats. Triple therapy, despite being effective on BP (blood pressure), offered neither attenuated cardiomyocyte hypertrophy nor anti-arrhythmia. The effects of DOCA-salt treatment on NGF expression, sympathetic hyperinnervation and arrhythmias were attenuated by NAC. Furthermore, the effects of NAC on NGF were abolished by administering BSO (L-buthionine sulfoximine), an inhibitor of glutamate-cysteine ligase. In conclusion, DOCA-salt treatment contributes to up-regulation of NGF proteins probably through a free radical-dependent pathway in a BP-independent manner. DOCA-salt rats treated with NAC attenuate sympathetic hyperinnervation and thus show a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

Differential effects of EPA and DHA on PPARγ-mediated sympathetic innervation in infarcted rat hearts by GPR120-dependent and -independent mechanisms.

The ω-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to attenuate inflammation processes, whereas the molecular mechanisms remain unclear. This study was aimed at figuring out the differential effects of EPA and DHA on fatal arrhythmias and whether the signaling pathway could be a target after myocardial infarction, an inflammatory status. Male Wistar rats after ligating coronary artery were randomized to either vehicle, EPA, or DHA for 4 weeks. Postinfarction was associated with increased myocardial norepinephrine levels and sympathetic innervation. Furthermore, infarction was associated with the activation of NLRP3 inflammasomes and increased the protein and expression of IL-1ß and nerve growth factor (NGF). These changes were blunted after adding either EPA or DHA with a greater extent of EPA than DHA. Immunoblotting and immunohistochemical analysis showed that EPA had significantly lower phosphorylation of PPARγ at Ser 112 compared with DHA. Arrhythmic severity during programmed stimulation in the infarcted rats treated with EPA was significantly lower than those treated with DHA. Specific inhibition of GPR120 by AH-7614 and PPARγ by T0070907 reduced the EPA-or DHA-related attenuation of IL-1ß and NGF release. Besides, AH-7614 treatment partially reduced the PPARγ levels, whereas T0070907 administration did not affect the GPR120 levels. These results suggest that EPA was more effective than DHA in prevention of fatal arrhythmias by inhibiting NLRP3 inflammasome and sympathetic innervation through activation of PPARγ-mediated GPR120-dependent and -independent signaling pathways in infarcted hearts.

Impact of elevated uric acid on ventricular remodeling in infarcted rats with experimental hyperuricemia.

Hyperuricemia is associated with cardiovascular disease, but it is usually considered a marker rather than a risk factor. Previous studies using uric acid-lowering drugs in normouricemic animals are not suitable to answer the effect of hyperuricemia on ventricular remodeling after myocardial infarction. The purpose of this study was to determine whether hyperuricemia adversely affects ventricular remodeling in infarcted rats with elevated uric acid. Male Wistar rats aged 8 wk were randomly assigned into either vehicle, oxonic acid, oxonic acid + allopurinol, oxonic acid + benzbromarone, oxonic acid + ABT-627, or oxonic acid + tempol for 4 wk starting 24 h after ligation. Postinfarction was associated with increased oxidant production, as measured by myocardial superoxide, isoprostane, xanthine oxidase activity, and dihydroethidium staining. Compared with normouricemic infarcted rats, hyperuricemic infarcted rats had a significant increase of superoxide production (1.7×) and endothelin-1 protein (1.2×) and mRNA (1.4×) expression, which was associated with increased left ventricular dysfunction and enhanced myocardial hypertrophy and fibrosis. These changes were all prevented by treatment with allopurinol. For similar levels of urate lowering, the uricosuric agent benzbromarone had no effect on ventricular remodeling. In spite of equivalent hyperuricemia, the ability of both ABT-627 and tempol to attenuate ventricular remodeling suggested involvement of endothelin-1 and redox pathways. Hyperuricemia is associated with unfavorable ventricular remodeling probably through a superoxide and endothelin-1-dependent pathway. Uric acid lowering without inhibition of superoxide and endothelin-1 may not have an effect on remodeling. Chronic administration of allopurinol, ABT-627, and tempol is associated with attenuated ventricular remodeling.