Inhibition of the INa/K and the activation of peak INa contribute to the arrhythmogenic effects of aconitine and mesaconitine in guinea pigs.

Aconitine (ACO), a main active ingredient of Aconitum, is well-known for its cardiotoxicity. However, the mechanisms of toxic action of ACO remain unclear. In the current study, we investigated the cardiac effects of ACO and mesaconitine (MACO), a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs. We showed that intravenous administration of ACO or MACO (25 µg/kg) to guinea pigs caused various types of arrhythmias in electrocardiogram (ECG) recording, including ventricular premature beats (VPB), atrioventricular blockade (AVB), ventricular tachycardia (VT), and ventricular fibrillation (VF). MACO displayed more potent arrhythmogenic effect than ACO. We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes, and observed that treatment with ACO (0.3, 3 µM) or MACO (0.1, 0.3 µM) depolarized the resting membrane potential (RMP) and reduced the action potential amplitude (APA) and durations (APDs) in a concentration-dependent manner. The ACO- and MACO-induced AP remodeling was largely abolished by an INa blocker tetrodotoxin (2 µM) and partly abolished by a specific Na+/K+ pump (NKP) blocker ouabain (0.1 µM). Furthermore, we observed that treatment with ACO or MACO attenuated NKP current (INa/K) and increased peak INa by accelerating the sodium channel activation with the EC50 of 8.36 ± 1.89 and 1.33 ± 0.16 µM, respectively. Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na+ and Ca2+ concentrations. In conclusion, the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak INa via accelerating sodium channel activation and inhibiting the INa/K. These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO, and identify potential novel therapeutic targets for Aconitum poisoning.

Cannabinoid type 2 receptor agonist JWH133 decreases blood pressure of spontaneously hypertensive rats through relieving inflammation in the rostral ventrolateral medulla of the brain.

OBJECTIVE: Neuroinflammation in the rostral ventrolateral medulla (RVLM) has been reported to be associated with hypertension. The upregulation and activation of the cannabinoid type 2 (CB2) receptor may be part of the active process of limiting or downregulating the inflammatory process. This study was designed to determine the role of the CB2 receptor in blood pressure (BP) through relieving neuroinflammation in the RVLM in spontaneously hypertensive rats (SHRs). METHODS: The long-term effects of intracerebroventricular injection of JWH133, a selective CB2 receptor agonist, on BP, heart rate (HR) and renal sympathetic nerve activity (RSNA) in SHR and Wistar-Kyoto (WKY) rats were determined. ELISA was used to measure the levels of proinflammatory cytokines, and western blotting was employed to detect protein expression of the CB2 receptor. Immunofluorescence staining was used to localize the CB2 receptor. Gene silencing of the CB2 receptor was realized by injecting adeno-associated virus (AAV) expressing CB2-specific shRNA (AAV2-r-CB2shRNA) into the RVLM. RESULTS: We found that SHRs exhibited higher levels of basal BP, HR, RSNA and proinflammatory cytokines (TNFα, IL-6 and IL-1ß) than those in WKY rats. The protein level of the CB2 receptor in the RVLM was robustly increased in SHRs. In addition, the CB2 receptor was mainly expressed on microglia cells of SHRs but not in WKY rats. No expression of the CB2 receptor was found on neurons of either WKY rats or SHRs. Furthermore, intracerebroventricular injection of JWH133 (1 mmol/l, 10 µl) for 28 days decreased the BP, HR, RSNA and proinflammatory cytokines significantly in SHRs, but it had no such effects in WKY rats. These effects were abolished by microinjection of 300 nl AAV2-r-CB2shRNA into the RVLM to knock down the CB2 receptor. CONCLUSION: Taken together, our results suggest that exciting the CB2 receptor relieves proinflammatory cytokine levels in the RVLM to decrease the BP, HR and RSNA in SHRs.

Protective effect of berberine on acute cardiomyopathy associated with doxorubicin treatment.

Doxorubicin (DOX) is a potent and broad-spectrum anthracycline chemotherapeutic agent, but dose-dependent cardiotoxic side effects limit its clinical application. This toxicity is closely associated with the generation of reactive oxygen species (ROS) radical during DOX metabolism. The present study investigated the effects of Berberine (Ber) on DOX-induced acute cardiac injury in a rat model and analysed its mechanism in cardiomyocytes in vitro. Serum creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and malondialdehyde (MDA) levels were significantly increased in the DOX group compared with the control group. This increase was accompanied by cardiac histopathological injury and a decrease in cardiomyocyte superoxide dismutase (SOD) and catalase (CAT). CK, CK-MB and MDA levels decreased and SOD and CAT levels increased in the Ber-treated group compared to the DOX group. Ber ameliorated the DOX-induced increase in cytosolic calcium concentration ([Ca2+]i), attenuated mitochondrial Ca2+ overload and restored the DOX-induced loss of mitochondrial membrane potential in vitro. These results demonstrated that Ber exhibited protective effects against DOX-induced heart tissue free radical injury, potentially via the inhibition of intracellular Ca2+ elevation and attenuation of mitochondrial dysfunction.

The regulation of FOXO1 and its role in disease progression.

Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic dysregulation are the basis of many diseases. Forkhead box transcription factor O1 (FOXO1) changes in response to cellular stimulation and maintains tissue homeostasis during the above-mentioned physiological and pathological processes. Substantial evidences indicate that FOXO1's function depends on the modulation of downstream targets such as apoptosis- and autophagy-associated genes, anti-oxidative stress enzymes, cell cycle arrest genes, and metabolic and immune regulators. In addition, oxidative stress, high glucose and other stimulations induce the regulation of FOXO1 activity via PI3k-Akt, JNK, CBP, Sirtuins, ubiquitin E3 ligases, etc., which mediate multiple signalling pathways. Subsequent post-transcriptional modifications, including phosphorylation, ubiquitination, acetylation, deacetylation, arginine methylation and O-GlcNAcylation, activate or inhibit FOXO1. The regulation of FOXO1 and its role might provide a significant avenue for the prevention and treatment of diseases. However, the subtle mechanisms of the post-transcriptional modifications and the effect of FOXO1 remain elusive and even conflicting in the development of many diseases. The determination of these questions potentially has implications for further research regarding FOXO1 signalling and the identification of targeted drugs.

Tert-butylhydroquinone ameliorates doxorubicin-induced cardiotoxicity by activating Nrf2 and inducing the expression of its target genes.

Oxidative stress plays an important role in doxorubicin (DOX)-induced cardiotoxicity. Nuclear factor E2-related factor-2 (Nrf2) is a transcription factor that orchestrates the antioxidant and cytoprotective responses to oxidative stress. In the present study, we tested whether tert-butylhydroquinone (tBHQ) could protect against DOX-induced cardiotoxicity in vivo and, if so, whether the protection was associated with the up-regulation of the Nrf2 pathway. The results showed that treatment with tBHQ significantly decreased the DOX-induced cardiac injury in wild-type mice. Moreover, tBHQ ameliorated the DOX-induced oxidative stress and apoptosis. Further studies suggested that tBHQ increased the nuclear accumulation of Nrf2 and the Nrf2-regulated gene expression, including heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxido-reductase-1 (NQO-1) expression. Knocking out Nrf2 in mice abolished the protective effect of tBHQ on the DOX-induced cardiotoxicity. These results indicate that tBHQ has a beneficial effect on DOX-induced cardiotoxicity, and this effect was associated with the enhanced expression of Nrf2 and its downstream antioxidant genes, HO-1 and NQO-1.

Mechanisms of isoform-specific Na/K pump regulation by short- and long-term adrenergic activation in rat ventricular myocytes.

BACKGROUND: Many stressful conditions, including cardiovascular diseases, induce long-term elevations in circulating catecholamines, thereby leading to changes of the Na/K pump and thus affecting myocardial functions. However, only short-term adrenergic regulation of the Na/K pump has been reported. The present study is the first investigation of long-term adrenergic regulation of the Na/K pump and the potential mechanism. METHODS: After acutely isolated Sprague-Dawley rat myocytes were incubated with noradrenaline or isoprenaline for 24 h, Na/K pump high- (IPH) and low-affinity current (IPL), α-isoform mRNA, and α-isoform protein were examined using patch-clamp, RT-PCR, and Western blotting techniques, respectively. RESULTS: After the short-term incubation, isoprenaline reduced the IPL through a PKA-dependent pathway that involves α1-isoform translocation from the membrane to early endosomes, and noradrenaline increased the IPH through a PKC-dependent pathway that involves α2-isoform translocation from late endosomes to the membrane. After long-term incubation, isoprenaline increased the IPL, α1-isoform mRNA, and α1-isoform protein, and noradrenaline reduced the IPH, α2-isoform mRNA, and α1-isoform protein through a PKA-or PKC-dependent pathway, respectively. CONCLUSIONS: These results suggest that long-term adrenergic Na/K pump regulation is isoform-specific and negatively feeds back on the short-term response. Furthermore, long-term regulation involves transcription and translation of the respective α-isoform, whereas short-term regulation involves the translocation of the available α-isoform to the plasma membrane.

Mitochondrial permeability transition pore plays a role in the cardioprotection of CB2 receptor against ischemia-reperfusion injury.

The aim of this study was to investigate whether the mitochondrial permeability transition pore (MPTP) opening was involved in the protective effects of CB2 receptor against ischemia-reperfusion (I-R) injury. For this, isolated perfused rat hearts were subjected to 30 min global ischemia followed by 120 min reperfusion, and left ventricle function was recorded. At the end of reperfusion, the infarct size in the hearts was measured by staining with triphenyltetrazolium chloride. MPTP opening and the mitochondrial membrane potential (ΔΨ(m)) were measured by flow cytometry. Western blot analysis of cytochrome c in the mitochondrion and cytosol, as well as ERK1/2 and p-ERK1/2 were performed. Administration of CB2 receptor agonist JWH133 before ischemia significantly improved the recovery of cardiac ventricular function during reperfusion, increased coronary flow, reduced infarct size, prevented the loss of ΔΨ(m) and MPTP opening, reduced the release of cytochrome c from mitochondria, and increased levels of p-ERK1/2. These effects of JWH133 were abolished by pretreatment with CB2 receptor antagonist AM630, or ERK1/2 inhibitor PD98059. Furthermore, JWH133 reversed the MPTP opening induced by atractyloside. The protective effect of JWH133 on the heart against I-R injury may be through increased ERK1/2 phosphorylation, inhibiting MPTP opening.

The protective effect of grape seed procyanidin extract against cadmium-induced renal oxidative damage in mice.

As an important environmental pollutant, cadmium (Cd) can lead to serious renal damage. Grape seed procyanidins extract (GSPE), a biological active component of grape seed, has been shown to possess antioxidative effects. Here, we assessed the protective effect of GSPE on Cd-induced renal damage using animal experiment. After 30 days, the oxidative damage of kidney was evaluated through measurement of superoxide dismutase (SOD), glutathione peroxidation (GSH-Px) and malondialdehyde (MDA). Since, oxidative stress could lead to apoptosis, the renal apoptosis was measured using flow cytometer. Moreover, the expression of apoptosis-related protein Bax and Bcl-2 was analyzed by immunohistochemistry and Western blot. The results showed that Cd led to the decrease of SOD and GSH-Px activities, and the increase of MDA level, induced renal apoptosis. However, the coadministration of GSPE attenuated Cd-induced lipid peroxidation, and antagonized renal apoptosis, probably associated with the expression of Bax and Bcl-2. These data suggested that GSPE has protective effect against renal oxidative damage induced by Cd, which provide a potential natural chemopreventive agent against Cd-poisoning.

[Protective effect of intermittent hypobaric hypoxia on cardiomyocytes injury induced by hydrogen peroxide].

OBJECTIVE: To observe the protective effect and mechanism of intermittent hypobaric hypoxia (IHH) on cardiomyocytes induced by hydrogen dioxide. METHODS: Male guinea pigs were divided randomly into two groups (n = 10): intermittent hypoxia group (IHH), and control group (non-IHH). The IHH guinea pigs were exposed to a simulated 5,000 m high altitude and hypoxia in hypobaric chamber for 28 d, 6 h/d. The control guinea pigs were kept in the same environment as IHH except hypoxia exposure. Cardiomyocytes were enzymatically isolated from left ventricle of non-CIHH or CIHH guinea pigs. The contractile was assessed in guinea pigs by a video-based motion edge-detection system. The contents and activities of malondialdehyde(MDA), lactate hydrogenase(LDH) and antioxidant enzymes were evaluated by using biochemical methods. RESULTS: 1. Hydrogen peroxide could induce contractile and diastole dysfunction, the latent period was longer in IHH cardiac myocytes. 2. After hydrogen peroxide(300 micromol/L, 10 min) perfusion, LDH and MDA contents in supernatant increased significantly in non-IHH and CIHH cardiomyocytes (P < 0.01), Whereas the contents of MDA and LDH in IHH cardiomyocytes were lower than those in non-IHH cardiomyocytes (P < 0.01). 3. The activities of superoxide dismutase (SOD) and catalase (CAT) were significantly increased in the myocardium of IHH guinea pigs, after hydrogen peroxide (300 micromol/L, 10 min) perfusion, SOD and CAT activities decreased significantly in non-IHH and CIHH cardiomyocytes (P < 0.01), whereas the activities of SOD and CAT in CIHH cardiomyocytes were still higher than those in non-IHH cardiomyocytes. CONCLUSION: IHH had a protective effect on cardiomyocytes injury induced by hydrogen peroxide, which might relate with its antioxidation effects.

The H1-H2 domain of the α1 isoform of Na+-K+-ATPase is involved in ouabain toxicity in rat ventricular myocytes.

The composition of different isoforms of Na+-K+-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H1-H2 domain of α1 and α2 isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α1 or α2 isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca²âº](i)), attenuated mitochondrial Ca²âº overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 µM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca²âº](i) and the contractility induced by 1 µM but not that induced by 1 mM OUA. These results indicate that the H1-H2 domain of the NKA α1 isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease.

Serotonin-mediated modulation of Na+/K+ pump current in rat hippocampal CA1 pyramidal neurons.

BACKGROUND: The aim of this study was to investigate whether serotonin (5-hydroxytryptamine, 5-HT) can modulate Na+/K+ pump in rat hippocampal CA1 pyramidal neurons. RESULTS: 5-HT (0.1, 1 mM) showed Na+/K+ pump current (Ip) densities of 0.40 ± 0.04, 0.34 ± 0.03 pA/pF contrast to 0.63 ± 0.04 pA/pF of the control of 0.5 mM strophanthidin (Str), demonstrating 5-HT-induced inhibition of Ip in a dose-dependent manner in hippocampal CA1 pyramidal neurons. The effect was partly attenuated by ondasetron, a 5-HT3 receptor (5-HT3R) antagonist, not by WAY100635, a 5-HT1AR antagonist, while 1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG), a 5-HT3R specific agonist, mimicked the effect of 5-HT on Ip. CONCLUSION: 5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in rat hippocampal CA1 pyramidal neurons. This discloses novel mechanisms for the function of 5-HT in learning and memory, which may be a useful target to benefit these patients with cognitive disorder.

[The effects of the cadmium chloride on the DNA damage and the expression level of gadd gene in HepG2 cell line].

OBJECTIVE: To investigate the effects of the cadmium chloride on the DNA damage and the expression of the gadd153 and gadd45beta promoter and mRNA in HepG2 cells. METHODS: DNA damage induced by cadmium chloride was detected by comet assay. The plasmids (pGADD153-Luc and pG45-Luc) containing DNA damage and repair inducible gene 153 and 45 (gadd153 and gadd45beta) promoter and luciferase and gadd45beta reporter gene were constructed. The activity of gadd153 and gadd45beta promoter were represented by the luciferase activity, the inducible luciferase activities was detected by bioluminescence. The expression of gadd153 and gadd45beta mRNA was detected by RT-PCR. RESULTS: The results of comet assay indicated that Olive Tail Moment induced by the cadmium chloride increased significantly at the dose of 100, 300 micromol/L, compared with the control (P < 0.05). The luciferase activity analysis showed that the expression levels of gadd153 promoter increased significantly in 1, 5, 10 micromol/L treatment group, compared with the control (P < 0.05). The expression levels of gadd45beta promoter in 5, 10 micromol/L treatment group were significantly higher than that in control group (P < 0.05). The expression levels of gadd153 mRNA induced by cadmium chloride at the doses of 1, 5, 10 micromol/L and the expression levels of gadd45beta mRNA induced at the doses of 5, 10 micromol/L were significantly higher than thoae in control group (P < 0.05). CONCLUSION: The cadmium chloride can induce the DNA damage and increase the expression levels of the gadd153 and gadd45beta promoters in HepG2 cells.

Enhancement of Na/K pump activity by chronic intermittent hypobaric hypoxia protected against reperfusion injury.

Chronic intermittent hypobaric hypoxia (CIHH) has been shown to attenuate intracellular Na(+) accumulation and Ca(2+) overload during ischemia and reperfusion (I/R), both of which are closely related to the outcome of myocardial damage. Na/K pump plays an essential role in maintaining the equilibrium of intracellular Na(+) and Ca(2+) during I/R. It has been shown that enhancement of Na/K pump activity by ischemic preconditioning may be involved in the cardiac protection. Therefore, we tested whether Na/K pump was involved in the cardioprotection by CIHH. We found that Na/K pump current in cardiac myocytes of guinea pigs exposed to CIHH increased 1.45-fold. The K(1) and f(1), which reflect the portion of α(1)-isoform of Na/K pump, dramatically decreased or increased, respectively, in CIHH myocytes. Western blot analysis revealed that CIHH increased the protein expression of the α(1)-isoform by 76%, whereas the protein expression of the α(2)-isoform was not changed significantly. Na/K pump current was significantly suppressed in simulated I/R, and CIHH preserved the Na/K pump current. CIHH significantly improved the recovery of cell length and contraction during reperfusion. Furthermore, inhibition of Na/K pump by ouabain attenuated the protective effect afforded by CIHH. Collectively, these data suggest that the increase of Na/K pump activity following CIHH is due to the upregulating α(1)-isoform of Na/K pump, which may be one of the mechanisms of CIHH against I/R-induced injury.

Chronic intermittent hypobaric hypoxia protects the heart against ischemia/reperfusion injury through upregulation of antioxidant enzymes in adult guinea pigs.

AIM: To investigate the protection and the anti-oxidative mechanism afforded by chronic intermittent hypobaric hypoxia (CIHH) against ischemia/reperfusion (I/R) injury in guinea pig hearts. METHODS: Adult male guinea pigs were exposed to CIHH by mimicking a 5000 m high altitude (p(B)=404 mmHg, p(O2)=84 mmHg) in a hypobaric chamber for 6 h/day for 28 days. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia and the reperfusion period. The activity and protein expression of antioxidant enzymes in the left myocardium were evaluated using biochemical methods and Western blotting, respectively. Intracellular reactive oxygen species (ROS) were assessed using ROS-sensitive fluorescence. RESULTS: After 30 min of global no-flow ischemia followed by 60 min of reperfusion, myocardial function had better recovery rates in CIHH guinea pig hearts than in control hearts. The activity and protein expression of superoxide dismutase (SOD) and catalase (CAT) were significantly increased in the myocardium of CIHH guinea pigs. Pretreatment of control hearts with an antioxidant mixture containing SOD and CAT exerted cardioprotective effects similar to CIHH. The irreversible CAT inhibitor aminotriazole (ATZ) abolished the cardioprotection of CIHH. Cardiac contractile dysfunction and oxidative stress induced by exogenous hydrogen peroxide (H(2)O(2)) were attenuated by CIHH and CAT. CONCLUSIONS: These data suggest that CIHH protects the heart against I/R injury through upregulation of antioxidant enzymes in guinea pig.Acta Pharmacologica Sinica (2009) 30: 947-955; doi: 10.1038/aps.2009.57; published online 22 June 2009.

Isoform-specific regulation of the Na+ -K+ pump by adenosine in guinea pig ventricular myocytes.

AIM: The present study investigated the effect of adenosine on Na(+)-K(+) pumps in acutely isolated guinea pig (Cavia sp.) ventricular myocytes. METHODS: The whole-cell, patch-clamp technique was used to record the Na(+)-K(+) pump current (I(p)) in acutely isolated guinea pig ventricular myocytes. RESULTS: Adenosine inhibited the high DHO-affinity pump current (I(h)) in a concentration-dependent manner, which was blocked by the selective adenosine A(1) receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists staurosporine, GF 109203X or the specific delta isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A(1) receptor agonist CCPA and a specific activator peptide of PKC-delta, PP114. In contrast, the selective A(2A) receptor agonist CGS21680 and A(3) receptor agonist Cl-IB-MECA did not affect I(h). Application of the selective A(2A) receptor antagonist SCH58261 and A(3) receptor antagonist MRS1191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced I(h) inhibition. CONCLUSION: The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of I(h) via adenosine A(1) receptors and the PKC-delta isoform.