TRPC6 promotes daunorubicin-induced mitochondrial fission and cell death in rat cardiomyocytes with the involvement of ERK1/2-DRP1 activation.

Daunorubicin (DNR-) induced cardiotoxicity seriously restricts its clinical application. Transient receptor potential cation channel subfamily C member 6 (TRPC6) is involved in multiple cardiovascular physiological and pathophysiological processes. However, the role of TRPC6 anthracycline-induced cardiotoxicity (AIC) remains unclear. Mitochondrial fragmentation greatly promotes AIC. TRPC6-mediated ERK1/2 activation has been shown to favor mitochondrial fission in dentate granule cells. The aim of the present study was to elucidate the effects of TRPC6 on daunorubicin- induced cardiotoxicity and identify the mechanisms associated with mitochondrial dynamics. The sparkling results showed that TRPC6 was upregulated in models in vitro and in vivo. TRPC6 knockdown protected cardiomyocytes from DNR-induced cell apoptosis and death. DNR largely facilitated mitochondrial fission, boosted mitochondrial membrane potential collapse and damaged debilitated mitochondrial respiratory function in H9c2 cells，these effects were accompanied by TRPC6 upregulation. siTRPC6 effectively inhibited these mitochondrial adverse aspects showing a positive unexposed effect on mitochondrial morphology and function. Concomitantly, ERK1/2-DRP1 which is related to mitochondrial fission was significantly activated with amplified phosphorylated forms in DNR-treated H9c2 cells. siTRPC6 effectively suppressed ERK1/2-DPR1 over activation, hinting at a potential correlation between TRPC6 and ERK1/2-DRP1 by which mitochondrial dynamics are possibly modulated in AIC. TRPC6 knockdown also raised the Bcl-2/Bax ratio, which may help to block mitochondrial fragmentation-related functional impairment and apoptotic signaling. These findings suggested an essential role of TRPC6 in AIC by intensifying mitochondrial fission and cell death via ERK1/2-DPR1, which could be a potential therapeutic target for AIC.

Preventive treatment with ginsenoside Rb1 ameliorates monocrotaline-induced pulmonary arterial hypertension in rats and involves store-operated calcium entry inhibition.

CONTEXT: Ginsenoside Rb1, the main active ingredient of ginseng, exhibits ex vivo depression of store-operated calcium entry (SOCE) and related vasoconstriction in pulmonary arteries derived from pulmonary hypertension (PH) rats. However, the in vivo effects of ginsenoside Rb1 on PH remain unclear. OBJECTIVE: This study explored the possibility of using ginsenoside Rb1 as an in vivo preventive medication for type I PH, i.e., pulmonary arterial hypertension (PAH), and potential mechanisms involving SOCE. MATERIALS AND METHODS: Male Sprague-Dawley rats (170-180 g) were randomly divided into Control, MCT, and MCT + Rb1 groups (n = 20). Control rats received only saline injection. Rats in the MCT + Rb1 and MCT groups were intraperitoneally administered single doses of 50 mg/kg monocrotaline (MCT) combined with 30 mg/kg/day ginsenoside Rb1 or equivalent volumes of saline for 21 consecutive days. Subsequently, comprehensive parameters related to SOCE, vascular tone, histological changes and hemodynamics were measured. RESULTS: Ginsenoside Rb1 reduced MCT-induced STIM1, TRPC1, and TRPC4 expression by 35.00, 31.96, and 32.24%, respectively, at the protein level. SOCE-related calcium entry and pulmonary artery contraction decreased by 162.6 nM and 71.72%. The mean pulmonary artery pressure, right ventricle systolic pressure, and right ventricular mass index decreased by 19.5 mmHg, 21.6 mmHg, and 39.50%. The wall thickness/radius ratios decreased by 14.67 and 17.65%, and the lumen area/total area ratios increased by 18.55 and 15.60% in intrapulmonary vessels with 51-100 and 101-150 µm o.d. CONCLUSION: Ginsenoside Rb1, a promising candidate for PH prevention, inhibited SOCE and related pulmonary vasoconstriction, and relieved MCT-induced PAH in rats.

Calcineurin/NFAT Signaling Modulates Pulmonary Artery Smooth Muscle Cell Proliferation, Migration and Apoptosis in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats.

BACKGROUND/AIMS: Pulmonary arterial hypertension (PAH) is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, which is driven by excessive proliferation and migration and apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs). The calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway is the most important downstream signaling pathway of store-operated Ca2+ entry (SOCE), which is increased in PAH. CaN/NFAT has been reported to contribute to abnormal proliferation in chronic hypoxia (CH)-induced PAH. However, the effect of CaN/NFAT signaling on PASMC proliferation, migration and apoptosis in monocrotaline (MCT)-induced PAH remains unclear. METHODS: PAH rats were established by a single intraperitoneal injection of MCT for 21 days. PASMCs were isolated and cultured in normal and MCT-induced PAH Sprague-Dawley rat. PASMCs were treated with CsA targeting CaN and siRNA targeting NFATc2-4 gene respectively by liposome. We investigated the expression of calcineurin/NFAT signaling by immunofluorescence, qRT-PCR and Western blotting methods. Cell proliferation was monitored using MTS reagent or by assessing proliferating cell nuclear antigen (PCNA) expression. Cell apoptosis was evaluated with an Annexin V - FITC/propidium iodide (PI) apoptosis kit by flow cytometry. PASMC migration was assessed with a Transwell chamber. RESULTS: MCT successfully induced PAH and pulmonary vascular remodeling in rats. CaN phosphatase activity and nuclear translocation of NFATc2-4 were increased in PASMCs derived from MCT-treated rats. In addition, CaNBß/NFATc2-4 expression was amplified at the mRNA and protein levels. PASMC proliferation and migration were markedly inhibited in a dosedependent manner by cyclosporin A (CsA). Furthermore, siRNA targeting NFATc2 and NFATc4 attenuated the excessive proliferation and migration and apoptosis resistance in PASMCs derived from both CON and MCT-treated rats, while NFATc3 knockdown specifically affected MCT-PASMCs. CONCLUSION: Our results demonstrate that CaN/NFAT signaling is activated and involved in the modulation of PASMC proliferation, migration and apoptosis in MCT-induced PAH.

[Changes of colonic permeability and its correlation with TNF-α, NF-κB p65 in ulceration colitis mice].

OBJECTIVE: To study the changes of colonic permeability and its correlation with TNF-α, NF-κB p65 in indextran sulphate sodium (DSS) -induced ulcerative colitis(UC) of mice. METHODS: Forty-eight ICR mice were randomly divided into the control group and the model group. The acute UC model was induced by quantified intragastric administration of 2.5% DSS in mice. The disease activity index(DAI), histopathology scores, colonic permeability, expression of TNF-α, NF-κB p65 in colonic tissue were determined. The change of colonic permeability and its correlation with DAI, TNF-α, NF-κB p65 were analyzed. RESULTS: Compareded with the control group, DAI colonic permeability of colonic tissue,and the expression of TNF-α NF-κB p65 in the model group were increased significantly (P<0.01, P<0.01). The increased colonic permeability correlated with DAI (P<0.01), and the expression of TNF-α(P<0.01), NF-κB p65(P<0.01) changed significantly. CONCLUSIONS: The alteration of colonic permeability and increased expression of TNF-α, NF-κB p65 may play important roles in the occurrence and development of UC.

Sodium Ferulate Prevents Daunorubicin--Induced Apoptosis in H9c2 Cells via Inhibition of the ERKs Pathway.

BACKGROUND: Daunorubicin (DNR)-induced cardiotoxicity, which is closely associated with cardiomyocyte apoptosis, limits the drug's clinical application. The activation of the extracellular regulated protein kinases (ERKs) pathway is responsible for the pro-apoptosis effect of DNR Sodium ferulate (SF) has recently been found to attenuate both DNR-induced cardiotoxicity and mitochondrial apoptosis in juvenile rats. Nonetheless, the precise mechanism underlying SF-induced cardio-protection remains unclear. METHODS: The DNR-injured H9c2 cell model was prepared by incubating the cells in 1 µM DNR for 24 h. Amounts of 15.6, 31.3 or 62.5 µM SF were simultaneously added to the cells. The effect of SF on the cytotoxic and apoptotic parameters of the cells was studied by monitoring apoptosis regulation via the ERKs pathway. RESULTS: SF attenuated DNR-induced cell death (particularly apoptotic death), cTnI and ß-tubulin degradation, and cellular morphological changes. SF reduced mitochondrial membrane potential depolarization, cytochrome c leakage, and caspase-9 and caspase-3 activation. SF also decreased ERK1/2, phospho-ERK1/2, p53 and Bax expression and increased Bcl-2 expression. These effects were similar to the results observed when using the pharmacological ERKs phosphorylation inhibitor, AZD6244. CONCLUSION: We determined that SF protects H9c2 cells from DNR-induced apoptosis through a mechanism that involves the interruption of the ERKs signaling pathway.

Ginsenoside Rb1 attenuates agonist-induced contractile response via inhibition of store-operated calcium entry in pulmonary arteries of normal and pulmonary hypertensive rats.

BACKGROUND: Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, enhanced vasoreactivity and vascular remodeling, which leads to right heart failure and death. Despite several treatments are available, many forms of PH are still incurable. Ginsenoside Rb1, a principle active ingredient of Panax ginseng, exhibits multiple pharmacological effects on cardiovascular system, and suppresses monocrotaline (MCT)-induced right heart hypertrophy. However, its effect on the pulmonary vascular functions related to PH is unknown. METHODS: We examined the vasorelaxing effects of ginsenoside Rb1 on endothelin-1 (ET-1) induced contraction of pulmonary arteries (PAs) and store-operated Ca(2+) entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs) from chronic hypoxia (CH) and MCT-induced PH. RESULTS: Ginsenoside Rb1 elicited concentration-dependent relaxation of ET-1-induced PA contraction. The vasorelaxing effect was unaffected by nifedipine, but abolished by the SOCE blocker Gd(3+). Ginsenoside Rb1 suppressed cyclopiazonic acid (CPA)-induced PA contraction, and CPA-activated cation entry and Ca(2+) transient in PASMCs. ET-1 and CPA-induced contraction, and CPA-activated cation entry and Ca(2+) transients were enhanced in PA and PASMCs of CH and MCT-treated rats; the enhanced responses were abolished by ginsenoside Rb1. CONCLUSION: Ginsenoside Rb1 attenuates ET-1-induced contractile response via inhibition of SOCE, and it can effectively antagonize the enhanced pulmonary vasoreactivity in PH.

[Decreased amplitude of [Ca²âº]i elevation induced by menthol in pulmonary arterial smooth muscle cells of pulmonary hypertensive rats].

The study was designed to explore the alteration of intracellular calcium concentration ([Ca²âº]i), induced by transient receptor potential melastatin 8 (TRPM8) channel-specific agonist menthol, in pulmonary arterial smooth muscle cells (PASMCs) between control and pulmonary hypertensive (PH) rats. PH rat models were established by means of chronic hypoxia (CH) and monocrotaline (MCT) injection, respectively. PASMCs from control and PH rats were cultured. The change of [Ca²âº]i in PASMCs induced by menthol, and the effect of TRPM8 channel-specific antagonist BCTC on the change of [Ca²âº]i, were observed. Cellular localization of TRPM8 was examined by using immunohistochemistry. Results showed that menthol increased [Ca²âº]i in the control PASMCs both in Ca²âº -normal and Ca²âº - free Tyrode's solutions, and at the same time BCTC could inhibit these two kinds of elevations. Compared with the control group, elevations of [Ca²âº]i were decreased notably in CH- and MCT-pretreated PASMCs superfused with 2 mmol/L Ca²âº - or 0 Ca²âº -Tyrode's solutions. Immunohistochemical localization experiments showed that the whole PASMCs were dyed brown except for the nucleus. This study verified that TRPM8 exists both in membrane and sarcoplasmic reticulum of PASMCs. In addition, CH- and MCT-pretreatment could independently down-regulate the Ca²âº influx and Ca²âº release mediated by TRPM8 channel.

Sodium ferulate protects against daunorubicin-induced cardiotoxicity by inhibition of mitochondrial apoptosis in juvenile rats.

Daunorubicin (DNR) is a widely used chemotherapeutic agent; however, its clinical use is limited because of its cardiotoxicity. This study was aimed to investigate the protective effect of sodium ferulate (SF), an effective component from traditional Chinese herbs, against DNR-induced cardiotoxicity in juvenile rats. DNR was administered intraperitoneally to rats at the dosage of 2.5 mg·kg(-1)·wk(-1) for 5 consecutive weeks (cumulative dose of 12.5 mg/kg) or in combination with intraperitoneal injection of SF at 50 mg·kg(-1)·d(-1) over a period of 30 days. The animals were killed 6 days after the last injection of DNR. SF significantly ameliorated the DNR-induced cardiac dysfunction, structural damage of the myocardium, and release of lactate dehydrogenase and creatine kinase. Treatment with SF also reversed DNR-induced oxidative stress as evidenced by a decrease in malondialdehyde levels with a concomitant increase in myocardical superoxide dismutase activities. Furthermore, SF afforded significant cardioprotection against DNR-induced apoptosis in vivo and effectively suppressed the complex mitochondrion-dependent apoptotic signaling triggered by DNR. This study indicates that SF may improve cardiac function by inhibition of oxidative stress and apoptosis, thus providing a beneficial effect on the prevention of DNR-induced cardiotoxicity.

[The application of isoprenaline provocation test on rat model of alcoholic cardiomyopathy].

OBJECTIVE: To study a simple and reliable method to produce the rat model of alcoholic cardiomyopathy, evaluating the model with isoprenaline provocation test (IPT) and morphological indicators. METHODS: Adult male rats were intragastrically infused of wine (52% v/v) at 20 g/(kg x d) for 15 days with the general condition, the change of eating amount and weight being observed. The levels of mean left ventricle systolic pressure (mLVSP), mean left ventricle diastolic pressure (mLVDP), mean left ventricle pressure (mLVP), heart ratio (HR), maximal rise velocity of left ventricular pressure (+ dp/dtmax), maximal fall velocity of left ventricular pressure (- dp/dtmax) and the reaction of isoprenaline were examined by left ventricular cannulation, and the morphological change was observed with optical microscope and transmission electron microscopy. RESULTS: Both diastolic and systolic function of the model rats was lower than that of the control group as well as the cardiac energy reserve induced by IPT. Pathological observation demonstrated myocardial hypertrophy, myocardiocyte necrosis and infiltration of inflammatory cells. The transmission electron microscopy showed that mitochondria became enlarged or crimpled with fused or disappeared cristae and myofibrils dissolved and fractured. CONCLUSION: The adult male SD model rats exhibit diabetic cardiomyopathy by intragastric infusion of wine (52% v/v) at 20 g/(kg x d) for 15 days. IPT can induce the cardiac energy reserve and evaluate that accurately, displaying the hidden heart dysfunction.

Enhanced store-operated Ca²+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats.

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that canonical transient receptor potential (TRPC) genes are upregulated and store-operated Ca(2+) entry (SOCE) is augmented in PASMCs of chronic hypoxic rats and patients of pulmonary arterial hypertension (PAH). Here we further examine the involvement of TRPC and SOCE in PH with a widely used rat model of monocrotaline (MCT)-induced PAH. Rats developed severe PAH, right ventricular hypertrophy, and significant increase in store-operated TRPC1 and TRPC4 mRNA and protein in endothelium-denuded pulmonary arteries (PAs) 3 wk after MCT injection. Contraction of PA and Ca(2+) influx in PASMC evoked by store depletion using cyclopiazonic acid (CPA) were enhanced dramatically, consistent with augmented SOCE in the MCT-treated group. The time course of increase in CPA-induced contraction corresponded to that of TRPC1 expression. Endothelin-1 (ET-1)-induced vasoconstriction was also potentiated in PAs of MCT-treated rats. The response was partially inhibited by SOCE blockers, including Gd(3+), La(3+), and SKF-96365, as well as the general TRPC inhibitor BTP-2, suggesting that TRPC-dependent SOCE was involved. Moreover, the ET-1-induced contraction and Ca(2+) response in the MCT group were more susceptible to the inhibition caused by the various SOCE blockers. Hence, our study shows that MCT-induced PAH is associated with increased TRPC expression and SOCE, which are involved in the enhanced vascular reactivity to ET-1, and support the hypothesis that TRPC-dependent SOCE is an important pathway for the development of PH.

[The effect of different dose of verapamil and propranolol in kalium cardioplegia on the function of immature rat heart].

AIM: To explore the appropriate dose of the verapamil and propranolol in kalium cardiaplegia (KVP) by observation of the effect on the function of ischemic immature rat heart and compared with ST. Thomas II cardiaplegia. METHODS: 48 isolated hearts from Sprague-Dawley rats of 60 to approximately 80 g body weight, 22 +/- 2 days, male or female are perfused by Langendorff method for 20 min, and assigned to 1 of the following 6 groups (n = 8): control (CON), continuously perfused for 150 min. Ischemia/reperfusion (I/R), perfused with Locke's solution without glucose and oxygen equilibration for 3 min then no perfusion 27 min, repeated 3 cycles (ischemia for 90 min), followed by reperfusion for 60 min. Ischemia protected with ST. Thomas II cardioplegia (ST), each 3 min perfusion with ST. Thomas II cardioplegia during ischemia. Ischemia protected with three dose KVP cardioplegia (L, M, and H), perfused with ST. Thomas II cardioplegia containing verapamil and propranolol (x 10(-7) mol L(-1)) respectively 2.0, 0.34 (L), 6.8, 1.1 (M), 20,3.4 (H) during each 3 min perfusion of ischemia. Heart rate (min (-1), tens on(g), contraction force(g), peak systolic velocity (g.s-1), peak diastole velocity (g.s-), coronary flow (ml x min(-1 ), re-beat time (s) were monitored during the ischemia/ reperfusion. RESULTS: Compared to CON group, heart tension was rose when ischemia for 40 min and kept higher and could not rebeat after reperfusion in I/R group, In ST group, heart tension was rose after ischemia for 60 min and could re-beat but the pulse was weaker. Compared with ST group, KVP decreased the ischemic cardiac tension in dose dependently and the re-beat was stronger in L, M, and H groups. While compared with CON group, in L group, heart tension was rose when ischemia for 60 min and the re-beat was weaker. In H group, the heart tension was maintained lower when ischemia for 40 min and the re-beat was delay and weaker. Only in M group, heart tension was maintained stable during ischemia for 90 min and re-beat was stronger after reperfusion. CONCLUSION: Kalium cardiaplegia containing verapamil 6.8 x 10(-7) mol x L(-1) and propranolol 1.1 x 10(-7) mol x L(-1) has the best effect to protect the immature heart from ischemic injury.