OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with HO, gastrodin, gastrodin+HO, cyclosporin A (CsA), or CsA+gas+HO group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in HO-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in HO-exposed cells. CONCLUSIONS Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.
Adenosine Triphosphate ; analysis ; Apoptosis ; drug effects ; Benzyl Alcohols ; antagonists & inhibitors ; pharmacology ; Caspase 3 ; analysis ; Cell Line ; Cell Survival ; drug effects ; Cyclosporine ; pharmacology ; Cytochromes c ; analysis ; Glucosides ; antagonists & inhibitors ; pharmacology ; Humans ; Hydrogen Peroxide ; antagonists & inhibitors ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

To discuss the protective effect of aralosides (AS) on I/R-induced rat myocardial injury. The adult rat ventricular myocyte ischemia model was established through perfusion with sodium lactate perfusate and reperfusion with Ca(2+) -containing Tyrode's solution simulation. The cell contraction and ion concentration synchronization determination system was applied to detect the effect of AS on single I/R cell contraction and Ca2+ transients. According to the findings, AS could increase resting sarcomere length, contraction amplitude, ± dL/dt(max), calcium transient amplitude and speed of post-reperfusion myocardial cells (P < 0.05, P < 0.01), and decrease in time for achieving 90.0% of maximum relaxation, time for achieving peak value, resting calcium ratio, contraction period [Ca2+] i, time for achieving 50.0% of maximum relaxation and attenuation rate of intracellular calcium transient (P < 0.05, P < 0.01). Therefore, it is suggested that AS improved the post-reperfusion cell contraction and injury of calcium homeostasis.
Animals ; Aralia ; chemistry ; Biological Transport ; drug effects ; Calcium ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Muscle Contraction ; drug effects ; Myocardial Ischemia ; drug therapy ; metabolism ; physiopathology ; surgery ; Myocardial Reperfusion ; Myocytes, Cardiac ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Saponins ; administration & dosage

To study the antiarrhythmic effect of the newly developed alpha/beta-blocker TJ0711, a variety of animal models of arrhythmia were induced by CaCl2, ouabain and ischemia/reperfusion. Glass microelectrode technique was used to observe action potentials of right ventricular papillary muscle of guinea pig. The onset time of arrhythmia induced by CaCl2 was significantly prolonged by TJ0711 at 0.75, 1.5 and 3 mg x kg(-1) doses. TJ0711 (1.5 and 3 mg x kg(-1)) can significantly shorten the ventricular tachycardia (VT) and ventricular fibrillation (VF) duration, the incidence of VF and mortality were significantly reduced. On ischemia-reperfusion-induced arrhythmic model, TJ0711 (0.25, 0.5, 1 and 2 mg x kg(-1)) can significantly reduce the ventricular premature contraction (PVC), VT, VF incidence, mortality, arrhythmia score with a dose-dependent manner. At the same time, rats serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities decreased significantly by TJ0711 (1 and 2 mg x kg(-1)). Ouabain could cause arrhythmia in guinea pigs, when TJ0711 (0.375, 0.75, 1.5 and 3 mg x kg(-1)) was given, the doses of ouabain inducing a variety of arrhythmia PVC, VT, VF, cardiac arrest (CA) were significantly increased with a dose-dependent manner. In the TJ0711 0.1-30 micromol x L(-1) concentration range, guinea pig right ventricular papillary muscle action potential RP (rest potential), APA (action potential amplitude) and V(max) (maximum velocity of depolarization) were not significantly affected. APD20, APD50 and APD90 had a shortening trend but no statistical difference with the increase of TJ0711 concentration. TJ0711 has antiarrhythmic effect on the sympathetic nerve excitement and myocardial cell high calcium animal arrhythmia model. Myocardial action potential zero phase conduction velocity and resting membrane potential were not inhibited by TJ0711. APD20, APD50 and APD90 were shortened by TJ0711 at high concentration. Its antiarrhythmic action mechanism may be besides the action of blocking beta1 receptor, may also have a strong selective blocking action on alpha1 receptor and reducing intracellular calcium concentration.
Action Potentials ; drug effects ; Adrenergic alpha-Antagonists ; administration & dosage ; pharmacology ; Adrenergic beta-Antagonists ; administration & dosage ; pharmacology ; Animals ; Anti-Arrhythmia Agents ; administration & dosage ; pharmacology ; Arrhythmias, Cardiac ; blood ; chemically induced ; etiology ; pathology ; physiopathology ; Calcium Chloride ; Creatine Kinase ; blood ; Dose-Response Relationship, Drug ; Female ; Guinea Pigs ; Heart Ventricles ; cytology ; Lactate Dehydrogenases ; blood ; Male ; Myocardial Reperfusion Injury ; complications ; Myocytes, Cardiac ; drug effects ; physiology ; Ouabain ; Papillary Muscles ; cytology ; Phenoxypropanolamines ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

This study examined the effect of cholic acid (CA) on cultured cardiac myocytes (CMs) from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intrahepatic cholestasis of pregnancy (ICP). Inverted microscopy was performed to detect the impact of CA on the beating rates of rat CMs. MTT method was used to study the effect of CA on the viability of CMs. CMs cultured in vitro were incubated with 10 μmol/L Ca(2+)-sensitive fluorescence indicator fluo-3/AM. The fluorescence signals of free calcium induced by CA were measured under a laser scanning confocal microscope. The results showed that CA decreased the beating rates of the CMs in a dose-dependent manner. CA could suppress the activities of CMs in a time- and dose-dependent manner. CA increased the concentration of intracellular free calcium in a dose-dependent manner. Our study suggested that CA could inhibit the activity of CMs by causing calcium overload, thereby leading to the sudden fetal death in ICP.
Animals ; Animals, Newborn ; Calcium ; metabolism ; Cells, Cultured ; Cholestasis, Intrahepatic ; complications ; metabolism ; physiopathology ; Cholic Acid ; metabolism ; pharmacology ; Death, Sudden ; etiology ; Dose-Response Relationship, Drug ; Female ; Fetal Death ; etiology ; Humans ; Microscopy, Confocal ; Myocardial Contraction ; drug effects ; Myocytes, Cardiac ; drug effects ; metabolism ; physiology ; Pregnancy ; Pregnancy Complications ; metabolism ; physiopathology ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo evaluate whether Shenfu injection (SFI) protects against cardiac myocyte injury induced by Fupian injection (FPI) in vitro.

METHODSH9c2 cells were separately treated with FPI, Renshen injection (RSI) and SFI. Cell viability, lactate dehydrogenase (LDH) release, spontaneous beating rate of primative cardical cells, caspase-3/7 activity, cell apoptosis, and cytochrome P450 2J3 (CYP2J3) mRNA expression were analyzed.

RESULTSThe viability of H9c2 cells treated with SFI (37 and 75 mg/mL) was significantly higher than that of H9c2 cells treated with FPI (25 and 50 mg/mL) (P<0.05, P<0.01, respectively). LDH activity of H9c2 cells treated with SFI (75 mg/mL) was significantly decreased (P<0.01) compared with that of H9c2 cells treated with FPI (50 mg/mL). SFI (150 mg/mL) significantly attenuated FPI (100 mg/mL)-induced spontaneous beating rate decrease in primary myocardial cells after 4-hour treatment. Compared with FPI (12 and 25 mg/mL), SFI (18 and 37 mg/mL) treatment could effectively reverse the change of caspase-3/7 activity (P<0.01 and P<0.01, respectively). Compared with FPI (6 and 25 mg/mL), apoptotic cells decreased significantly (P<0.05, P<0.01, respectively) when H9c2 cells were incubated with SFI (9 and 37 mg/mL). The expression of CYP2J3 mRNA was down-regulated by FPI, while RSI and SFI could up-regulate the expression of CYP2J3 (P<0.01), which suggested the potential mechanism of protection of RSI against cardiac myocyte damage induced by FPI treatment.

CONCLUSIONThese observations indicate that SFI has the potential to exert cardioprotective effects against FPI toxicity. The effect was possibly correlated with the activation of CYP2J3.

Animals ; Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Drugs, Chinese Herbal ; pharmacology ; L-Lactate Dehydrogenase ; secretion ; Myocytes, Cardiac ; drug effects ; enzymology ; Rats

OBJECTIVETo study the effects of erythropoietin (EPO) on cardiomyocyte apoptosis and endoplasmic reticulum stress (ERS)-related proteins, glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), in neonatal rats with asphyxia.

METHODSA total of 120 newborn Sprague-Dawley rats (7 days old) were randomly divided into sham-operated (n=40), asphyxia (n=40) and EPO-treated asphyxia groups (n=40). A neonatal rat model of normobaric asphyxia was established in the asphyxia and EPO-treated asphyxia groups. The rats in the EPO-treated asphyxia group received intraperitoneal injection of recombinant human erythropoietin (500 U/mL) immediately after the model was established, while the other two groups received the same volume of normal saline (0.9%). Heart blood and myocardial tissue samples were collected from 8 rats in each group at 2, 6, 12, 24 or 48 hours after the model was established. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels were measured; cardiomyocyte apoptosis was evaluated, and expression of myocardial GRP78 and CHOP was measured.

RESULTSCompared with the sham-operated and EPO-treated asphyxia groups, the asphyxia group had significantly increased serum CK and LDH levels, number of apoptotic cells, and expression of myocardial GRP78 and CHOP at each time point (P<0.01), and all the indices were significantly higher in the EPO-treated asphyxia group than in the sham-operated group (P<0.01). At 24 hours after asphyxia, the expression of myocardial CHOP was positively correlated with the myocardial apoptosis index (r=0.944, P<0.01).

CONCLUSIONSEPO exerts a protective effect on the myocardium of neonatal rats with hypoxic-ischemic injury by regulating ERS-related proteins GRP78 and CHOP and reducing cardiomyocyte apoptosis.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Asphyxia Neonatorum ; drug therapy ; pathology ; Creatine Kinase ; blood ; Endoplasmic Reticulum Stress ; physiology ; Erythropoietin ; pharmacology ; Heat-Shock Proteins ; analysis ; L-Lactate Dehydrogenase ; blood ; Myocytes, Cardiac ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Transcription Factor CHOP ; analysis

Action Potentials ; drug effects ; physiology ; Animals ; Cadmium ; toxicity ; Calcium Channels, L-Type ; physiology ; Myocytes, Cardiac ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism. METHODS: P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined. RESULTS: P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells. CONCLUSION Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.
Animals ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; Dimethyl Sulfoxide ; pharmacology ; Embryonal Carcinoma Stem Cells ; pathology ; Hedgehog Proteins ; metabolism ; Hepatocyte Nuclear Factor 3-beta ; physiology ; Homeodomain Proteins ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; Nanog Homeobox Protein ; Proteins ; metabolism ; Transfection