In order to investigate the effects of aconitine on [Ca2+] oscillation patterns in cultured myocytes of neonatal rats, fluorescent Ca2+ indicator Fluo-4 NW and laser scanning confocal microscope (LSCM) were used to detect the real-time changes of [Ca2+] oscillation patterns in the cultured myocytes before and after aconitine (1.0 micromol/L) incubation or antiarrhythmic peptide (AAP) and aconitine co-incubation. The results showed under control conditions, [Ca2+] oscillations were irregular but relatively stable, occasionally accompanied by small calcium sparks. After incubation of the cultures with aconitine, high frequency [Ca2+] oscillations emerged in both nuclear and cytoplasmic regions, whereas typical calcium sparks disappeared and the average [Ca2+] in the cytoplasm of the cardiomyocyte did not change significantly. In AAP-treated cultures, intracellular [Ca2+] oscillation also changed, with periodic frequency, increased amplitudes and prolonged duration of calcium sparks. These patterns were not altered significantly by subsequent aconitine incubation. The basal value of [Ca2+] in nuclear region was higher than that in the cytoplasmic region. In the presence or absence of drugs, the [Ca2+] oscillated synchronously in both the nuclear and cytoplasmic regions of the same cardiomyocyte. It was concluded that although oscillating strenuously at high frequency, the average [Ca2+] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. The observations indicate that aconitine induces the changes in [Ca2+] oscillation frequency other than the Ca2+ overload.
Aconitine/*pharmacology ; Animals, Newborn ; Calcium Signaling/*drug effects ; Cells, Cultured ; Myocytes, Cardiac/cytology ; Myocytes, Cardiac/*metabolism ; Rats, Sprague-Dawley

AIMTo study the effect of Dehydrocorydaline and Verapamil (Ver) on intracellular free calcium concentration of myocardial cell ([Ca2+]i) under hypoxic condition.

METHODSWe adopted guinea-pig heart Langendorff instillation. The myocardial cells were isolated by collagenase (Type I, sigma)and marked by fluorescence ratio imaging. The suspension of myocardial cells was assigned to six groups: DHC, Ver, and control were each two. Each three groups was exposed to hypoxia and normoxia before determination of [Ca2+]i.

RESULTS(1) In normoxia state, [Ca2+]i was 120.5-8.3 nml/L (n = 20).( 2) In hypoxia state, the increased [Ca2+]i of myocardial cells was proportional to the time (degree) of hypoxia. Correlation coefficient (r) was about 0.98. (3) Under the condition of normoxia DHC and Ver decreased [Ca2+]i. (4) DHC was obviously slow the increase of [Ca2+]i after hypoxia.

CONCLUSIONIn normoxia and hypoxia, DHC decreases the increased [Ca2+]i. It can prevent intracellular calcium overload. We believe DHC may improve self-protected performance of myocardial cells.

Alkaloids ; pharmacology ; Animals ; Calcium ; metabolism ; Cell Hypoxia ; Female ; Guinea Pigs ; Male ; Myocytes, Cardiac ; drug effects ; metabolism

AIMTo study the effect of thyroid hormone on protein kinase C activity and isoprotein expressions in cardiac myocytes and fibroblasts of rats in vitro.

METHODSCardiac myocytes and fibroblasts were cultured according to the method of Simpson. Cells were pretreated with 1% newborn calf serum (NCS) or Angiotensin II (Ang II) for 24 hours, then Triiodothyronine (T3) was added to the culture medium and the culture was kept for another 48 hours. The protein kinase C activation were measured by PepTaga non-radioactive PKC assay, and the expressions of PKC alpha and PKC epsilon were detected by Western blot method.

RESULTSAt the condition of 1% NCS culture medium, T3 could inhibit PKC activity and PKC epsilon expression in cardiac myocytes significantly, but the expression of PKC alpha in cardiac myocytes was not influenced by T3. In cardiac fibroblasts, neither PKC activity nor PKC alpha and PKC epsilon expressions was influenced by T3. When cells were pretreated with Ang II for 24 hours, PKC activities in cardiac myocytes and fibroblasts were increased significantly, and PKC epsilon expressions in cardiac myocytes were also markedly increased. Following a T3 treatment, PKC activity and PKC epsilon expression in cardiac myocytes were markedly decreased, but PKC activity in cardiac fibroblasts was not changed.

CONCLUSIONWhether at the condition of 1% NCS medium or in a pretreatment with Ang II, thyroid hormone could inhibit the PKC activity and PKC epsilon expression in cardiac myocytes. The influence of thyroid hormone on the PKC signal pathway in cardiac myocyte may be involved in many pathophysiological progress of myocardium.

Animals ; Cells, Cultured ; Myoblasts, Cardiac ; drug effects ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thyroid Hormones ; pharmacology

OBJECTIVETo investigate the protective effects of glycyl-glutamine dipeptide supplement on the function of myocardial dynamics in severely burned rats, and to explore its mechanism.

METHODSOne hundred and thirty-six Wistar rats were randomly divided into five groups: i. e, control group (C, n = 8, without burns), burn group (B, n = 32), Gln group (Gln, n = 32), Gly group (Gly, n = 32) and Gly-Gln group (Gly-Gln, n = 32). The rats in the latter four groups were respectively treated with tyrosine (1.5 g x kg(-1) x d(-1)), glutamine (1.0 g x kg(-1) x d(-1)) and tyrosine (0.5 g x kg(-1) x d(-1)), glycine (0.5 g x kg(-1) x d(-1)) and tyrosine (1.0 g x kg(-1) x d(-1)), and Glycyl-glutamine dipeptide (1.5 g x kg(-1) x d(-1)) after receiving a 30% TBSA full-thickness burn on the back. Glutathione (GSH), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cell energy charge (EC) and the index of myocardial dynamics (ASOP, AODP, LVSP, + dp/dtmax) were measured at 12, 24, 48, 72 post-burn hours (PBH).

RESULTSThe content of GSH, ATP, EC and the level of aortic systolic pressure (ASOP), aortic diastolic blood pressure (AODP), left ventricular end diastolic pressure (LVEDP) and maximum rate of intraventricular pressure rise/down (+ dp/dtmax) in B, Gln, Gly, Gly-Gln groups were obviously lower than those in C group (P < 0.01), while the levels of AMP and ADP showed an opposite tendency. Compared with B group, the above indices were ameliorated. The content of GSH (72.7 +/- 1.7) micromol/g in Gly-Gln group at 12 PBH was obviously higher than that in Gln group (67.8 +/- 3.8) micromol/g (P < 0.01). The levels of EC and AOSP were obviously higher in Gly-Gln group than that in Gln group (P < 0.01). The level of GSH, EC, AOSP in Gly-Gln groups were obviously higher than those in Gly group at 48 PBH.

CONCLUSIONGlycyl-glutamine dipeptide, Gly and Gln supplementation after burns can improve the content of GSH and high energy phosphate compound, and suppress the decline of myocardial dynamics function. The effects of Glycyl-glutamine dipeptide is better than single Gly or Gln, indicating that the protective effect on myocardial function after severe burns by Gln and Gly is synergistic.

Animals ; Burns ; drug therapy ; metabolism ; Dipeptides ; pharmacology ; Glutathione ; metabolism ; Glycine ; Myocytes, Cardiac ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

BackgroundShensong Yangxin Capsule (SSYX), traditional Chinese medicine, has been used to treat arrhythmias, angina, cardiac remodeling, cardiac fibrosis, and so on, but its effect on cardiac energy metabolism is still not clear. The objective of this study was to investigate the effects of SSYX on myocardium energy metabolism in angiotensin (Ang) II-induced cardiac hypertrophy.

MethodsWe used 2 μl (10 mol/L) AngII to treat neonatal rat cardiomyocytes (NRCMs) for 48 h. Myocardial α-actinin staining showed that the myocardial cell volume increased. Expression of the cardiac hypertrophic marker-brain natriuretic peptide (BNP) messenger RNA (mRNA) also increased by real-time polymerase chain reaction (PCR). Therefore, it can be assumed that the model of hypertrophic cardiomyocytes was successfully constructed. Then, NRCMs were treated with 1 μl of different concentrations of SSYX (0.25, 0.5, and 1.0 μg/ml) for another 24 h. To explore the time-depend effect of SSYX on energy metabolism, 0.5 μg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h. Mitochondria was assessed by MitoTracker staining and confocal microscopy. mRNA and protein expression of mitochondrial biogenesis-related genes - Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), energy balance key factor - adenosine monophosphate-activated protein kinase (AMPK), fatty acids oxidation factor - carnitine palmitoyltransferase-1 (CPT-1), and glucose oxidation factor - glucose transporter- 4 (GLUT-4) were measured by PCR and Western blotting analysis.

ResultsWith the increase in the concentration of SSYX (from 0.25 to 1.0 μg/ml), an increased mitochondrial density in AngII-induced cardiomyocytes was found compared to that of those treated with AngII only (0.25 μg/ml, 18.3300 ± 0.8895 vs. 24.4900 ± 0.9041, t = 10.240, P < 0.0001; 0.5 μg/ml, 18.3300 ± 0.8895 vs. 25.9800 ± 0.8187, t = 12.710, P < 0.0001; and 1.0 μg/ml, 18.3300 ± 0.8895 vs. 24.2900 ± 1.3120, t = 9.902, P < 0.0001; n = 5 per dosage group). SSYX also increased the mRNA and protein expression of PGC-1α (0.25 μg/ml, 0.8892 ± 0.0848 vs. 1.0970 ± 0.0994, t = 4.319, P = 0.0013; 0.5 μg/ml, 0.8892 ± 0.0848 vs. 1.2330 ± 0.0564, t = 7.150, P < 0.0001; and 1.0 μg/ml, 0.8892 ± 0.0848 vs. 1.1640 ± 0.0755, t = 5.720, P < 0.0001; n = 5 per dosage group), AMPK (0.25 μg/ml, 0.8872 ± 0.0779 vs. 1.1500 ± 0.0507, t = 7.239, P < 0.0001; 0.5 μg/ml, 0.8872 ± 0.0779 vs. 1.2280 ± 0.0623, t = 9.379, P < 0.0001; and 1.0 μg/ml, 0.8872 ± 0.0779 vs. 1.3020 ± 0.0450, t = 11.400, P < 0.0001; n = 5 per dosage group), CPT-1 (1.0 μg/ml, 0.7348 ± 0.0594 vs. 0.9880 ± 0.0851, t = 4.994, P = 0.0007, n = 5), and GLUT-4 (0.5 μg/ml, 1.5640 ± 0.0599 vs. 1.7720 ± 0.0660, t = 3.783, P = 0.0117; 1.0 μg/ml, 1.5640 ± 0.0599 vs. 2.0490 ± 0.1280, t = 8.808, P < 0.0001; n = 5 per dosage group). The effect became more obvious with the increasing concentration of SSYX. When 0.5 μg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h, the expression of AMPK (6 h, 14.6100 ± 0.6205 vs. 16.5200 ± 0.7450, t = 3.456, P = 0.0250; 12 h, 14.6100 ± 0.6205 vs. 18.3200 ± 0.9965, t = 6.720, P < 0.0001; 24 h, 14.6100 ± 0.6205 vs. 21.8800 ± 0.8208, t = 13.160, P < 0.0001; and 48 h, 14.6100 ± 0.6205 vs. 23.7400 ± 1.0970, t = 16.530, P < 0.0001; n = 5 per dosage group), PGC-1α (12 h, 11.4700 ± 0.7252 vs. 16.9000 ± 1.0150, t = 7.910, P < 0.0001; 24 h, 11.4700 ± 0.7252 vs. 20.8800 ± 1.2340, t = 13.710, P < 0.0001; and 48 h, 11.4700 ± 0.7252 vs. 22.0300 ± 1.4180, t = 15.390; n = 5 per dosage group), CPT-1 (24 h, 15.1600 ± 1.0960 vs. 18.5800 ± 0.9049, t = 6.048, P < 0.0001, n = 5), and GLUT-4 (6 h, 10.2100 ± 0.9485 vs. 12.9700 ± 0.8221, t = 4.763, P = 0.0012; 12 h, 10.2100 ± 0.9485 vs. 16.9100 ± 0.8481, t = 11.590, P < 0.0001; 24 h, 10.2100 ± 0.9485 vs. 19.0900 ± 0.9797, t = 15.360, P < 0.0001; and 48 h, 10.2100 ± 0.9485 vs. 14.1900 ± 0.9611, t = 6.877, P < 0.0001; n = 5 per dosage group) mRNA and protein increased gradually with the prolongation of drug action time.

ConclusionsSSYX could increase myocardial energy metabolism in AngII-induced cardiac hypertrophy. Therefore, SSYX might be considered to be an alternative therapeutic remedy for myocardial hypertrophy.

Angiotensin II ; metabolism ; Animals ; Cardiomegaly ; drug therapy ; Energy Metabolism ; Medicine, Chinese Traditional ; Myocardium ; Myocytes, Cardiac ; drug effects ; Rats

OBJECTIVETo investigate the influence of microtubule intervention drugs on the energy metabolism of myocardial cells after hypoxia.

METHODSThe primary passage of cultured myocardial cells from neonatal rats were divided into A (with hypoxia), B (with hypoxia and administration of 10 micromol/ml colchicine), C (with hypoxia and administration of 5 micromol/ml taxol), D (with hypoxia and administration of 10 micromol/ml taxol) and E (with hypoxia and administration of 15 micromol/ml taxol) groups. The creatine kinase (CK) activity and contents of ATP and ADP were assayed with colorimetry and HPLC, respectively, and the vitality of myocardial cells were determined by trypan blue method at 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 post-hypoxia hours (PHH).

RESULTSThe mortality was obviously higher in B and E groups than those in A group( P < 0.05) at each time-points, but that in C and D groups were markedly lower than those in A group during 6.0 to 24.0 PHH (P < 0.01). The CK activity was significantly higher in B group than that in A group during 1.0 to 24.0 PHH, while that in E group was evidently higher, but it was lower in C and D groups than that in A group at each time-points (P < 0.05 or 0.01). The ATP contents in C group during 0.5 to 6.0 PHH were [(49.9 +/- 2.8), (40.7 +/- 2.0), (25.8 +/- 1.9), (19.1 +/- 1.2) microg/10(6) cells, respectively], which were obviously higher than those in A group [(42.9 +/- 5.8), (29.5 +/- 1.8), (18.2 +/- 0.9), (14.1 +/- 0.7) microg/10(6) cells, respectively, P < 0.05 or P < 0.01, and those in E group at each time-point were significantly lower than those in A and D groups (P < 0.01). The changes in the contents of ADP were on the contrary to the above.

CONCLUSIONMicrotubule-destabilizing drugs and high concentration microtubule-stabilizing drugs can sharply decrease ATP content in myocardiocytes under hypoxic conditions, while suitable amount of microtubule-stabilizing drugs can protect myocardiocytes by promoting its energy production.

Animals ; Cell Hypoxia ; Cells, Cultured ; Colchicine ; pharmacology ; Energy Metabolism ; drug effects ; Microtubules ; drug effects ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Paclitaxel ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the protective effect of reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), against H9c2 cardiomyocytes from injuries induced by chemical hypoxia.

METHODSH9c2 cells were treated with cobalt chloride (CoCl2), a chemical hypoxia-mimetic agent, to establish the chemical hypoxia-induced cardiomyocyte injury model. NAC was added into the cell medium 60 min prior to CoCl2 exposure. The cell viability was evaluated using cell counter kit (CCK-8), and the intercellular ROS level was measured by 2', 7'- dichlorfluorescein-diacetate (DCFH-DA) staining and photofluorography. Mitochondrial membrane potential (MMP) of the cells was observed by Rhodamine123 (Rh123) staining and photofluorography, and the ratio of GSSG/ (GSSG+GSH) was calculated according to detection results of the GSSG kit.

RESULTSExposure of H9c2 cardiomyocytes to 600 micromol/L CoCl2 for 36 h resulted in significantly reduced cell viability. Pretreatment with NAC at the concentrations ranging from 500 to 2000 micromol/L 60 min before CoCl2 exposure dose-dependently inhibited CoCl2-induced H9c2 cell injuries, and obviously increased the cell viability. NAC at 2000 micromol/L obviously inhibited the oxidative stress induced by CoCl2, decreased the ratio of GSSG/(GSSG+GSH), increased ROS level, and antagonized CoCl2-induced inhibition on MMP.

CONCLUSIONNAC offers obvious protective effect on H9c2 cardiomyocytes against injuries induced by chemical hypoxia by decreasing in the ratio of GSSG/(GSSG+GSH) and ROS level and ameliorating MMP.

Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Embryo, Mammalian ; Free Radical Scavengers ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Oxidative Stress ; drug effects ; Rats ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the effects of endogenous and exogenous hydrogen sulfide (H(2)S) on the K(ATP) current in isolated rat ventricular myocytes.

METHODSVentricular myocytes were isolated from rat heart by modified Langendorff perfusion with collagenase. K(ATP) current of single rat ventricular myocytes was recorded by whole-cell patch-clamp technique.

RESULTSThe density of K(ATP) current was significantly reduced by 200 micromol/L DL-propargylglycine (PPG, an irreversible inhibitor of the H(2)S) [(5.3258 +/- 0.7556) pA/pF vs. (3.7856 +/- 0.4312) pA/pF, P < 0.01] in a time-dependent way. The density of K(ATP) current could be significantly increased by NaHS (a H(2)S donor, 9.375, 18.75, 37.5, 75, 150 micromol/L) in a concentration-dependent manner [(6.6310 +/- 0.6092) pA/pF vs. (9.0949 +/- 1.0259) pA/pF at 150 micromol/L, P < 0.01].

CONCLUSIONBoth endogenous and exogenous H(2)S could open K(ATP) channels and enhance the K(ATP) current in rat ventricular myocytes.

Animals ; Cells, Cultured ; Hydrogen Sulfide ; pharmacology ; KATP Channels ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques ; Rats ; Rats, Wistar

AIMTo observe the effects of adenosine on intracellular calcium concentration ([Ca2+]i) level in guinea pig ventricular myocytes and to define the possible mechanisms involved.

METHODSThe effects of adenosine on [Ca2+]i were investigated in guinea pig ventricular myocytes. [Ca2+]i was detected by laser confocal microscopy and represented by relative fluorescent intensity ((FI-FI0)/FI0, %, FIo: control, FI: administration of drugs).

RESULTS(1) Adenosine (10, 50, 100 micromol/L) reduced [Ca2+]i of ventricular myocytes in both normal Tyrode's solution and Ca(2+) -free Tyrode's solution in a concentration-dependent manner. (2) Tyrode's solution containing 30 mmol/L KCl (high K+ Tyrode's solution) induced [Ca2+]i elevation in ventricular myocytes, while adenosine (10, 50, 100 micromol/L) markedly inhibited the increase in [Ca2+]i induced by KCl. (3) Pretreatment with DPCPX (1 micromol/L) significantly reduced the effects of adenosine (100 micromol/L) in high K+ Tyrode's solution. The effects of adenosine (100 micromol/L) on [Ca2+]i in high K+ Tyrode's solution were also partially attenuated by pretreatment with L-NAME (1 mmol/L). (4) Adenosine (100 micromol/L) markedly inhibited the low concentration of ryanodine-induced [Ca2+]i increase in Ca(2+) -free Tyrode's solution. (5) When the propagating waves of elevated [Ca2+]i (Ca2+ waves) were produced by increasing extracellular Ca2+ concentration from 1 mmol/L to 10 mmol/L, adenosine (100 micromol/L) could block the propagating waves of elevated [Ca2+]i, reduce the frequency and duration of propagating waves, and reduce [Ca2+]i as well.

CONCLUSIONAdenosine may reduce the [Ca2+]i in isolated guinea pig ventricular myocytes via inhibiting Ca2+ influx and alleviating Ca2+ release from sarcoplasmic reticulum(SR). The reduction of Ca2+ influx might be due to the inhibition of voltage-dependent Ca2+ channel via adenosine A1 receptor, and NO might be involved in this process.

Adenosine ; pharmacology ; Animals ; Calcium ; metabolism ; Cells, Cultured ; Guinea Pigs ; Heart Ventricles ; cytology ; Myocytes, Cardiac ; drug effects ; metabolism

OBJECTIVETo evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius (TGRJ).

METHODSNeonatal rat cardiomyocytes were cultured and hypertrophy was induced by administrating isoproterenol (ISO, 10 µmol/L) or angiotensin 2 (Ang 2, 1 µmol/L) for 48 hours. In the treatment groups, cells were pretreated with TGRJ (0.3 g/L) for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca(2+) concentration ([Ca(2+)]i) was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) protein expression levels were measured by Western blotting . SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method.

RESULTSIncreased cell size, total protein content, and protein synthesis following ISO or Ang 2 stimulation were significantly inhibited by pretreatment with TGRJ (all P<0.05). This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and β-MHC. In addition, TGRJ inhibited ISO or Ang 2 induced up-regulation of [Ca(2+)]i under chronic but not acute conditions. And ISO or Ang 2 induced down-regulation of SERCA2a expression and activity was also effectively rectified by TGRJ pretreatment.

CONCLUSIONSThe results of present study suggested that TGRJ could prevent ISO or Ang 2 induced cardiac hypertrophy through improving chronic [Ca(2+)]i disorder, might via normalizing SERCA2a expression and activity.

Animals ; Animals, Newborn ; Calcium ; metabolism ; Cells, Cultured ; Glycosides ; analysis ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Ranunculus ; chemistry ; Rats