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Objective·To observe mitochondria permeability transition pore (mPTP) opening and apoptosis of H9c2 myocardial cell stimulated by lipopolysaccharide (LPS),and to explore the anti-apoptotic effect of combined application of cyclosporine A (CsA) and ryanodine (Rya).Methods·The H9c2 cells were divided into Control group,LPS group,LPS+CsA group,LPS+Rya group,and LPS+CsA+Rya group.The mPTP opening state,Ca2+ concentration within cell and mitochondrial,mitochondrial membrane potential (AΦm),cell apoptosis,expression of Bax and Bcl-2 at mRNA and protein levels,and activity of caspase 3 were determined respectively.Results·mPTP opened after being stimulated by LPS for 24 h,which increased the fluorescence intensity for Ca2+in cytosolic and mitochondria by 298％ and 231％ respectively,induced about 1/3 cell apoptosis,improved the activity of caspase 3 approximately twice,and enhanced expression ofBax mRNA (P=0.008).The combined use of CsA and Rya effectively inhibited mPTP opening,increased the enhancement of fluorescence intensity for Ca2+in both cytosolic and mitochondria,maintained normal AΦrn,reduced LPS-induced apoptosis,inhibited the activity of caspase 3,and decreased Bax mRNA expression level induced by LPS in the myocardial cells.Conclusion·mPTP plays an important role in in LPS-induced myocardial apoptosis,whereas the combination of CsA and Rya can alleviate it effectively.
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Neuregulin-1 (NRG-1) and the ErbBs family of receptor tyrosine kinases are widely expressed in the cardiovascular system.NRG-1/ErbBs signaling plays an essential role in physiology and pathophysiology of the heart,including stabilization of cardiac myocyte structure and function,promotion of cardiac myocyte proliferation and survival,inhibition of cardiac myocyte apoptosis,reduction of myocardial interstitial fibrosis,regulation of energy utilization,and enhancement of angiogenesis and so on.Therefore,NRG-1/ErbBs signaling is involved in the development and treatment of chronic heart failure(CHF).In this review,we bring the growing literature on NRG-1/ErbBs signaling and its significance in cardiovascular development and heart failure.
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Aim To observe the cytochrome 450 effect of ginsenoside Re on H9c2 cells, in order to clarify the molecular mechanism of ginsenoside Re. Methods H 9 c 2 cells were separately treated with ginsenoside Re for 1, 5, 10, 50, 100 μmol·L-1 or 6, 24, 36, 48, 60 h. CYP2C11, 2J3, 4A1, 4A3, 4F4 and ANP mR-NA expressions were analyzed by Real time PCR, and CYP4 A1 , 2 J3 protein expressions were detected by Western blot. Results Compared with control group, ginsenoside Re could effectively upregulate CYP2 C11 , CYP2 J3 , ANP mRNA expression to 1. 6 , 1. 8 , 3. 2 fold, and downregulate CYP4A1, CYP4A3, CYP4F4 mRNA expression to 0. 4, 0. 15, 0. 3 fold. Ginsen-oside Re could decrease CYP4 A1 protein expression in a concentration-dependent manner, while ginsenoside Re could increase CYP2 J3 protein expression in a con-centration-dependent manner. Conclusion Ginsen-oside could regulate CYP450 enzyme and change ANP gene expression, which might be the molecular mecha-nism of ginsenoside Re.
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OBJECTIVE@#To observe the regulatory effects of RhoA/ROCK pathway on the apoptosis of cardiac myocyte induced by anoxia and its mechanism.@*METHODS@#The model of cardiac myocyte anoxia was established. The beat pulsations and apoptosis rates after 1 h, 3 h, 6 h, 9 h and 12 h of anoxia were recorded and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspae-3 were detected, too. The apoptosis and the expressions of related proteins were detected after RNAi of RhoA and the inhibition of ROCK by Y-27632.@*RESULTS@#The beat pulsations after 1 h, 3 h, 6 h, 9 h and 12 h decreased gradually but the apoptosis rates increased gradually, and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspase-3 were increasing along with the increasing duration of anoxia. The apoptotic rates after 1 h, 3 h, 6 h, 9 h and 12 h of anoxia were (4.360.98)%, (8.362.12)%, (15.323.62)%, (18.684.83)% and (24.566.22)%, respectively and decreased more significantly than control group in different time points of anoxia (P<0.05), and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspase-3 decreased significantly (P<0.05). The apoptosis rate and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspase-3 decreased significantly (P<0.05) after the inhibition of ROCK by Y-27632 (P<0.05).@*CONCLUSIONS@#RhoA/ROCK pathway plays a critical role in the regulation of the apoptosis of cardiac myocyte induced by anoxia, which may be accompanied by regulating the activity of PI3K/AKT/Caspase-3 pathway.
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Objective: To observe the regulatory effects of RhoA/ROCK pathway on the apoptosis of cardiac myocyte induced by anoxia and its mechanism. Methods: The model of cardiac myocyte anoxia was established. The beat pulsations and apoptosis rates after 1 h, 3 h, 6 h, 9 h and 12 h of anoxia were recorded and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspae-3 were detected, too. The apoptosis and the expressions of related proteins were detected after RNAi of RhoA and the inhibition of ROCK by Y-27632. Results: The beat pulsations after 1 h, 3 h, 6 h, 9 h and 12 h decreased gradually but the apoptosis rates increased gradually, and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspase-3 were increasing along with the increasing duration of anoxia. The apoptotic rates after 1 h, 3 h, 6 h, 9 h and 12 h of anoxia were (4.36[U+4F9D]0.98)%, (8.36[U+4F9D]2.12)%, (15.32[U+4F9D]3.62)%, (18.68[U+4F9D]4.83)% and (24.56[U+4F9D]6.22)%, respectively and decreased more significantly than control group in different time points of anoxia (. P<0.05), and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspase-3 decreased significantly (. P<0.05). The apoptosis rate and the expressions of RhoA, ROCK1/2, p-PI3K, p-AKT and caspase-3 decreased significantly (. P<0.05) after the inhibition of ROCK by Y-27632 (. P<0.05). Conclusions: RhoA/ROCK pathway plays a critical role in the regulation of the apoptosis of cardiac myocyte induced by anoxia, which may be accompanied by regulating the activity of PI3K/AKT/Caspase-3 pathway.
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It has been previously demonstrated that dystrophin is cleaved in the cardiac myocyte by the viral protease 2A following infection with Coxsackievirus B3 (CVB3). The viral protease 2A mediated cardiomyopathy can be prevented by inhibiting cleavage of dystrophin. However, it is less clear whether uncleaved dysdrophin have other heart protective effect in coxsackievirus infection. To address this, we generated a Balb/C background mouse that had a point mutation in dystrophin that prevents cleavage by protease 2A (KI). We show here that when mice expressing cleavage-resistant dystrophin were infected with CVB3, there was increased cardiac myocyte apoptosis. Bax and Bcl-X(L) mRNA ratio was significantly increased in KI mice heart compare to wild type mice heart. We found cleavage-resistant dystrophin induced the apoptosis related enzyme capspase-3 and caspase-8 activity. In addition, TUNEL stain was observed many TUNEL positive cardiac myocyte in KI mice heart compare to wild type mice heart (3.7% vs 0.3%). However, zVAD treatment for apoptosis blocking was significantly decreased myocardium damage and fibrosis in KI mice heart. These findings indicated that uncleaved dystrophin may have a critical role in cardiac myocyte viral propagation. Uncleaved dystrophin mutant induced cardiac myocyte apoptosis. It delayed coxsackievirus propagation in cardiac myocyte and could prevent cardiac myocyte death.
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Animals , Mice , Apoptosis , Cardiomyopathies , Caspase 8 , Coxsackievirus Infections , Dystrophin , Fibrosis , Heart , In Situ Nick-End Labeling , Myocardium , Myocytes, Cardiac , Point Mutation , RNA, MessengerABSTRACT
OBJECTIVES: The present study was performed to investigate 1) whether aerobic exercise training prior to myocardial infarction would prevent cardiac dysfunction and structural deterioration and 2) whether the potential cardiac benefits of aerobic exercise training would be associated with preserved morphological and contractile properties of cardiomyocytes in post-infarct remodeled myocardium. METHODS: Male Wistar rats underwent an aerobic exercise training protocol for eight weeks. The rats were then assigned to sham surgery (SHAM), sedentary lifestyle and myocardial infarction or exercise training and myocardial infarction groups and were evaluated 15 days after the surgery. Left ventricular tissue was analyzed histologically, and the contractile function of isolated myocytes was measured. Student's t-test was used to analyze infarct size and ventricular wall thickness, and the other parameters were analyzed by the Kruskal-Wallis test followed by Dunn's test or a one-way analysis of variance followed by Tukey's test (p<0.05). RESULTS: Myocardial infarctions in exercise-trained animals resulted in a smaller myocardial infarction extension, a thicker infarcted wall and less collagen accumulation as compared to myocardial infarctions in sedentary animals. Myocardial infarction-induced left ventricular dilation and cardiac dysfunction, as evaluated by +dP/dt and -dP/dt, were both prevented by previous aerobic exercise training. Moreover, aerobic exercise training preserved cardiac myocyte shortening, improved the maximum shortening and relengthening velocities in infarcted hearts and enhanced responsiveness to calcium. CONCLUSION: Previous aerobic exercise training attenuated the cardiac dysfunction and structural deterioration promoted by myocardial infarction, and such benefits were associated with preserved cardiomyocyte morphological and contractile properties. .
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Animals , Male , Rats , Heart/physiopathology , Myocardial Infarction/physiopathology , Myocardial Infarction/prevention & control , Myocytes, Cardiac/physiology , Physical Conditioning, Animal/physiology , Blood Pressure/physiology , Hemodynamics/physiology , Myocardial Contraction/physiology , Random Allocation , Rats, Wistar , Time Factors , Ventricular Function, Left/physiologyABSTRACT
Autophagy is induced by a variety of signals during ischemia and reperfusion.Autophagy has been shown to protect cardiac cells and to reduce the cell loss,but it also has been shown that enhanced autophagy contributes to cell death during I/R.
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Objective To study the safety and effect of the umbilical cord blood(UCB)-derived mesenchymal stem cells(MSCs)on apoptosis of human cardiomyocytes(HCM). MethodsUCB was collected at the time of delivery with informed consent obtained from 10donors.The UCB-derived MSCs were treated with 5-azaserube(5-AZA)and were further induced to differentiate into cardiomyocytes.Telomerase activity,G-banding patterns of chromosomal karyotypes,tumor formation in nude mice,RT-PCR,and the effect of inhibiting apoptosis of HCM were investigated. ResultsMSCs derived from UCB were differentiated into cardiomyocytes in vitro,which possessed telomerase activity after 5-AZA induction,and no abnormal chromosomal karyotypes were observed.Expression of p53,cyclin A,cdk2,β-actin,C-fos,h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment.There was no tumor formation in nude mice after injection of UCB-derived MSCs.UCB-derived MSCs significantly inhibited apoptosis of HCM. ConclusionUCB-derived MSCs are a valuable,safe and effective source of cell-transplantation treatment.
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Objective To investigate the effect of ligustrazine on the expression of platelet-derived growth factor-?(PDGF-?) receptor and extracellular signal regulated kinase(ERK1/2) induced by angiotensin Ⅱ(AngⅡ) in cardiac myocytes,and explore the mechanism of therapeutic.Methods Cultured cardiac myocytes of neonatal rats were treated with 10-7 mol/L AngⅡ as Ang Ⅱ group,10-7 mol/L AngⅡ plus 10 mg/L ligustrazine as ligustrazine group,the normally cultured neonatal rat cardiac myocytes as control group.Protein synthesis was measured by -leucine incorporation,and the expression of PDGF-? receptor and ERK1/2 was detected by Western blot.SPSS 11.0 software was used to analyze the data.Results There were significant differences among 3 groups(F=20.71 P
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The Ca2+ wave is a chain reaction of intracellular Ca2+ release channels through a Ca2+-induced Ca2+ release mechanism. In cardiac myocytes, Ca2+ wave has drawn much attention because it is found to induce arrhythmia genesis. To investigate the microscopic process of wave propagation, Ca2+ imaging was performed with high spatial and temporal resolution via a laser-scanning confocal microscope combined with loose-seal patch clamp. These observation and analysis revealed that Ca2+ waves originated from a stochastic recruiting of Ca2+ release units (CRUs) by a pioneer Ca2+ spark, which had a low possibility in normal cells. During wave propagation, the 'waiting' time that the wave propagate between two neighboring CRUs along propagation direction distributed normally, and cells with a lower speed had a more dispersive distribution of 'waiting' time. To study the cause of the randomicity, the wave propagation was simulated with a numerical model. The simulation showed that the intrinsic stochastic open process of CRUs can fully explain the above phenomenon. Increasing the maximal open probability of CRUs reduced the randomness of wavefront propagation and enhanced the average velocity of wave meantime. These experimental and numerical results provided an unequivocal quantification for the stochastic behavior of wave initiation and propagation.
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Objective To observe whether mesenchymal stem cells(MSCs) which have been induced by 5-azacytidine(5-aza) can differentiate into cardiac like cells. To find out the role of TITIN playing during the development of cardiomycytes among structure proteins. Methods To establish a recombinant plasmid vector involving a shRNA which matching the base pair of rat TITIN N2B region mRNA perfectly,transfect it into normal neonatal cardiomyocytes and MSCs which have been induced by 5-aza respectively and investigate the expression of TITIN Z band,MHC,ACTIN as well as cTnT by immunofluorescence. Results The expression of TITIN was weakened after recombinant plasmid has been transfected into neonatal cardiomyocytes.The same thing happened upon MSCs that have been induced by 5-aza. The expression of cTnT was weakened after TITIN been silenced by small interfering RNA (siRNA). But there was obvious change in MHC and ACTIN. Conclusion Our results demonstrate that MSCs can be induced into cardiomyocyte-like cells by 5-aza in vitro,although the degree of differentiation is still lower and can not form intact contractive structure. TITIN plays an important role in the development of structure proteins.
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To investigate the neuronal migration and synthesis of peptidic neurotransmitters in vitro, neuromuscular co-cultures of nodose ganglia and cardiac muscle cells of Wistar rats were established. The living cells of co-cultures were observed with an inverted contrast microscope at different culture age. At 72 h and 96 h of culture age, the neuromuscular co-cultures were examined by Holmes' reduced silver staining technique for observing the neuronal migration. Immunohistochemical staining method was used for investigating the synthesis of peptidic neurotransmitters- substance P (SP) and calcitonin gene-related peptide (CGRP). The neuronal morphorlogical maturation in neuromuscular co-cultures seems at 72 h of culture age in terms of the observation of living cells and Holmes' reduced silver staining technique. SP- and CGRP-immunoreactive neurons were observed in neuromuscular co-cultures at 96 h of culture age but not 72 h. The results indicate that the neuronal morphological maturation can not represent the maturation of neurotransmitter synthesis. The synthesis of neurotransmitters is not maturated in the neuromuscular co-cultures until 96 h of the culture age.
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Aim To observe the protective effects and its mechanisms of Garlic Polysaccharide(GP) on toxic cardiac myocyte induced by adriamycin (ADR). Methods Primary culture neonatal SD rat cardiac myocyte and ADR injury model were established. The activities of several superior fluid and cells enzymes were measured. Using MTT assay and flow cytometry, the apoptotic cardiac myocyte was shown. Results ADR increased the superior fluid creatine kinase (CK), lactate dehydrogenase(LDH), glutamic oxaloacetic transaminase (GOT), and augmented myocardial malondialdehyde (MDA) content, while decreased the superoxide dismutase (SOD) activities (P
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Objective:To discuss how the more convenient primary culture method of rats'cardiac myocytes to increase the quantity,livability and purity of isolated cells.Methods:Neonatal rats'cardiac myocytes were isolated by co-digestion of trypsin and collagenase Ⅱ,and then cultured in vitro.Morphocytology changes were observed and cell purity was evaluated by immunofluorescence.Results:Isolation of cardiomyocytes was with a good quantity and high livability.Cells adhered and impulsed well.Immunofluorescence staining confirmed the cell with purity higher than 95%.Conclusion:The modified cell culture method can obtain the Cardiomyocytes with good growth state.
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Objective To explore the mechanism by which urotensin Ⅱ induces hypertrophy of the cultured rat cardiomyocytes. Methods The cultured cardiomyocytes from neonatal SD rats were treated with urotensin Ⅱ, also with cyclosporine A for its blocking effect on urotensin Ⅱ induced cardiomyocytes hypertrophy. The mRNA and protein levels of ?-MHC and CaN were evaluated by real-time PCR and Western blotting, respectively. Results In the cells treated with 10-8 and 10-7 mol/L urotensin Ⅱ, the mRNA and protein levels of ?-MHC and CaN were significantly higher than that of control (P
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BACKGROUND AND OBJECTIVES: The dominant mode of cell death in cardiomyocytes under acute ischemic insult, either necrosis or apoptosis, remains to be clearly shown. MATERIALS AND METHODS: Cultured neonatal rat ventricular myocytes (NRVM) were incubated under hypoxic (mixture gas of 95%N2/5%CO2 in glucose containing media), ischemic (hypoxia plus glucose depletion in the media), ischemic and acidic conditions (ischemia with media pH 7.1). The level of cell death was assessed by trypan blue staining. To differentiate the mode of cell death, genomic DNA extraction and electrophoresis, Annexin V/propodium iodide staining, western blot for caspase activation and transmission electron microscopy were employed. RESULTS: The number of cell deaths in the NRVM cultured under hypoxic conditions was similar to that of the controls. The rate of cell death was significantly increased in the NRVM cultured under ischemic conditions, and was accelerated further in an acidic milieu, which simulated the accumulation of metabolic byproducts in ischemia. No signs of apoptotic cell death were observed in the NRVM cultured under ischemic conditions. The morphological examination of the cells in ischemia mostly revealed necrotic death. CONCLUSION: The presence of glucose protected the cardiomyocytes from cell death under hypoxic conditions. Incubation of the NRVM in ischemia resulted in increased cell deaths, which was accelerated in an acidic milieu. In our model of acute ischemia, without reoxygenation, the cardiomyocyte cell deaths appeared to be primarily induced via necrosis rather than apoptosis.
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Animals , Rats , Apoptosis , Blotting, Western , Cell Death , DNA , Electrophoresis , Glucose , Hydrogen-Ion Concentration , Ischemia , Microscopy, Electron, Transmission , Muscle Cells , Myocytes, Cardiac , Necrosis , Trypan BlueABSTRACT
AIM: To establish a PCR method for investigating the expression of clock genes in cultured rattus cardiac myocytes. METHODS: PCR was carried out using 3 primer pairs based on the published sequences of dbp, bmal1 and per2 genes of rattus. The conditions of PCR were optimized and the specificity of amplication was tested. RESULTS: In a volume of 20 ?l, the optimal PCR mixture of bmal1 gene contains 0.5 U Taq polymerase, 0.006 ?mol dNTP and 0.035 ?mol Mg 2+; the annealing temperature being 57 ℃; and circle times being 30. In a same volume, the optimal PCR mixture of dbp gene contains 0.5 U Taq polymerase, 0.006 ?mol dNTP and 0.03 ?mol Mg 2+; the annealing temperature being 58 ℃; and circle times being 32. The optimal PCR mixture of per2 gene contains 0.5 U Taq polymerase, 0.006 ?mol dNTP and 0.05 ?mol Mg 2+; the annealing temperature being 57 ℃; circle times being 30. The specificity of amplication was very high. CONCLUSION: The PCR method can successfully detect mRNA expression of clock genes in cultured rattus cardiac myocytes.
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In the article the effect of thyroid hormones was reviewed on cardiac ion channels and transports, so as to explain the changes of the action current on cardiac myocyte. Based on the molecular biology, the hormonal regulation of cardiac and transports were discussed and explained.
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PURPOSE: Calcium-activated potassium channels(KCa) may be involved in the transient outward current of the first phase of cardiac action potential. But it is still not clear whether cardiac myocytes express any Kca. We try to identify here the types of Kc, expressed in rat caridac myocytes. METHODS: We isolated total heart RNA from 50 rats(Spague-Dawley) and performed reverse transcription-polymerase chain reaction(RT-PCR) using specifically designed synthetic oligonucleotide primer sets. From the pure culture of cardiac myocyte, Kc, gene expression was detected by Southern blot analysis. RESULTS: RT-PCR revealed expressions of BKca(large-conductance Kca, rSlo) and S&,(small-conductance Kca, rSK1). We prepared cardiac myocytes pure culture(>9596 pure myocyte) using pure culture technique. RT-PCR and Southern blot analysis of rat cardiac myocyte showed only rSK1-specific band, but no rSlo-specific was detected. CONCLUSION: The expressions of more than one type of Kca are detected from rat heart. A sub-type of SKcrSK1, was expressed in cardiac myocyte, while the main subunit of BKca(rSlo) was found in cells other than myocytes, most likely in the smooth muscle of cardiac blood vessels.