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Objective:To evaluate the effects of different densities of rat cardiac fibroblasts (RCF) subjected to hypothermic hypoxia-reoxygenation on cardiomyocyte injury and intercellular coupling.Methods:RCF was cultured in vitro and divided into 3 groups ( n=12 each) using a random number table method: RCF density 0.5×10 5 cells/ml group (T 0.5 group), RCF density 1.0×10 5 cells/ml group (T 1.0 group), and RCF density 2.0×10 5 cells/ml group (T 2.0 group). The three groups were placed in an anoxic device, into which 95% N 2 + 5% CO 2 was continuously blown at the speed of 5 L/min for 15 min, and then placed in a 4 ℃ refrigerator for 1 h for low temperature treatment.After completion of culture, cells were placed in a incubator containing 95% air + 5% CO 2 at 37 ℃ for 4 h of reoxygenation.After the end of culture, RCF in three groups were indirectly co-cultured with cardiomyocytes of the same density (1.0×10 5 cells/ml) in a Transwell chamber for 16 h, cardiomyocytes were seeded in the lower chamber of Transwell, and RCF were seeded in the upper chamber of Transwell.After the end of co-culture, cardiomyocytes were collected for determination of the cell viability (by CCK8 method), apoptosis rate (by flow cytometry), expression of connexin 43 (Cx43) mRNA (by real-time fluorescence quantitative polymerase chain reaction), and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:Compared with T 0.5 group, the cell viability, apoptosis rate and expression of Cx43, p-Cx43 and Cx43 mRNA were significantly decreased in T 1.0 and T 2.0 groups ( P<0.01). Compared with T 1.0 group, the cell viability, apoptosis rate and expression of Cx43 and p-Cx43 were significantly decreased ( P<0.01), and no significant change was found in expression of Cx43 mRNA in cardiomyocytes in T 2.0 group ( P>0.05). Conclusions:RCF subjected to hypothermic hypoxia-reoxygenation induces cardiomyocyte injury in a density-dependent manner in a certain range, and the mechanism may be related to down-regulation of the expression of Cx43 and reduction of the activity of Cx43.
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Objective:To evaluate the effect of rat cardiac fibroblasts (RCF) on the expression of connexin43 (Cx43) in H9c2 cells during hypothermic hypoxia/reoxygenation.Methods:H9c2 cells cultured in vitro were divided into 4 groups ( n=12 each) using the random number table method: control group (group C), hypothermic hypoxia/reoxygenation group (group HHR), RCF co-culture group (group Co) and RCF co-culture plus hypothermic hypoxia/reoxygenation group (group Co+ HHR). Group C was incubated at 37℃ in 5% CO 2 + 95% air for 5 h. Group HHR was incubated at 4 ℃ in 5% CO 2 + 95% N 2 for 1 h and then at 37 ℃ in 5% CO 2 + 95% air for 4 h. In group Co and group Co+ HHR, H9c2 cells 0.3×10 5 cells/well were inoculated in the lower chamber and RCF 0.6×10 5 cells/well in the the upper chamber of a transwell ? culture dish.Group Co was incubated at 37 ℃ in 5% CO 2 + 95% air for 5 h. Group Co+ HHR was incubated at 4℃ in 95% N 2 + 5% CO 2 for 1 h, and then incubated at 37 ℃ in 5% CO 2 + 95% air for 4 h. The mortality rate of H9c2 cells was measured by trypan blue staining, the expression of Cx43 and extracellular signal-regulated protein kinases 1/2 (ERK1/2) by immunofluorescence, and the expression of Cx43, phosphorylated Cx43, ERK1/2 and phosphorylated ERK1/2 by Western blot. Results:Compared with group C, the mortality rate of H9c2 cells was significantly increased, the expression and phosphorylation of Cx43 were decreased, and the expression and phosphorylation of ERK1/2 were increased in group HHR ( P<0.05), and no significant change was found in the mortality rate of H9c2 cells or expression and phosphorylation of Cx43 and ERK1/2 in group Co ( P>0.05). Compared with group Co, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Compared with group HHR, the mortality rate of H9c2 cells was significantly increased, and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+ HHR ( P<0.05). Conclusions:RCFs can decrease the expression and activity of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation, and the mechanism may be related to the down-regulation of ERK1/2 expression and inhibition of ERK1/2 activity.
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Objective:To evaluate the effects of different density rat fibroblasts on the expression of conjunctin 43 (Cx43) in cardiomyocytes and cell viability.Methods:Cardiomyocytes and fibroblasts were co-cultured using Transwell, cardiomyocytes were inoculated into the lower chamber of Transwell and fibroblasts into the upper chamber of Transwell.The cells were divided into 3 groups ( n=12 each) by a random number table method: fibroblast density 0.5×10 5 cells/ml group (group C 0.5), fibroblast density 1×10 5 cells/ml group (group C 1), and fibroblast density 2×10 5 cells/ml group (group C 2), with the density of cardiomyocytes 1×10 5 cells/ml in three groups.Cardiomyocytes and fibroblasts were co-cultured for 20 h in three groups.Cardiomyocytes were collected after co-culture for determination of cell viability (by CCK8 method), apoptosis rate (by flow cytometry), and expression of Cx43 mRNA (by quantitative real-time polymerase chain reaction) and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:There was no significant difference in the apoptosis rate of cardiomyocytes among the three groups ( P>0.05). Compared with group C 0.5, the expression of Cx43 protein and mRNA and p-Cx43 was significantly up-regulated in group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Compared with group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Conclusion:Rat fibroblasts up-regulate the expression of Cx43 and enhance the activity of Cx43 in cardiomyocytes and enhance cell viability in a density-dependent manner in a certain range.
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Objective:To determine the changes in the expression of myocardial miRNA and the target genes in the rats with hypothermic ischemia-reperfusion (I/R) arrhythmia.Methods:Clean-grade healthy male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized, the chest was opened, and the heart was taken to establish an isolated heart perfusion model.Six successfully perfused isolated hearts were divided into 2 groups ( n=3 each) using a random number table method: control group (group C) and heart I/R group (IR group). The model of hypothermic global I/R injury was established by interrupting perfusion for 60-min followed by 30-min reperfusion in chloral hydrate-anesthetized rats.The arrhythmia score was recorded during reperfusion.High-throughput sequencing was used to identify the differentially expressed miRNAs in two groups.The RNAhybrid and miRanda databases were used to predict the target genes of mRNA regulated by the differentially expressed miRNAs, and the enrichment for target genes was performed by Gene Ontology and KEGG databases, and the miRNAs closely related to arrhythmia and with higher expression were selected to carry out the real-time polymerase chain reaction detection. Results:The results of high-throughput sequencing showed that there were 7 differentially expressed miRNAs (novel-miR-17, novel-miR-19, novel-miR-30, novel-miR-43, rno-miR-122-5p, novel-miR-16 and rno-miR-429) in group IR as compared with group C. There were 4 miRNAs that were closely related to arrhythmia and had higher expression: the expression of novel-miR-17, novel-miR-30 and rno-miR-122-5p was significantly up-regulated, and the expression of rno-miR-429 was down-regulated in group IR when compared with group C ( P<0.05). The miRNA-mRNA correlation analysis revealed that GJA1 gene was the target of novel-miR-17. Conclusion:Myocardial novel-miR-17 is involved in the occurrence of hypothermic I/R arrhythmia probably by acting on GJA1 gene in rats.
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Objective:To evaluate the changes in the electrical conduction of ventricular myocardium during hypothermic ischemia-reperfusion (I/R) in rats with arrhythmia.Methods:Healthy clean-grade adult male Sprague-Dawley rats, aged 2-3 months, weighing 200-300 g, were studied.The hearts were removed and retrogradely perfused with oxygenated K-H solution in a Langendorff apparatus. Sixteen isolated hearts were divided into 2 groups ( n=8 each) using a random number table method: normal control group (group C) and hypothermic I/R group (group I/R). In group C, the heart was perfused with K-H solution at 37 ℃ for 120 min.In group I/R, the heart was perfused with K-H solution at 37 ℃ for 30 min, and then perfusion was stopped, cardiac arrest was induced through injecting Thomas solution (4 ℃), the area around the heart was protected with low temperature (4 ℃) Thomas solution, and hearts were perfused with 4 ℃ Thomas solution at 30 min after cardiac arrest and with 37 ℃ K-H solution for 30 min staring from 60 min after cardiac arrest.The rats in group I/R were further divided into high-risk subgroup (I/R-H subgroup) and low-risk subgroup (I/R-L subgroup). The time of spontaneous recovery of heart beat and development of arrhythmia were recorded.At the end of reperfusion, the atrioventricular conduction 2∶1 block point (2∶1B) and ventricular electrical conduction velocity (CV) were measured and recorded by program-controlled electrical stimulation. Results:Compared with group C, CV and 2∶1B were significantly decreased in IR-L and IR-H subgroups ( P<0.05). Compared with IR-L subgroup, the time for restoration of spontaneous heart beat was significantly prolonged, the incidence of ventricular fibrillation and arrhythmia score were increased, and CV and 2∶1B were decreased in IR-H subgroup ( P<0.05). Conclusion:The electrical CV of ventricular myocardium is decreased during hypothermic I/R, which may be the mechanism of reperfusion-induced ventricular arrhythmia in rats with arrhythmia.
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Objective:To evaluate the relationship between decreased atrial myoelectric conduction and gap junction protein 40 (Cx40) and Cx43 in rats with reperfusion atrial arrhythmia.Methods:Sixteen Langendorff-isolated heart perfusion models were randomly divided into control group (group C) and ischemia-reperfusion group (group IR), with 8 rats in each group.According to whether the atrial arrhythmia occurred after reperfusion, group IR was further divided into reperfusion non-atrial arrhythmia subgroup (group R-NAA) and reperfusion atrial arrhythmia subgroup (group R-AA). Group C was balanced perfusion with K-H solution (37 ℃) for 120 min.In group IR, hearts were perfused with K-H solution (37 ℃) for 30 min, perfusion was then stopped, Thomas solution (4 ℃, 20 ml/kg) was injected to induce cardiac arrest for 60 min, the surrounding of the heart was protected with 4 ℃Thomas solution, and hearts were perfused with Thomas solution (4 ℃, 10 ml/kg) again after 30 min of cardiac arrest and then with K-H solution 37 ℃ for 30 min.At 120 min of equilibration or 30 min of reperfusion, the effective refractory period (ERP) and conduction velocity (CV) of the right atrium were measured, the expression of Cx40 and Cx43 in the right atrial myocardium was detected by Western blot, and ratio of Cx40 to Cx40+ Cx43 and the ratio of Cx43 to Cx40+ Cx43 were calculated.Results:The incidence of reperfusion atrial arrhythmia was 38% in group IR.Compared with group C, ERP was significantly prolonged, CV was decreased, the expression of Cx40 and Cx43 was down-regulated, the ratio of Cx40 to Cx40+ Cx43 was increased, and the ratio of Cx43 to Cx40+ Cx43 was decreased in R-NAA and R-AA groups ( P<0.05). Compared with group R-NAA, ERP was significantly prolonged, CV was decreased, the expression of Cx40 and Cx43 was down-regulated, the ratio of Cx40 to Cx40+ Cx43 was increased, and the ratio of Cx43 to Cx40+ Cx43 was decreased in group R-AA ( P<0.05). Conclusion:The decreased atrial myoelectric conduction may be related to the down-regulation of Cx40 and Cx43 expression in rats with reperfusion atrial arrhythmia.
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Objective:To observe the changes in the expression of microRNAs in ventricular myocardium in a rat model of hypothermic ischemia-reperfusion (I/R).Methods:Healthy clean-grade male Sprague-Dawley rats, aged 2-3 months, weighing 300-400 g, were anesthetized with intraperitoneal chloral hydrate.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%O 2-5%CO 2.Sixteen Langendorff-perfused hearts were prepared and divided into 2 groups ( n=8 each) by a random number table method: control group (group C) and hypothermic I/R group (group I/R). The hearts were made globally ischemic for 60 min followed by 30-min hypothermic (4 ℃) reperfusion to establish the model of hypothermic I/R injury.The type and duration of arrhythmia and time of recovery of spontaneous heartbeats were recorded during reperfusion.The rats in group I/R were further divided into low-risk group (I/R-L group) and high-risk group (I/R-H group). The left ventricular myocardium was collected after the end of perfusion for high throughput sequencing to screen the differentially expressed microRNAs, and the reliability of the sequencing results was verified by quantitative real-time polymerase chain reaction.Gene Ontology and KEGG databases were used to analyze the biological regulatory pathways of differentially expressed target genes. Results:Compared with group C, there were 437 up-regulated microRNAs and 242 down-regulated microRNAs in group I/R-L and 419 up-regulated microRNAs and 260 down-regulated microRNAs in group I/R-H.Compared with group I/R-L, 392 microRNAs were up-regulated, and 287 microRNAs were down-regulated in group I/R-H.There were 84 microRNAs with absolute value of fold change ≥2 and significantly differential expression ( P<0.01) among the three groups.Subsequently, 4 microRNAs were randomly selected for validation using quantitative real-time polymerase chain reaction, confirming that the sequencing results were reliable.These differentially expressed target genes were involved in 11 biological processes and 6 KEGG pathways which were related to reperfusion arrhythmia.Potassium ion transmembrane transport and the adrenergic receptor signaling pathway in cardiomyocytes were enriched by the largest number of target genes. Conclusion:The expression of microRNAs in ventricular myocardium changes significantly after heart hypothermic I/R.These differentially expressed microRNAs regulate potassium ion transmembrane transport probably and mainly through the adrenergic receptor signaling pathway in the cardiomyocytes and thus are involved in the occurrence and development of hypothermic I/R arrhythmias.
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Objective To evaluate the electrophysiological changes of atrial myocardium in a rat model of hypothermic ischemia-reperfusion (I/R).Methods Sixteen isolated Sprague-Dawley rat hearts successfully perfused in the Langendorff apparatus were divided into control group (group C) and hypothermic I/R group (group IR) using a random number table method,with 8 heats in each group.Heats in group IR were further divided into reperfusion-non-atrial arrhythmia subgroup (group R-NAA) and reperfusion-atrial arrhythmia subgroup (group R-AA) depending on whether atrial arrhythmia occurred after reperfusion.In group C,the heart was perfused with K-H solution at 37 ℃ for 120 min.In group IR,the heart was perfused with K-H solution at 37 ℃ for 30 min and then perfusion was stopped,cardiac arrest was induced for 60 min through injecting Thomas solution (4 ℃,20 ml/kg),the area around the heart was protected with low temperature (4 ℃℃) Thomas solution,and hearts were resuscitated with 4 ℃ Thomas solution (10 ml/kg) at 30 min after cardiac arrest and with 37 ℃ K-H solution for 30 min staring from 60 min after cardiac arrest.At 30 min of equilibration (T0),105 min of equilibration/15 min of reperfusion (T1),and 120 min of equilibration/30 min of reperfusion (T2),right atrial monophasic action potentials,maximal velocity of phase zero,monophasic action potential amplitude (MAPA) and MAP duration at 50％ and 90％ of repolarization (MAPDs0and MAPDg0) were measured.Right-atrium conduction velocity and effective refractory period were recorded at T2,and the ratio of ERP to MAPD90 (ERP/MAPD90) was calculated.Atrial fibrillation was induced by programmed electrical stimulation,and the maximum pacing cycle length of inducing atrial fibrillation (AF-PCLm=) was recorded.Results Compared with C and R-NAA groups,the maximal velocity of phase zero was significantly decreased and MAPDg0 was increased at T1,the right-atrium conduction velocity and ERP/MAPDg0 ratio were decreased and MAPD90,effective refractory period and AF-PCLmax were increased at T2 in group R-AA (P＜ 0.05).Conclusion The decrease in depolarization velocity,prolongation of repolarization duration and decrease in conduction velocity,excitability and electrical stability may be the electrophysiological mechanism of reperfused atrial arrhythmia in rats.
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Objective@#To evaluate the electrophysiological changes of atrial myocardium in a rat model of hypothermic ischemia-reperfusion (I/R).@*Methods@#Sixteen isolated Sprague-Dawley rat hearts successfully perfused in the Langendorff apparatus were divided into control group (group C) and hypothermic I/R group (group IR) using a random number table method, with 8 heats in each group.Heats in group IR were further divided into reperfusion-non-atrial arrhythmia subgroup (group R-NAA) and reperfusion-atrial arrhythmia subgroup (group R-AA) depending on whether atrial arrhythmia occurred after reperfusion.In group C, the heart was perfused with K-H solution at 37 ℃ for 120 min.In group IR, the heart was perfused with K-H solution at 37 ℃ for 30 min and then perfusion was stopped, cardiac arrest was induced for 60 min through injecting Thomas solution (4 ℃, 20 ml/kg), the area around the heart was protected with low temperature (4 ℃) Thomas solution, and hearts were resuscitated with 4 ℃ Thomas solution (10 ml/kg) at 30 min after cardiac arrest and with 37 ℃ K-H solution for 30 min staring from 60 min after cardiac arrest.At 30 min of equilibration (T0), 105 min of equilibration/15 min of reperfusion (T1), and 120 min of equilibration/30 min of reperfusion (T2), right atrial monophasic action potentials, maximal velocity of phase zero, monophasic action potential amplitude (MAPA) and MAP duration at 50% and 90% of repolarization (MAPD50 and MAPD90) were measured.Right-atrium conduction velocity and effective refractory period were recorded at T2, and the ratio of ERP to MAPD90 (ERP/MAPD90) was calculated.Atrial fibrillation was induced by programmed electrical stimulation, and the maximum pacing cycle length of inducing atrial fibrillation (AF-PCLmax) was recorded.@*Results@#Compared with C and R-NAA groups, the maximal velocity of phase zero was significantly decreased and MAPD90 was increased at T1, the right-atrium conduction velocity and ERP/MAPD90 ratio were decreased and MAPD90, effective refractory period and AF-PCLmax were increased at T2 in group R-AA (P<0.05).@*Conclusion@#The decrease in depolarization velocity, prolongation of repolarization duration and decrease in conduction velocity, excitability and electrical stability may be the electrophysiological mechanism of reperfused atrial arrhythmia in rats.
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Objective To investigate the electrophysiological characteristics of myocardium after hypothermic ischemia-reperfusion (I/R) in rats with different degrees of arrhythmia using an in vitro experiment.Methods Healthy clean-grade male Sprague-Dawley rats,aged 2-3 months,weighing 300-400 g,were used in this study.The rats were sacrificed after anesthesia,and their hearts were rapidly excised.Sixteen Langendorff-perfused hearts were prepared and divided into 2 groups (n=8 each) by a random number table method:control group (group C) and hypothermic I/R group (group I/R).The hearts were made globally ischemic for 60 min followed by 30-min hypothermic (4 ℃) reperfusion to establish the model of hypothermic I/R injury.The occurrence and duration of arrhythmia and time of recovery of spontaneous heartbeat were recorded during reperfusion.The rats in group I/R were further divided into low-risk group (I/R-L group,ventricular arrhythmia score≤3 points) and high-risk group (I/R-H group,ventricular arrhythmia score＞3 points) according to the arrhythmia score.Monophasic action potential amplitude (MAPA),monophasic action potential (MAP) duration at 50％ and 90％ repolarization (MAPDs0 and MAPD90) and maximum ascending velocity (Vmax) of phase 0 in the endocardium,myocardium and epicardium of the left ventricular anterior wall were recorded at 30 min of equilibration (T0) and 15 and 30 min of reperfusion (T1,2).The effective refractory period (ERP) and ventricular fibrillation threshold (VFT) of the left ventricle were measured by programmed electrical stimulation,and the ERP/MAPD90 ratio was calculated.Results Compared with the baseline at T0,MAPA in the three layers was significantly decreased,and MAPD50 and MAPD90 were prolonged at T1,2 in I/R-L and I/R-H groups,and V in the three layers was decreased at T1,2 in I/R-H group (P＜0.05).MAPD50 and MAPD90 in the three layers were significantly shorter at T2 than at T1 in I/R-L and I/R-H groups (P＜0.05).Compared with group C,MAPDs0,MAPDg0 and ERP in the three layers were significantly prolonged at T1,2,the ERP/MAPDg0 ratio was decreased,and VFT was increased in I/R-L and I/R-H groups (P ＜ 0.05).Compared with I/R-L group,the duration of arrhythmia and MAPD90 and ERP in the three layers were significantly prolonged at T2,the ERP/MAPDg0 ratio was decreased,and VFT was increased in group I/R-H (P＜0.05).Conclusion Myocardial depolarization is inhibited,repolarization duration is prolonged,and electrophysiological stability is decreased after hypothermic I/R in the rats with arrhythmia,and the prolongation of myocardial repolarization and decrease in electrophysiological stability are more obvious in the rats at high risk of arrhythmia.