Tumor Necrosis Factor α In Mouse Atrial Myocyte HL-1Electrical Remodeling Mechanism

With the aging of the population and the average life expectancy, the increasing incidence of atrial fibrillation (atrial fibrillation, AF), and the population mortality, morbidity is very significant. Is expected that China’s population will reach1.45billion in2020, accounting for about20%of the total population60years of age or older, health-care workers faced with the daunting atrial fibrillation prevention and treatment. Atrial fibrillation research in recent years, the rapid progress of drug therapy and radiofrequency catheter ablation has made some progress, but many poor patient outcomes, it is necessary to further study its pathogenesis, seeking new therapeutic approaches.Atrial fibrillation initiating many factors, with the prolonged duration of AF, atrial electrophysiology and cell characteristics changed sustained atrial fibrillation, a process called electrical remodeling, the main features of the action potential duration (APD) and atrialshorten the effective refractory period (ERP) and the increase in dispersion, reduced rate adaptive atrial conduction velocity, wavelength of the atrial contraction atrial fibrillation easily induced and increased stability. While the atrial refractory period and action potential shortening, net decrease of flow inward ion (Ca2+or Na+), net increase in outward (K+) ion flow, the gap junctions decrease closely relatedAtrial electrical remodeling occurred basis of ion channels damage or functional changes, the mechanism of complex, early studies suggest that atrial natriuretic peptide, atrial muscle ischemia with ATP-sensitive potassium channel, autonomic nervous system, calcium overload and metabolic disorders may be roomflutter electrical remodeling contacts in recent years focused on the pathway of oxidative stress and damage. Number of studies have confirmed that patients with AF right atrial appendage tissue, plasma high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF-alpha), interleukin-6(IL-6) compared with sinus rhythm group was significantly higher, TNF-alpha serum concentrations in paroxysmal, persistent and permanent atrial fibrillation was progressive increment that oxidative stress and inflammation can directly lead to atrial fibrillation pathophysiological changes. The animal was found that TNF-alpha can affect the the rat ventricular L-type calcium current, CX40, the role of other ion channels and gap link is not clear.Inflammatory cytokines TNF-alpha, IL-6, IL-4, involved in the activation and regulation of the JAK-STAT signaling pathway, JAK-STAT signaling pathway can upregulate cyclooxygenase (COX-2), nitric oxide synthase (NOS2), vascularendothelial growth factor (VEGF), antioxidant manganese superoxide dismutase (MnSOD), metallothionein (MT1and MT2), matrix metalloproteinase, thus affecting cardiac hypertrophy and cardiac remodeling. The study found that resveratrol by blocking JAK/STAT1passage so as to achieve the control of interferon-mediated macrophage migration can reduce the inflammatory response of vascular damage, and reduces the formation of arterial thrombus and plaque. STAT3gene knockout mice found increased interstitial fibrosis in the heart, increased TNF-alpha secretion, dilated cardiomyopathy and impaired cardiac function; knockout of STAT3gene or JAK2inhibitor AG490reduced expression of STAT3, myocardial infarct size can increase; Adawi study found that in the rat model of myocardial infarction, to the AG490Kv4.3mRNA expression shows that the JAK-STAT signaling pathway is involved in the pathophysiology of cardiovascular disease.Examined the effects of TNF-alpha chronic stimulation HL-1cells, to explore the effects of TNF-alpha on the part of the ion channel and gap connected JAK-STAT signaling pathway involved in the regulation of inflammatory cytokines TNF-alpha under the mechanisms of electrical remodeling. This experiment is divided into two parts.Part Ⅰ TNF-alpha on the HL-1atrial myocytes electrical remodelingObject:TNF-alpha involved in coronary heart disease, heart failure, rheumatic heart disease, congenital heart disease and a variety of cardiovascular disease pathological processes in the pathogenesis of atrial fibrillation is unclear. This part of the experiment in HL-1cells after stimulation by different concentrations of TNF-alpha, by detecting electrical remodeling related genes, to explore the effects of TNF-alpha on the HL-1cells electrical remodeling.Methods:TNF-alpha25ng/ml,50ng/ml stimulate HL-1cells after24hours, total RNA was extracted and total protein, using real-time polymerase chain reaction (RT-PCR) to detect CACNA1C, KCNJ2, KCNH2, KCNQ1, GJA5GJA1SLC8A1mRNA expression was measured by Western blot (Western bloting) Cav1.2and ERG expression, and the control group and the experimental group Ica, L, IKR current and APD recorded with the patch-clamp method. PCLAMP10.2software on a single whole-cell recording data and graphics conversion, Prism3.0software for data curve fitting and draw ion channel current density and APD, current density (pA/pF)=current intensity/capacitor.Results:RT-PCR, found1.CACNA1c mRNA levels decreased with the increase of TNF-alpha concentration, control group and the experimental group had significant difference (p<0.01), while the experimental group TNF-a50ng/ml group was significantly lower than25ng/ml (p<0.01).2.KCNJ2mRNA levels increase with the increase of TNF-alpha concentration, but control group and25ng/ml were no significant differences have a significant difference (p<0.05) with50ng/ml was no significant difference between the experimental group.3.KCNH2mRNA experimental group significantly higher than those in the control group (p<0.05), but no significant difference between the experimental groups. No significant difference between the the4.KCNQ1mRNA experimental and control groups. No significant difference between the the5.GJA5mRNA experimental and control groups.6.GJA1mRNA levels increase with the increase of TNF-alpha concentration, but the control group and25ng/ml group no significant difference between the control group,25ng/ml,50ng/ml significant difference (p<0.05).7.SLC8A1mRNA levels increase with the increase of TNF-alpha concentration, there is a significant difference the control group with the experimental group25ng/ml and50ng ng/ml(p<0.05and p <0.01), and25ng/ml group between50ng/ml group there are significant differences.Western blot found calcium channel Cavl.2decrease with the increase in TNF-alpha concentration and potassium ion channel ERG With the increase in TNF-alpha increase.By the the patch clamp detected with the control group compared to the decrease in TNF-alpha group L-type calcium ion current density, current density in the command potential-10mv,0mv,10mv,20mv,30mv,40mv decreased there were significant differences. Compared with the control group, TNF-alpha group quickly activate the delayed rectifier potassium current plateau and the tail current increase in current density, the current density of the plateau in the command potential-40,-30,-20,-10,30,40,50mv there are significant differences,30,40,50mV significantly increased. Compared with the control group, TNF-alpha group APD shortened APD50shortening of43.6%, APD90shortened to40%, the APD50-APD90shortening37.9%were significantly different.Conclusion:TNF-alpha can change the HL-1cells CACNA1C of KCNJ2, KCNH2, GJA1,, SLC8A1mRNA expression to reduce Cav1.2and ERG of protein to reduce the L-type calcium ion current to increase rapidly activating delayed rectifier potassium current currentand shorten the APD, TNF-alpha involved in electrical remodeling of atrial myocytes and in which there is an important role.Part Ⅱ Role of Jak-Stat signal pathway on ERGObject:JAK-Stat signaling pathway is cytokine-stimulated signal transduction pathways involved in cell proliferation, differentiation, apoptosis and immune regulation and many other important biological processes, play an important role in the development of the heart, myocardial hypertrophy and myocardial fibrosis. TNF-alpha TNFR2activates the JAK-Stat signaling pathway in atrial fibrillation electrical remodeling is unclear. This part of the study was to explore TNF-alpha stimulation HL-1atrial myocytes after the JAK-Stat signaling pathway of its electrical remodeling whether or not to have an impact.Methods:different concentrations of TNF-alpha stimulation of HL-1cells for24hours, with the western blot assay JAK1, STAT3expression, siRNA knock STAT3, was detected using Western blot STAT3and ERG level was observed in the control group, while using the patch-clamp methodand the experimental group (siRNA-STAT3) IKR current and APD.Results:Western blot analysis found that TNF-a Ong/ml,25ng/ml,50ng/ml, the chronic irritation after24hours, JAK1and STAT3expression increased. To siRNA48hour, TNF-a24hours after the STAT3and ERG of siRNA-negative group was significantly higher than that siRNA-STAT3-1group, siRNA-STAT3-2group, siRNA-STAT3-3group, siRNA-STAT3-pool group, The STAT3and ERG decreased expression.Patch-clamp observed compared with the control group, siRNA group rapid activation of the delayed rectifier potassium current platform and the current density of the tail current decreases, the current density of the plateau in the command potential significant difference-30,30,40,50mv,30,40,50mV significantly reduce the current density of the tail current at the command potential-30,-20,-10,10,20,30,40,50mV was significantly reduced, there are significant differences. Compared with the control group, siRNA group APD50extend the69%APD90extend the60%, the APD50-APD90extended to57.1%. Statistical methods, we found the siRNA group plateau current density, compared to the the tail current current density and the normal cells decreased significantly, APD50, APD90still shorter than normal cells, but no significant difference.Conclusion:TNF-alpha HL-1cells can JKA1and STAT3expression increased, while inhibiting siRNA to significantly reduce ERG expression and reduce the HL-rapid delayed rectifier potassium current platform and the current density of the tail current, and less than no interventionl cells, the same make the APD50and APD90close to the intervention HL-1cells. Description the STAT3ERG impact, and play a role in the process of reconstruction of TNF-alpha on the HL-1cells power, suggesting that futuremay be a target for the treatment of atrial fibrillation.