| Background:Hyperthyroidism is one of the most common cause of atrial fibrillation (AF), but the cell and molecular mechanisms of high thyroid hormone leading to AF attack and continuance is unclear, which limit the more effective treatment of AF. Thyroid hormone receptor is located in the nucleus, it combines with the receptor on target organs and play a directly effect. However, the study also found that some function of the thyroid hormone is indirectly effect on target organs by activating other systems, but not directly effect.Objective:Establish the rabbit AF cause of hyperthyroidism susceptible model by intraperitoneal injection and filling irbesartan by stomach tube, which doubly intervence the rabbit model; primary culture of left atrial cardiomyocyte in vitro by drug intervention at the same time; observation the left atrial electrophysiological characteristics and susceptible of AF, and the apoptosis phenomenon of left atrial cardiomyocyte, detection the relevant cytokines expression level of the Ang II etc in the RAS, detection the ASK1-p38MAPK signaling pathway with apoptosis related gene and protein expression, which research the mechanisms between the thyriod hormone, RAS and AF cause of hyperthyroidism with the left atrial cardiomyocyte apoptosis, then providing the scientific basis to treatment AF cause of hyperthyroidism by new methods.Methods:(1) Animals study1. Establish the rabbit model of AF cause of hyperthyroidism.70 adult healthy rabbits were randomly divided into 5 groups:control group, two months group, withdrawal group, continuous dosing group, irbesartan group; control group (10, injection of saline), two months dosing(10, injection of levo-thyroxine 50μg/kg, compared with continuous dosing group), withdrawal group (20, injection of levo-thyroxine 50μg/kg, change to inject isodose saline after two months), continuous dosing group (20, injection of levo-thyroxine 50μg/kg everyday), irbesartan group(10, injection of levo-thyroxine 50μg/kg and filling irbesartan 50μg/kg by stomach tube).The time lasted four months.2. Electrophysiological study of the left atrialDetection the left atrial electrophysiological characteristics change by program stimulation. Collected the data of left atrial effective refractory period(AERP), conduction velocity(CV), wavelength(WL) and AF induced rate after two months and four months.Then to take the blood in ear marginal vein and left atrial myocardial.3. Detection the rabbit left atrial cardiomyocyte apoptosis by TUNEL.4. Detection the relevant cytokines expression level of the RASDetection the left atrial myocardial ACEmRNA, ACE2mRNA relatively expression quantity by real-time fluorescent quantitative PCR. Assay the plasma concentration and left atrial myocardial expression quantity of ACE, ACE2, Ang II.5. Detection the ASK1-p38MAPK signaling pathway with apoptosis related gene and protein expressionDetection the left atrial myocardial BaxmRNA, Bcl-2mRNA relatively expression quantity by real-time fluorescent quantitative PCR. Assay the left atrial myocardial protein relatively expression quantity of p-ASK1, ASK1, p-p38MAPK, p38MAPK, Bax, Bcl-2, PARP cleaved, caspase3 cleaved.(2) Cardiomyocyte study1. Primary culture of left atrial cardiomyocyte in vitroTo Isolate, purify, culture and identify the neonatal rabbit single left atrial cardiomyocytes.2. Intervention left atrial cardiomyocyte by drugTaken the second generation of cultivation after 7 days left atrial cardiomyocyte were randomly divided into 6 groups:control group(physiological saline), NQDI group (ASK1 inhibitor,5×10-7mol/L), AngⅡ group(1×10-6mol/L), AngⅡ+NQDI group (1×10-6mol/L+5×10-7mol/L), thyroxine group(1×10-7mol/L), thyroxine+NQDI group (1×10-7mol/L+5 x 10"7mol/L), which intervented for 24 hours.3. Detection the left atrial cardiomyocyte apoptosisDetection the rabbit left atrial cardiomyocyte apoptosis by flow cytometry with Annexin V-FITC/PI.4. Detection the ASK1-p38MAPK signaling pathway with apoptosis related gene and protein expressionDetection the left atrial cardiomyocyte BaxmRNA, Bcl-2mRNA relatively expression quantity by real-time fluorescent quantitative PCR. Assay the left atrial cardiomyocyte protein relatively expression quantity of p-ASK1, ASK1, p-p38MAPK, p38MAPK, Bax, Bcl-2, PARP cleaved, caspase3 cleaved.Results(1) Animals study1. The left atrial electrophysiological characteristics change1.1 The AERP200 of control group, withdrawal group, continuous dosing group and irbesartan group were 129.46±5.21,104.55±5.12,105.78±4.10,123.40±6.71ms after two months, The AERP200 of withdrawal group, continuous dosing group and irbesartan group were shorter than the control group after two months, P<0.05; withdrawal group and continuous dosing group were significantly shorter than the irbesartan group, P< 0.05. The AERP150 of control group, withdrawal group, continuous dosing group and irbesartan group were 103.68±5.21,79.64±5.22,80.76±5.58,100.54±6.39ms after two months, The AERP150 of withdrawal group, continuous dosing group were shorter than the control group and irbesartan group, P<0.05. The AERP200 of control group, withdrawal group, continuous dosing group and irbesartan group were 128.75±5.05, 120.55±5.12,92.78±4.20,125.16±8.03ms after four months, The AERP200 of continuous dosing group was shorter than the control group, withdrawal group and irbesartan group after four months, P<0.05; the withdrawal group was shorter than the control group and irbesartan group, P<0.05. The AERP150 of control group, withdrawal group, continuous dosing group and irbesartan group were 104.32±5.20,98.08±5.36, 67.34±5.02,102.52±7.05ms after four months, The AERP)5o of continuous dosing group was shorter than the control group, withdrawal group and irbesartan group after four months, P<0.05; withdrawal group was shorter than the control group, P<0.05. 1.2 The CV of control group, withdrawal group, continuous dosing group and irbesartan group were 85.42±3.81,76.50±4.25,75.12±4.37,81.61±1.71 cm/s after two months, The CV of withdrawal group, continuous dosing group and irbesartan group were slower than the control group after two months, P<0.05; withdrawal group and continuous dosing group were significantly slower than the irbesartan group, P<0.05. The CV of control group, withdrawal group, continuous dosing group and irbesartan group were 84.59±3.81,85.66±4.08,70.94±4.07,82.26±2.30 cm/s after four months, The CV of continuous dosing group was slower than the control group, withdrawal group and irbesartan group after four months, P<0.05.1.3 The WL of control group, withdrawal group, continuous dosing group and irbesartan group were 10.99±2.75,7.95±3.11,7.93±2.67,9.76±2.98 cm after two months, The WL of withdrawal group and continuous dosing group were shorter than the control group and irbesartan group after two months, P<0.05. The WL of control group, withdrawal group, continuous dosing group and irbesartan group were 10.89±2.91,10.32±2.59, 6.57±2.80,9.57±3.14 cm after four months, The WL of continuous dosing group was shorter significantly than the control group, withdrawal group and irbesartan group after four months, P<0.01.1.4 The AF induced rate of control group, withdrawal group, continuous dosing group and irbesartan group were 0%,35%,31.6%,0% after two months, The AF induced rate of the continuous dosing group and withdrawal group were higher than the control group and irbesartan group after two months, P<0.05. The AF induced rate of control group, withdrawal group, continuous dosing group and irbesartan group were 0%,15%, 57.9%,11.1% after four months, The AF induced rate of the continuous dosing group was higher than the control group, withdrawal group and irbesartan group after four months, P<0.05.2. The apoptosis rate of left atrial cardiomyocyteThe AF induced rate of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 4.58±1.75%,26.10±6.36%,20.49±6.74%, 37.51±9.57%,17.60±4.45%; The apoptosis rate of withdrawal group, continuous dosing group and irbesartan group were higher than the control group,P<0.05;the withdrawal group and irbesartan group were lower than the continuous dosing group, P <0.05;the continuous dosing group was higher than two months group,.P=0.003.3. The relevant cytokines expression level of the RAS3.1 The left atrial myocardial ACEmRNA relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.0180±0.0035,0.0270±0.0118,0.0593±0.0284,0.0321±0.0084, 0.0354±0.0080; The ACEmRNA relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P< 0.05;the continuous dosing group was higher than the two months group, P<0.05.3.2 The left atrial myocardial ACE2mRNA relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.0171±0.0025,0.0061±0.0025,0.0024±0.0013,0.0116±0.0038, 0.0047±0.0010; The ACE2mRNA relatively expression quantity of withdrawal group, continuous dosing group and irbesartan group were lower the control group, P< 0.05;the withdrawal group and irbesartan group were higher than continuous dosing group,P<0.05; irbesartan group was higher than withdrawal dosing group, P<0.05; the continuous dosing group was significantly lower than the two months group, P< 0.001.3.3 The ACE plasma concentration of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 3.616±0.582,6.413±0.771, 13.402±1.991,6.723±0.914,8.559±0.854ng/ml; The ACE plasma concentration of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than continuous dosing group, P<0.05; the continuous dosing group was significantly higher than the two months group, P< 0.001. The left atrial myocardial ACE expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 517.91±85.26,758.38±73.42, 1392.4±153.84,880.13±106.94,1006.4±103.71ng/100mg; The myocardial ACE expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than continuous dosing group, P<0.05; the irbesartan group was higher than withdrawal group, P<0.05; the continuous dosing group was significantly higher than the two months group, P<0.001.3.4 The ACE2 plasma concentration of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 755.52±54.03, 511.91±47.10,352.27±43.16,710.81±31.07,554.54±43.73pg/ml; The ACE2 plasma concentration of withdrawal group, continuous dosing group and irbesartan group were lower than the control group, P<0.05; the withdrawal group and irbesartan group were higher than the continuous dosing group, P<0.05; the irbesartan group was higher than the withdrawal group, P<0.05; the continuous dosing group was significantly lower than the two months group, P<0.001. The left atrial myocardial ACE2 expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 255.36±19.38,156.78±17.38,79.67±11.60,187.60±9.06, 137.23±12.92pg/100mg; The myocardial ACE2 expression quantity of withdrawal group, continuous dosing group and irbesartan group were lower than the control group, P<0.05; the withdrawal group and irbesartan group were higher than the continuous dosing group, P<0.05; the irbesartan group was higher than the withdrawal group, P< 0.05; the continuous dosing group was significantly lower than the two months group, P<0.001.3.5 The AngⅡ plasma concentration of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.321±0.093,0.590±0.142, 1.284±0.197,0.530±0.092,0.940±0.271ng/ml; The AngⅡ plasma concentration of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than continuous dosing group, P<0.05;the continuous dosing group was higher than the two months group, P=0.001. The left atrial myocardial Ang Ⅱ expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.166±0.053,0.291±0.068,0.644±0.092,0.264±0.039,0.452±0.099 ng/100mg; The myocardial Ang Ⅱ expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than continuous dosing group, P< 0.05; the continuous dosing group was significantly higher than the two months group, P<0.001.4. The ASK1-p38MAPK signaling pathway with apoptosis related gene and protein expression4.1 The left atrial myocardial BaxmRNA relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.0202±0.0060,0.0197±0.0090,0.0392±0.0199,0.0217±0.0032,0.0213±0.0087; The BaxmRNA relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the continuous dosing group was higher than the two months group, P<0.05.4.1 The left atrial myocardial Bcl-2mRNA relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.0015±0.0003,0.0008±0.0005,0.0004±0.0002,0.0010±0.0003, 0.0006±0.0001; The Bcl-2mRNA relatively expression quantity of withdrawal group, continuous dosing group and irbesartan group were lower than the control group, P< 0.05; the withdrawal group and irbesartan group were higher than continuous dosing group, P<0.05; the continuous dosing group was lower than the two months group, P <0.05.4.3 The left atrial myocardial p-ASK1/ASK1 relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.5825±0.0608,0.5276±0.0537,0.8746±0.1010,0.6298±0.0383, 0.8344±0.0511; The p-ASK1/ASK1 relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P< 0.05; the irbesartan group was higher than the withdrawal group, P<0.05; the continuous dosing group and the two months group were difference, P=0.273.4.4 The left atrial myocardial p-p38MAPK/p38MAPK relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.3609±0.0596,0.7245±0.0919,1.0473±0.1310,0.8062±0.0931, 0.4838±0.0776; The p-p38MAPK/p38MAPK relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than the continuous dosing group, P<0.05;the continuous dosing group was significantly higher than the two months group,P<0.001.4.5 The left atrial myocardial Bax relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.8274±0.0864,0.8375±0.0638,1.1232±0.1347,0.8265±0.0642,0.8054±0.1117; The Bax relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the continuous dosing group was significantly higher than the two months group, P<0.001.4.6 The left atrial myocardial Bcl-2 relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.5429±0.0870,0.3844±0.0471,0.2305±0.0488,0.4142±0.0330,0.3752±0.0827; The Bcl-2 relatively expression quantity of withdrawal group, continuous dosing group and irbesartan group were lower than the control group, P<0.05; the withdrawal group and irbesartan group were higher than the continuous dosing group,P<0.05; the continuous dosing group was significantly lower than the two months group, P<0.001.4.7 The left atrial myocardial PARP cleaved relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.4841±0.0995,0.6129±0.0938,0.8318±0.0620,0.6292±0.0640, 0.6499±0.0873; The PARP relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than the continuous dosing group, P <0.05; the continuous dosing group was significantly higher than the two months group, P< 0.001.4.8 The left atrial myocardial caspase3 cleaved relatively expression quantity of control group, withdrawal group, continuous dosing group, irbesartan group and two months group were 0.3861±0.0699,0.5262±0.1095,0.6884±0.0929,0.5451±0.0815, 0.6036±0.0763; The caspase3 relatively expression quantity of continuous dosing group was higher than the control group, withdrawal group and irbesartan group, P<0.05; the withdrawal group and irbesartan group were lower than the continuous dosing group, P <0.05; the continuous dosing group was higher than the two months group, P=0.025.(2) Cardiomyocyte study1. Primary culture of left atrial cardiomyocyte1.1 Under microscopic:few cardiomyocytes adhered wall after 2-3h; all cardiomyocytes adhered wall after 16-24h;the morphology of cardiomyocytes was fusiform and triangular, the single cardiomyocyte had beated, the frequency was slow; some cardiomyocytes connected after 48h;most cardiomyocytes connected after 72h, cardiomyocytes beat had synchronized; 70% cardiomyocytes had covered the bottom of the bottle after 4 days, interconnection clusters and the rhythm synchronization; 80-90% cardiomyocytes had covered the bottom of the bottle after 7 days, cardiomyocytes had arranged radial in concentric circles.1.2 By trypan blue staining, the cell survival rate was 85.2% after each digestion.1.3 The left atrial cardiomyocytes was maintaining high vital in a long time, the time of inoculation 2-5 days was the logarithmic growth, then got into the ceiling.1.4 The purity was 95% by identification.2. The apoptosis rate of left atrial cardiomyocyteBy Annexin V-FITC/PI staining, the left atrial cardiomyocyte apoptosis rate of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 0.0400±0.0100%,0.7000±0.2291%,51.3067±6.0515%, 31.8300±13.3143%,6.1200±2.8319%,0.7000±0.1967%; the apoptosis rate of AngⅡ group was significantly higher than other group, P<0.001; AngⅡ+NQDI group was lower than AngⅡ group, P<0.05; other group were no difference each other.3. The ASK1-p38MAPK signaling pathway with apoptosis related gene and protein expression3.1 The left atrial cardiomyocyte BaxmRNA relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 1.0000±0.0000,1.1542±0.3566,3.4350±0.7997, 1.2908±0.5254,1.0825±0.3835,1.1341±0.3269; The BaxmRNA relatively expression quantity of AngⅡ group was significantly higher than other group, P<0.001; other group were no difference each other.3.2 The left atrial cardiomyocyte Bcl-2mRNA relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 1.0000±0.0000,1.0210±0.1609,0.2455±0.0699, 0.5417±0.1792,0.8915±0.2128,0.8796±0.1859; The Bcl-2mRNA relatively expression quantity of AngⅡ group was lower than other group, P<0.05; AngⅡ+NQDI group was higher than AngⅡ group, P<0.05; other group were no difference each other.3.3 The left atrial cardiomyocyte p-ASK1/ASK1 relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 0.9142±0.1208,0.9481±0.2169,1.2989±0.2754, 0.7840±0.0963,0.9391±0.1281,0.9284±0.1904; The p-ASK1/ASK1 relatively expression quantity of Ang II group was higher than other group, P<0.05; other group were no difference each other.3.4 The left atrial cardiomyocyte p-p38MAPK/p38MAPK relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 0.7341±0.1689,0.7467±0.1254,1.1603±0.1413, 0.9261±0.1447,0.7565±0.1434,0.7523±0.2065; The p-p38MAPK/p38MAPK relatively expre-ssion quantity of AngⅡ group was higher than other group, P<0.05; the AngⅡ+NQDI group was higher than other group except AngⅡ group, P<0.05; other group were no difference each other.3.5 The left atrial cardiomyocyte Bax relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 1.1658±0.0945,1.1103±0.1082,1.4355±0.1280,0.8288±0.0644, 1.1231±0.1179,1.1030±0.1415; The Bax relatively expression quantity of AngⅡ group was higher than other group, P<0.05; AngⅡ+NQDI group was lower than other group, P<0.05; other group were no difference each other.3.6 The left atrial cardiomyocyte Bcl-2 relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group,thyroxine+NQDI group were 0.9816±0.1284,0.9675±0.1605,0.6059±0.0949,0.7452±0.1258, 0.7983±0.1407,0.7496±0.1331; The Bcl-2 relatively expression quantity of AngⅡ group was lower than other group, P<0.05; the control group, NQDI group were higher than other group; other group were no difference each other.3.7 The left atrial cardiomyocyte PARP cleaved relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 0.5456±0.0525,0.5574±0.0696,0.9876±0.0800, 0.7921±0.1113,0.5795±0.0679,0.5911±0.0624; The PARP relatively expression quantity of AngⅡ group was higher than other group, P<0.05; the AngⅡ+NQDI group was higher than other group except Ang Ⅱ group, P<0.05; other group were no difference each other.3.8 The left atrial cardiomyocyte caspase3 cleaved relatively expression quantity of control group, NQDI group, AngⅡ group, AngⅡ+NQDI group, thyroxine group, thyroxine+NQDI group were 0.4322±0.0569,0.6229±0.0882,0.7437±0.1075, 0.6328±0.0955,0.6230±0.1278,0.6460±0.1139; The caspase3 relatively expression quantity of AngⅡ group was higher than other group,P<0.05; the control group was lower than other group,P<0.05; other group were no difference each other.Conclusion:1. It’s simple, feasible and high success rate to establish the rabbit model of AF cause of hyperthyroidism, electrophysiological characteristics can be used qualitative and quantitative, which provides the basis of the pathogenesis research about AF cause of hyperthyroidism.2. The apoptosis of left atrial cardiomyocyte had occurred in the process of AF cause of high thyroxine.3. Excessive activation RAS by high thyroxine may be one of the mechanisms about AF cause of hyperthyroidism, then AngⅡ high expression in the circulation and myocardial tissue, which further induce left atrial cardiomyocyte apoptosis, and left atrial electrical and anatomical remodeling.4. The ASK1-p38MAPK signaling pathways may involve in the process of AF cause of hyperthyroidism occurrence and continuance, the mechanism may be excessive thyro-xine through indirect effect to induce and deteriorate left atrial cardiomyocyte apopto-sis by mitochondria pathway. |
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