Experimental Study On RAS Inhibitors Improved The Substrate Of Atrial Fibrillation Underlying Hyperthyroid

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice representing an independent risk factor for cardiovascular events. AF often results in cardiac insufficiency and artery embolism with characters of high morbidity, high disability, high mortality and high cost, which seriously affect the quality of the patients'life. With the population aging, the harm of AF will continue to grow and bring great social and economic burden.RAS plays an important role in the promotion and maintainance of AF. Experimental and clinical study suggested RAS inhibitor could reduce the incidence of AF. ACEI could reduce the atrial pressure, the atrial premature beat, the atrial fibrosis and the recurrence after cardioversion.AF is the most common cardiac complication of hyperthyroidism. AF occurs in 10%-15% of patients with hyperthyroidism. 13% of AF patients without definite cause suffer from disorder of thyroxine related biochemistry index. Some patients can convert to sinus rhythm after thyroid function resume normal. But for some patients, AF still exists even three months later after thyroid function resume normal, which suggests there exists the AF substrate. Therefore, make clear the mechanism has the vital significance in the prevention and treatment of AF underlying hyperthyroidStudies suggested that thyroxine could result in RAS activation by many pathway. However, present studies were mainly concentrated on direct toxic effects of thyroxine. whether RAS activation indirectly involved in the substrate AF was seldom reported. There were no study on whether RAS inhibitors could improve the substrate of AF underlying hyperthyroid.Based on the above mentioned theoretical basis and problems, this study was designed as follows: firstly, hyperthyroid rabbit model was established by intraperitoneal injection levorotatory thyroxine(L-Thy), and given RAS inhibitors: benazepril or irbesartan; Then atrial electrophysiological properties were detected to identify whether RAS inhibitors could improve the abnormal electrophysiological properties underlying hyperthyroid, thus reducing the occurrence of AF; Meanwhile, the atrial structural remodeling, ion channels remodeling and gap junctions remodeling were evaluated to explore the potential mechanism of RAS activation influence on hyperthyroid rabbit; Based on above, proteomics study was performed to comprehensive analysis of the mechanism of hyperthyroid susceptibility to AF and the role of RAS.Overall,The study was composed of the following three parts. PARTâ… THE EFFECTS OF RAS INHIBITORS ON ABNORMAL ELECTROPHY- SIOLOGICAL PROPERTIES UNDERLYING HYPERTHYROIDObjective: To explore the effects of RAS inhibitors on abnormal electrophysiological properties underlying hyperthyroid and the occurrence of induced AF.Methods: 40 New Zealand white rabbit were randomly divided into four groups (n = 10) : sham group, thyroxine group, benazepril group and irbesartan group. The rabbits in sham group and thyroxine group only received peritoneal injection saline or peritoneal injection L-Thy 50ug/kg/day for four weeks respectively. The rabbits in benazepril and irbesartan group received peritoneal injection L-Thyroxine (50 ug/kg/day) and benazepril (1 mg/kg/day) or irbesartan (10 mg/kg/day) orally for four weeks. 4w later, intracardiac electrophysiologic study was performed to evaluate AERP, physiologic rate adaptation of AERP and the occurrence of induced AF.Results: the occurrence of induced AF in thyroxine group was significantly higher than sham group (75% vs 10%), and administered benazepril or irbesartan significantly reduced it (37% and 44%,respectively). Compared with the sham group, the AERP200 in thyroxine group was significantly shortened (97.10±4.75ms vs 75.13±5.41ms, P<0.01). However, no significant difference was found in AERP200 among thyroxine group, benazepril group and irbesartan group (75.13±5.41ms vs 76.63±4.44ms, 79±4.95ms, P=0.28). AERP150 and AERP130 in each group had the same trend with AERP200. The physiologic rate adaptation of the AERP was normal. The AERP130 shortened for 12.70±2.95ms in contrast to AERP200. But the difference in thyroxine group was significantly shorter compared with sham group(6.25±2.55ms vs 12.70±2.95ms). Administered benazepril or irbesartan significantly inhibited the shorten.Conclusion: Benazepril or irbesartan could partly improve abnormal electrophysiological properties underlying hyperthyroid and thus reduce the occurrence of AF. PARTâ…¡THE EFFECTS OF RAS INHIBITORS ON ATRIAL REMODELING UNDERLYING HYPERTHYROIDSECTIONâ… : THE EFFECTS OF RAS INHIBITORS ON ATRIAL STRUCTURAL REMODELING UNDERLYING HYPERTHYROIDObjective: To investigate the effects of RAS inhibitors on atrial structural remodeling underlying hyperthyroid.Methods: 40 New Zealand white rabbits were randomly divided into four groups. The group and treatment strategy were the same to part 1. 4w later, left atrial free wall was carefully dissected from all tissue preparations, and immersed in 10% phosphate-buffered formalin for 24h. After dehydration, each section was cut into 4μm-thick slices. Deparaffinized sections were stained with HE and Masson. Observed the pathological structure changes by microscopy and calculated collagen volume fraction in each groups to evaluate atrial fibrosis.Results: Atrial myocyte from sham rabbits showed a normal composition of sarcomeres distributed throughout the cell, and the intracellular space also appeared normal. Hyperplasia, disturbance, fragment of myocyte fiber, widen intracellular space, inflammation cell filtration and obvious fibrous connective tissue accrementition were observed in thyroxine group. However, these changes were significantly attenuated by benazepril or irbesartan. Masson dyeing showed blue collagen fiber in thyroxine group significantly increased compared with sham group (collagen volume fraction: 17.1±2.2% vs 7.3±1.3%, P<0.01, for sham group and thyroxine group respectively). Benazepril or irbesartan group could significantly restrain the increase (collagen volume fraction: 12.3±1.8% , 11.7±1.2% vs 17.1±2.2%,P<0.01, for benazepril group, irbesartan group and thyroxine group respectively).Conclusion: Benazepril or irbesartan could improve atrial structural remodeling underlying hyperthyroid.SECTIONâ…¡: THE EFFECTS OF RAS INHIBITORS ON ATRIAL ION CHANNEL REMODELING UNDERLYING HYPERTHYROIDObjective: To investigate the effects of RAS inhibitors on atrial ion channel remodeling underlying hyperthyroid.Methods: 40 New Zealand white rabbits were randomly divided into four groups. The group and treatment strategy were the same to part 1. 4w later, left atrial free wall was carefully dissected from all tissue preparations. Extracted RNA and protein with routine methods. The real-time PCR and western blot was performed to detect the expression of L-type Ca²⁺ channel and Ito current related subunit.Results: The mRNA expression of Cav1.2 and Cav1.3 in thyroxine group significantly reduced compared with sham group. Benazepril and irbesartan could significantly inhibite these reductions. The result of western blot demostated the protein expression of Cav1.2 parallelled with the mRNA expression. The mRNA expression of kv1.4 in thyroxine group was higher than in sham group. However, no significant difference were seen for the mRNA expression of kv4.2 and kv4.3. Benazepril and irbesartan significantly increased these mRNA expression. The result of western blot showed that the protein expression of kv4.2 was consistent with its mRNA expression.Conclusion: Benazepril and irbesartan could inhibit the reduction of L-type Ca²⁺ channels related subunit, but couldn't inhibit the changes of Ito related subunit underlying hyperthyroid. SECTIONâ…¢: THE EFFECTS OF RAS INHIBITORS ON ATRIAL GAP JUNCTION REMODELING UNDERLYING HYPERTHYROIDObjective: To investigate the effects of RAS inhibitors on atrial gap junction remodeling underlying hyperthyroid.Methods: 40 New Zealand white rabbits were randomly divided into four groups. The group and treatment strategy were the same to part 1. Four weeks later, left atrial free wall was carefully dissected from all tissue preparations. The real-time PCR and western blot was performed to detect the expression of Cx43 and Cx40. The fluorescent immunohistochemistry was performed to detect the distribution of Cx43 and Cx40.Results: The mRNA expression of Cx43 in thyroxine group significantly increased compared with sham group, but no obvious difference was shown for Cx40. Benazepril and irbesartan increased the mRNA expression of Cx43 and Cx40. In the protein level, the expression of Cx40 in thyroxine group significantly lower than sham group. Benazepril and irbesartan significantly increased the protein expression of Cx40. The protein expression of Cx43 was consistent with the mRNA expression. Fluorescent immunohistochemistry found that thyroxine group Cx43 and Cx40 displayed with a heterogeneous distribution, arranging disorderly and obvious reduction of polar connection. Benazepril and irbesartan significantly extenuated the gap junctions heterogeneity distribution and arranging disorderly, and increased the proportion of polar connection.Conclusion: Benazepril or irbesartan could improve the abnormal expression and distribution of Cx43and Cx40.PARTâ…¢PROTEOMICS STUDY ON THE SUBSTRATE OF THE ATRIAL FIBRILLATION UNDERLYING HYPERTHYROIDObjective: To comprehensive explore the mechanism of hyperthyroid susceptibility to AF and the role of RAS activation in it.Methods: 30 New Zealand white rabbits were randomly divided into three groups (n = 10) : sham group, thyroxine group and irbesartan group The treatment strategy were the same to part 1. Four weeks later, the right atrial free wall was carefully dissected from all tissue preparations;Prepared sample with routine methods; Performed 2-DE and silver stain; Took picture; Detected differential proteins points with PDQest soft; Identificated proteins with MALDI-TOF-MS-MS; Then, analyzed the construction and function of these protein, and evaluated the association between these protein and hyperthyroid susceptibility to atrial fibrillation.Results:The results of 2-DE had high repeatability and the matching rate was 88.6%. The protein spot number was 1314±58, 1364±69 and 1287±47 in sham group, thyroxine group and irbesartan group respectively. Matched and analyzed with PDQuest soft and found 25 differential protein spots. 8 protein spots increased, 10 protein spots decreased and 4 protein spots disappeared were seen in thyroxine group compared with sham group. There were 7 protein spots increased, 13 protein spots decreased and 1 new protein spots disappeared in irbesartan group compared with thyroxine group. 21 protein spots (19 protein) was successfully identified , and we believed there were 13 protein being associated with the substate of AF underlying hyperthyroid.Conclusion:Many proteins were involved in the substrate of AF underlying hyperthyroid. RAS activation was associated with part of these substrates.