Expression Of Connexin In Atrium Of Atrial Fibrillation And Its Signal Transduction Pathway

Objective Atrial fibrillation (AF) is currently the most common sustained clinical arrhythmia and is responsible for a substantial proportion of hospital costs incurred in the treatment of cardiac rhythm disorders. AF is associated with a variety of complications, including thromboemboli, a loss of the fine adjustment of ventricular rate to the body's precise metabolic needs, potential impairment of cardiac function and subjective symptoms like palpitations, dizziness, breathlessness and chest pain. Although initation of AF has been intensively investigated ,theories dealing with determinants of AF do not explain the trend of this arrythmia to become persistent.AF is primarily characterized by electrical remodeling and structural remodeling, it has recently been shown that changes in intercellular coupling at gap junctions may play a critical role in the short-term electrical remodeling and structural remodeling of atrial fibrillation.The purpose of this study is to investigate whether atrial expression of connexin 40(Cx40) and connexin43(Cx43) is altered and whether the signal transduction systems were activated at the molecular atrial tissue level in patients with AF.MethodsHuman studies We investigated 63 patients undergoing cardiac surgery .Right appendages were obtained from 21 patients with chronic AF , 13 patients with documented episodes of paroxysmal AF , 16 patients with sinus rhythm and without valve disease and 13 patients with sinus rhythm with valve disease. Left atrium tissues were obtaimed from 16 patients with chronic AF , 11 patients with documented episodes of paroxysmal AF and 10 patients with sinus rhythmwith valve disease.RNA extration and RT-PCRTotal RNA was extrated from the tissues according to the Tri-zol Reagent Protocol (Gibco).RT-PCR was performed as follows: cDNA was synthesized from 1.5 g of total RNA using M-MLV reverse transcriptase (Promega) and random primers. Single-stranded cDNA was incubated on ice with a specific pair of primers designed to amplify cDNA coding for Calcineurin B, MKP-1, and P 2-MG as an internal control. Amplification were performed individely. Amplified products were electrophoresized in 1.5% ethidium bromide-stained agarose gel. Theintensities of the amplified cDNA fragment were estimated using a video-densitometer. Western-blot immunoblot AnalysisAfter boiling for 3 mins, 30 g of the sample was subjected to SDS-PAGE in a 10% SDSgel and transferred to PVDF membrane, which was followed by blocking for 1 h with 5 % non-fat milk in TBST. The blots were incubated for 12h at 4 with the primary antibodies (at a1 : 1000 dilution for Cx40,Cx43,ERK1,P-ERKl and GAPDH) followed by incubation for2 h with secondary antibody (horseradish peroxidase conjugated,at a 1 : 1000 dilution).Immunoreactive bands were visualized with enhanced chemiluminescence reagents. Immunohistochemistry Fixed cryostat section of atrial right appendages and left atrium were stained withanti-Cx40(1:100 dilution ) and anti-Cx43(1:100 dilution ) antibody . Samples were incubated with secondary antibodies.ResultsClinical characteristics:Preoperative data for the all groups are listed in Table (4) .There were no significantdiference in left ventricular (LV)diameter, LV ejection fraction, LV posterior wall thickness and LV septal thickness . left atrial diameters were higher in patients with chronic AF or paroxysmal AF than in patients with NSR with valve disease or (and) NSR without valve disease.Analysis of calcineurin B mRNA, MKP-1 mRNA by RT-PCRIn the right appendages .Increased amounts of Calcineurin B mRNA, MKP-3 mRNA(Chronic AF: 1.38 0.29,1.12 0.26; paroxysmal AF: 1.29 0. 54, 1.06 0.28) were found patients with Chronic AF or paroxysmal AF than those of NSR with valve disease or and NSR without valve disease. (P<0.05). In the left atrium,the mRNA expression of Calcineurin B, MKP-1 in the patients with Chronic AF or paroxysmal AF were higher than those of patients withNSR with valve disease(P<0...