| Background Atrial fibrillation (AF) is the most common arrhythmia with an overall prevalence of almost 1%, which increases significantly with age, and affects about 5% of individuals older than 65 years. The growing prevalence of AF together with the increased risk of stroke, congestive heart failure and mortality will bring great suffering to patients, and increase the burden on the society. However, there are no effective medical treatments to AF at present. It's a hot subject to study the pathophysiological mechanisms underlying AF, in order to develop safe and more effective treatments. AF is primarily characterized by electrical remodeling and structural remodeling, and calcium overload is likely the primary factor mediating both phenomena. Na(+)/Hwexchanger isoform 1 (NHE1) indirectly participates in the regulation of intracellular calcium, and it has recently been shown that the activation of NHE1 is one mechanism underlying short-term electrical remodeling and structural remodeling. However, the cellular metabolisms of short-term AF and chronic AF are not complete the same. It has been reported that short-term AF is associated with the imbalance between oxygen supply and demand, which may potentially lead to atrial ischemia ; conversely, the balance will be restored in chronically fibrillating atria. The present study aims toelucidate the expression of NHE1 in chronic AF, and explore the molecular basis of electrical remodeling and structural remodeling.Materials and Methods1. Rabbit Na(+)/H<+) exchanger-1 polyclonal antibody(primary antibody): CHEMICON INTERNATIONAL Lot number:210819652. Goat anti-rabbit horseradish-peroxidase coupled IgG antibody (secondary antibody): Beijing Zhongshan corporation Lot number.505193. Nitrocellulose membrane: Amersham, Life Science, Hybond-C4. Atrial samples: lOOmg atrial samples were obtained from 24 patients' right appendages during the open heart surgery. The number of cases in both AF group and sinus rhythm group is 12.5. Protein extraction and Western Blotting analysis: Each atrial tissue sample was homogenized in 1 ml of ice cold lysis buffer for 10 minutes followed by centrifugation at 14000 rpm at 4癈 for another 10 minutes. The supernatants were collected, and then protein concentration was determined by the method of Lowry . Aliquots were mixed with loading buffer, and boiled for 3 minutes at 100癈. For classic Western Blotting, 20ug of total protein per lane was separated by an 8% SDS-PAGE gel. Proteins were transferred onto the nitrocellulose membrane. The membrane was washed for only about 1 minute in TBST, blocked for 1 hour in 5% nonfat milk/TBST, and then it was washed three times for 30 minutes in TBST, incubated with primary rabbit polyclonal antibody for the cardiac NHE1(1:1000) overnight at 4癈 in the refrigerator. After three washes for 30 minutes in TBST, the membrane was incubated with 1:1000 dilution of the goat anti-rabbit horseradish-peroxidase coupled secondaryantibody for 2 hours at room temperature. After four washes for 2 hours inTBST, the membrane was incubated with components A and B of theenhanced chemiluminescence for 1 minute, exposed to x-ray film for 1minute, and the signals were developed. Quantification of western blotswas performed on a computer using Kodak image analysis system.6. Statistics: Results are expressed as mean盨EM, and comparison wasdetermined by student's t test, performed on SPSS statistics soft package.ResultsThere was no significant difference in the protein expression of NHE1between chronic AF group and sinus rhythm group(0.90?.29 vs 0.86?0.26,t=0.382,p>0.05).ConclusionThe protein expression of NHE1 in chronic AF is not significantlydifferent from that in sinus rhythm , suggesting that NHE1 may notparticipate in electrical remodeling and structural remodeling in chronicAF.
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