Role Of Protein Phosphatase Inhibitoroa On SK2Channels In Right Atrial Myocardium Of Patients With Atrial Fibrillation

Objective:Atrial fibrillation (Af) is the most frequent sustained arrhythmia in the clinical practice, occurring in1%-2%of the general population. Atrial fibrillation is an important risk factor for heart failure and other adverse cardiac events. Atrial electrical remodeling and intracellular calcium regulation abnormalities play an important role in the incidence of atrial fibrillation and maintenance. Small-conductance calcium-activated potassium channels (SK.2) is the recent discovery of an important class of calcium-dependent potassium channels. The SK2is expressed differentially in atria compared with ventricles. Because of the marked differential expression of SK2channels in the heart, specific ligands for SK2currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes. From another perspective, atrial dysfunction (such as atrial fibrillation) may also indicate the change of the SK2channels. Kinase and protein phosphatase enzymes in myocardial cells will be abnormal in atrial fibrillation. This becomes of particular importance in the Atrial fibrillation, where abnormalities in the activity of enzymes in the kinase and phosphatase families disturb this fine equilibrium of phosphorylation. SK.2channels are multiprotein complexes and, in addition to the pore-forming subunits and CaM, have constitutively bound protein kinase CK2and protein phosphatase2A (PP2A). Abnormal protein phosphatase enzymes likely will have an impact to SK2channel function. Changes of SK2channels in atrial fibrillation and protein phosphatase on the SK2channels remains unclear. Therefore, we studied normal and Af patients myocytes to test the change of SK2channels current and the regulation of OA (a kind of protein phosphatase inhibitors) on SK2channels with whole-cell patch clamp in Atrial myocytes. Explore the role of SK2current in atrial fibrillation, and to clarify the influence of the protein phosphatase inhibitors on the SK2current from the cellular level.Methods:Human right atrial appendages were obtained from patients undergoing extracorporeal circulation cardiac surgery were used for patch clamp studies.Cutted the samples into small pieces (1mm3) in oxygenated cardioplegic solution. Single myocytes were enzymatically dissociated by modified procedure of enzymatic dissociation with protease and collagenase. Choose the smooth, well-striated and rod-shaped myocytes for the experiments. Currents were recorded with the whole-cell patch-clamp technique at room temperature. A voltage clamp protocol incuding preconditioning depolarizing clamp steps (-130to+60mV, duration of the pulse is200ms). Conventional patch clamp experiments were performed with an Axopatch700B amplifier. The amplified and filtered (1KHz) signals were sampled and stored on a computer. Clampex10.1software was used for data acquisition and Clampfit10.1was used for data analysis. The whole-cell patch clamp was used to observe the regulation of SK2channels (Af and SR group) by OA. Results:We recorded macroscopic currents of atrial myocytes in whole-cell patch.The macroscopic currents was voltage-dependent and inwardly rectifying.The macroscopic current density at-130mV was-21.32±2.84pA/pF in SR (n=23) vs.41.51±5.38pA/pF in Af (n=11,P<0.05).The I-V curve showed SK2channels were inwardly rectifying. The SK2current density at-130mV was3.67±0.37pA/pF in SR (n=9) vs.9.81±2.54pA/pF in Af (n=9,P<0.01). This experiment by adding the protein phosphatase inhibitor OA After accession to Apamin. In the-130mV to join the Apamin,the current density was-14.80±4.03pA/pF. Then re-join the OA, the current density was-12.12±4.18pA/pF(n=6), P=0.20,which indicating that OA main effect on SK2, less impact on other current. OA inhibition on the Af group inward rectifier mixed current rate is greater than the SR group, inhibition of the SR group was12.51%±4.02(n=6), the Af group, the current inhibition rate was32.88%±9.20(n-4, P<0.05).Conclusions:Af group SK2current is greater than the SR group;PP2A involved in the regulation of SK2channels, inhibition of PP2A allows SK2current density decreases, and the lower of the Apamin sensitivity; SK2channel enhanced calcium ion sensitivity in Af group; Af group the current inhibition rate of OA larger than the SR group.