Protein Kinase A Regulates SK2Channels In Human Chronic Atrial Fibrillation

Objective:Atrial fibrillation (AF) is one of the most common and serious arrhythmia in clinical practice, contributes to overall cardiovascular morbidity and mortality.According to epidemiological data,1%～2%of the general population suffered from AF, with a5-hold increased risk for stroke. Patients with AF have a wide range of potential complications (stroke, heart failure, et al.) that seriously affected life quality of people. However, the mechanisms of AF are complicated and incompletely understood recently. Evidence suggests that the occurrence of AF has important links with the intracellular calcium overlaod and the alteration of ion channel currents in atrial myocytes. Biochemical evidence indicates that the subtype of Small conductance Ca2+-activated K+channels--SK2channels are more abundantly distribute in human atrial versus ventricular myocytes. Electrophysiological properties of SK2channels are solely activated by alteration of intracellular Ca2+([Ca2+]i) and can be specially blocked by apamin. SK2channels are highly sensitive to [Ca2+]i that alter the membrane potential accompany with [Ca2+]i change.SK2channels are considered as a new candidate in human chronic AF pathogenesis and a novel potential drug target for AF therapy recently. Some reports showed that the overload of [Ca2+]i and the increase sensitivity to [Ca2+]i of SK2channels occur in AF.Thus, we hypothesis that the overload of [Ca2+]i as well as the inceresed sensitiveness to [Ca2+]i may alter the function of SK channels which is an interesting target in AF and the abnormal function of SK2channels may invoves the occurrence and maintaince of AF. Besides, data showed that protein kinase A (PKA) involved in the Ca2+cycling of atrial myocytes and countributes to the overload of [Ca2+]i in AF. The regulation of SK2channels by PKA were reported in nervous system, but never in AF.This study mainly investigates the alteration of SK2channel currents, as well as the functional relationship between PKA and SK2channels in human AF with patch clamp technique. Methods:(1) Electrophysiological Recording30patients undergoing extracorporeal circulation cardiac surgery were divided into2groups:20patients with SR and10patients with AF. Cardiomyocytes isolation:Human right atrial appendages were quickly immersed in oxygenated, normally Ca2+-free cardioplegic solution and transported to the laboratory quickly. Then the samples were washed and chopped into small pieces (1mm3) in oxygenated cardioplegic solution. Single myocytes were enzymatically dissociated by our laboratory modified procedure of enzymatic dissociation with protease XXIV (12.2U/mL) and collagenase V (150U/mL). SK2channel currents recording:Chose the smooth, well-striated and rod-shaped myocytes for the experiments. Patch clamp experiments were performed with EPC10amplifier. The amplified and filtered (2kHz) signals were sampled and stored in a computer. PatchMaster software was used for data acquisition and Clampfit10.1as well as OriginPro8.0software was used for data analysis.The experiment including:(1) Recording the SK2channel currents with whole-cell patch clamp experiments in isolated human atrial cardiomyocytes.(2) Compare the alteration of SK2channel currents in AF groups.(3)The regulation of SK2channels by PKA were detected in isolated human atrial cardiomyocytes with whole-cell patch clamp experiments from SR and AF groups.(2)Protein levels detecting The total protein levels and protein kinase A levels were dected by BCA and ELISA methods from20tissues in SR patients and10tissues in AF patients.Results:(1) The functional characteristics of SK2channel currents in human atrial cardiomyocytes:①The integrated inward currents before and after application of apamin(10-7mol/L) were recorded; the apamin-sensitive SK2currents were obtained with digital subtraction.②In the whole-cell patch clamp,10-7mol/L apamin blocks integrated inward currents partly that can recovery to the basic state when apamin was washed out. The SK2channel currents recorded in this experiment are inwardly rectifying and can be specially blocked by apamin.(2) The difference of SK2channel currents between SR and AF groups:The experiment was performed at room temperature. The currents were recorded when the concentration of free [Ca2+] in the internal solution was5×10-7mol/L. In AF, the SK2channel current density (pA/pF) as well as the ration in the integrated inward currents is significantly upregulated in AF groups.The SK2channel current density at-130mV was-2.61±0.14pA/pF in SR group vs.-6.21±0.59PA/pF in AF group(nSR=15, nAF±6,p<0.05); The ration of SK2channel currents in the integrated inward currents was20.01±1.44%vs.42.87±1.79%(nSR=15, nAF=6, p<0.05).(3)The effect of H-89on SK2channel currents PKA-selective inhibitor H-89(10μmol/L) reduced SK2currents in the cardiomyocytes from SR and AF patients, with a bigger magnitude of reduction in AF group. H-89reduced SK2channel current density at-130mV by39.27±4.08%in SR group vs.76.32±2.94%in AF group (nSR=6, nAF=5, p<0.05); H-89reduced the ration of SK2channel in the integrated inward currents at-130mV by37.48±4.77%in SR group vs.56.40±3.66%in AF group(nSR=6, nAF=5,p<0.05). Morever, the ration of SK2currents in the integrated inward currents can nearly be normalized to the SR levels after the application of H-89.(4) The alteration of PKA levels The PKA levels was511.91±7.22pg/mL in SR group vs.444.09±7.88pg/mL in AF atrial tissues (nSR=11,nAF=13, p<0.05).Conclusions:(1) The SK2channel currents recorded in this experiment are inwardly rectifying and can be specifically blocked by apamin.(2) When the internal solution [Ca2+]i is5×10-7mol/L, SK2channel current density and the ration in the integrated inward currents in AF group were significantly larger than that in SR group which suggested that the increase of SK2channel currents underlies the occurrence and maintainance of AF.(3) SK2channels can be regulated through PKA-dependent way, PKA-slective inhibitior H-89reduced SK2channel current density in human atrial cardimyocytes from SR and AF patients, with a stronger effect in AF group.(4) The alteration of PKA levels ocurres in the atrial tissues from AF patients that involves the cardiomyocytes electric remodeling in AF.