Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca2+-Activated K+ Current in Patients with Chronic Atrial Fibrillation

Figure 1. Patch clamp results showing the IKAS in patients with SR and AF. (A) Representative currents recorded in atrial myocytes from SR and AF using voltage steps at a holding potential of −60 mV, step from −130 to +60 mV and in the absence or presence of 100 nmol/l apamin at 500 nmol/l [Ca2+]i. IKAS was obtained using digital subtraction of the currents recorded in absence or presence of apamin. (B) Current-voltage relation curves of SK currents in atrial myocytes from SR (n=35 cells from 12 patients) and AF (n=24 cells from 9 patients). *P < 0.05, ** P < 0.01 vs. SR.

Figure 2. qRT-PCR and Western blotting results showing KCNN 1–3 mRNA levels and KCa 2.1/2.2/2.3 proteins levels in the atria of SR controls (n=20) and AF patients (n=32). (A) mRNA levels of KCNN1, KCNN2, and KCNN3 in SR and AF. (B) mRNA expression differences of KCNN1, KCNN2, and KCNN3 in SR group. (C) mRNA expression differences of KCNN1, KCNN2, and KCNN3 in AF group. (D) KCa 2.1–2.3 (SK1–3) proteins expression changes in SR (n=20) and AF (n=32). * P<0.05 vs. SR.

Figure 3. Steady-state Ca2+ sensitivity curve of SK currents in SR and AF. SK current was normalized to maximal currents with a free Ca2+ of 1 μmol/l. Data were fitted with Hill equation: y=1/(1+[Kd/χ]n), where y represented the normalized IKAS and x was the concentration of intracellular free Ca2+; Kd value was the concentration of intracellular free Ca2+ at half-maximal activation; n was the Hill coefficient. Numbers in parentheses indicated the number of atrial myocytes with successful recording.

Figure 4. Diastolic free cytosolic Ca2+ levels in SR and AF. (A) Atrial myocytes loaded with 10 μmol/l Fura-2 AM measured by epifluorescence. (B) Diastolic free Ca2+ levels in SR (n=8 atrial myocytes from 4 patients) and in AF (n=10 atrial myocytes from 5 patients. * P<0.05 vs. SR.

Figure 5. Effects of CaMKII inhibitor KN-93 on IKAS in atrial myocytes from SR and AF groups. (A) Total K+ currents recorded in the absence and presence of KN-92 (1 μmol/L), KN-93 (1 μmol/L), and KN-93 with apamin (0.1 μmol/L) in the extracellular solution with ramp-pulse protocol (test pulse from −130 mV to +60 mV, with holding membrane potential −60 mV). (B) Time-course of total K+ current recorded with ramp protocol treated with absence and presence KN-92, KN-93, or KN-93 with apamin. (C) Inhibited percentage of KN-93 on IKAS in SR and AF with holding potential at −110mV. ** P<0.01 vs. SR.

Figure 6. Effects of specific membrane-permeable CaMKII peptide inhibitor AIP on IKAS in atrial myocytes from SR and AF groups. (A) Total K+ currents recorded in the absence and presence of AIP (0.5 μmol/L), AIP with apamin (0.1 μmol/L) in the extracellular solution with ramp-pulse protocol (test pulse from −130 mV to +60 mV, with holding membrane potential −60 mV). (B) Time-course of total K+ current recorded with ramp-pulse protocol treated with absence and presence AIP or AIP with apamin. (C) Inhibited percentage of AIP on IKAS in SR and AF group with holding potential at −110 mV. ** P<0.01 vs. SR.

Figure 7. Protein levels of CaM, total CaMKII, and autophosphorylation of CaMKII at Thr286 and Thr287 phosphorylation sites (both n = 6 for SR and AF). (A) and (B) Note that CaM and CaMKII expression were increased in AF. (C) Phosphorylated CaMKII ((Thr286)p-CaMKII) expression in AF. (D) and (E) Phosphorylated CaMKII ((Thr287)p-CaMKII) expression and the proportion of autophosphorylation of CaMKII at Thr287 in total CaMKII in AF. * P < 0.05 represented the difference between SR and AF.

Figure 8. Protein expressions of (Thr287)p-CaMKII and SKca2.2 before and after treatment with KN-92 and KN-93 in neonatal rat atrial myocytes. (A) Effects of KN-92 and KN-93 on the expression of (Thr287)p-CaMKII in neonatal rat atrial myocytes (n=4). (B) Effects of KN-92 and KN-93 on the expression of SK2 protein. * P<0.05 vs. KN92.