Heterogeneous upregulation of apamin-sensitive potassium currents in failing human ventricles

Abstract

Background: We previously reported that IKAS are heterogeneously upregulated in failing rabbit ventricles and play an important role in arrhythmogenesis. This study goal is to test the hypothesis that subtype 2 of the small-conductance Ca(2+) activated K(+) (SK2) channel and apamin-sensitive K(+) currents (IKAS) are upregulated in failing human ventricles.

Methods and results: We studied 12 native hearts from transplant recipients (heart failure [HF] group) and 11 ventricular core biopsies from patients with aortic stenosis and normal systolic function (non-HF group). IKAS and action potential were recorded with patch-clamp techniques, and SK2 protein expression was studied by Western blotting. When measured at 1 μmol/L Ca(2+) concentration, IKAS was 4.22 (median) (25th and 75th percentiles, 2.86 and 6.96) pA/pF for the HF group (n=11) and 0.98 (0.54 and 1.72) pA/pF for the non-HF group (n=8, P=0.008). IKAS was lower in the midmyocardial cells than in the epicardial and the endocardial cells. The Ca(2+) dependency of IKAS in HF myocytes was shifted leftward compared to non-HF myocytes (Kd 314 versus 605 nmol/L). Apamin (100 nmol/L) increased the action potential durations by 1.77% (-0.9% and 7.3%) in non-HF myocytes and by 11.8% (5.7% and 13.9%) in HF myocytes (P=0.02). SK2 protein expression was 3-fold higher in HF than in non-HF.

Conclusions: There is heterogeneous upregulation of IKAS densities in failing human ventricles. The midmyocardial layer shows lower IKAS densities than epicardial and endocardial layers of cells. Increase in both Ca(2+) sensitivity and SK2 protein expression contributes to the IKAS upregulation.

Figure 1.

I_KAS densities of isolated ventricular myocytes. A, Representative K⁺ current traces obtained from a non‐HF (top) and an HF (bottom) ventricular myocyte. The K⁺ currents were recorded with an intrapipette free‐Ca²⁺ of 1 μmol/L in the absence (I_K‐baseline) and the presence of 100 nmol/L apamin (I_K‐apamin). I_KAS was calculated as the difference between I_K‐apamin and I_K‐baseline. B, Current‐voltage (I‐V) relationship of I_KAS obtained from failing ventricles (n=22 cells from 11 patients) and nonfailing ventricles (n=16 cells from 8 patients). Current densities were presented as median [25th percentile; 75th percentile]. C, Transmural distribution of I_KAS in failing ventricles. I_KAS density at 0 mV with an intrapipette free‐Ca²⁺ of 1 μmol/L recorded from 3 different layers (Epi, epicardium; Mid, midmyocardium; Endo, endocardium). The midmyocardium had lower current density than the other 2 layers (P=0.005 for Kruskal–Wallis test; *P<0.05 for post hoc Mann–Whitney–Wilcoxon test). D, The graph shows the time course of total K⁺ currents measured at 0 mV with ramp‐pulse protocol (test pulse: between +20 and −140 mV; holding membrane potential: −80 mV; pulse frequency: every 6 seconds) in a cardiomyocyte isolated from a failing heart. The current density during apamin (b) and after washout (c) showed only slight differences, indicating incomplete washout of apamin. HF indicates heart failure.

Figure 2.

Steady‐state Ca²⁺ dependency of I_KAS in non‐HF and HF ventricular myocytes. I_KAS was normalized to the maximal I_KAS with a free Ca²⁺ of 10 μmol/L and plotted as a function of Ca²⁺ concentration. The data were fitted with Hill equation: y=1/(1+[K_d/x]ⁿ), where y indicates the normalized I_KAS and x is the intrapipette free calcium; K_d is the concentration of intrapipette free calcium at half‐maximal activation of I_KAS; and n is the Hill coefficient. Error bars represent SEM. Numbers in parentheses indicate the number of cells patched. Normalized currents were presented as mean±SEM. HF indicates heart failure.

Figure 3.

Effects of apamin on APD in ventricular cardiomyocytes. A, Action potentials recorded in 1 non‐HF and 2 HF ventricular myocytes at baseline (solid line) and in the presence of 100 nmol/L apamin (dotted line). B, The box plots depict APD₈₀ in various conditions (*P<0.05 for Mann–Whitney–Wilcoxon test). APD₈₀ were presented as median [25th percentile; 75th percentile]. HF indicates heart failure.

Figure 4.

SK2 proteins in HF and non‐HF (NF) ventricles. A, Left, Western blot analysis of atrial and ventricular tissues from a 57‐year‐old male patient with paroxysmal atrial fibrillation and normal LVEF. Right, Western blot analysis of SK2 and glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) in non‐HF (n=5) and HF (n=6) human ventricles. B, Aggregated results of SK2 protein (66 kDa) and presumed SK2 short form protein (31 kDa) expression in non‐HF and HF groups. The signal intensity was normalized to the GAPDH. Data are presented as median [25th percentile; 75th percentile]. HF indicates heart failure; LVEF, left ventricular ejection fraction.

Figure 5.

Anti‐KCNN2 immune reactivity in human cardiac tissue. A, Color and black‐and‐white panels show confocal fluorescence images and differential interference contrast (DIC) images, respectively, taken from nonfailing and failing hearts stained for KCNN2. Arrows denote areas of yellow fluorescence that were also detectable in the absence of the secondary antibody and result from the overlap of unspecific red and green autofluorescence in the tissue from nonfailing and failing hearts. B, Confocal fluorescence and DIC images taken from sections that were incubated with the secondary antibody only. C, Anti‐KCNN2 immunofluorescence in small‐artery walls. Scale bar, 20 μm.