Altered Repolarization Reserve in Failing Rabbit Ventricular Myocytes: Calcium and β-Adrenergic Effects on Delayed- and Inward-Rectifier Potassium Currents

Abstract

Background: Electrophysiological remodeling and increased susceptibility for cardiac arrhythmias are hallmarks of heart failure (HF). Ventricular action potential duration (APD) is typically prolonged in HF, with reduced repolarization reserve. However, underlying K⁺ current changes are often measured in nonphysiological conditions (voltage clamp, low pacing rates, cytosolic Ca²⁺ buffers).

Methods and results: We measured the major K⁺ currents (I_Kr, I_Ks, and I_K1) and their Ca²⁺- and β-adrenergic dependence in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched controls). APD was significantly prolonged only at lower pacing rates (0.2-1 Hz) in HF under physiological ionic conditions and temperature. However, when cytosolic Ca²⁺ was buffered, APD prolongation in HF was also significant at higher pacing rates. Beat-to-beat variability of APD was also significantly increased in HF. Both I_Kr and I_Ks were significantly upregulated in HF under action potential clamp, but only when cytosolic Ca²⁺ was not buffered. CaMKII (Ca²⁺/calmodulin-dependent protein kinase II) inhibition abolished I_Ks upregulation in HF, but it did not affect I_Kr. I_Ks response to β-adrenergic stimulation was also significantly diminished in HF. I_K1 was also decreased in HF regardless of Ca²⁺ buffering, CaMKII inhibition, or β-adrenergic stimulation.

Conclusions: At baseline Ca²⁺-dependent upregulation of I_Kr and I_Ks in HF counterbalances the reduced I_K1, maintaining repolarization reserve (especially at higher heart rates) in physiological conditions, unlike conditions of strong cytosolic Ca²⁺ buffering. However, under β-adrenergic stimulation, reduced I_Ks responsiveness severely limits integrated repolarizing K⁺ current and repolarization reserve in HF. This would increase arrhythmia propensity in HF, especially during adrenergic stress.

Keywords: action potential; calcium/calmodulin-dependent protein kinase II; electrophysiology; heart failure; potassium channels.

Figure 1. Frequency- and Ca²⁺-dependent changes of AP parameters in HF

(A–B) Representative action potentials (APs) recorded at 1 Hz and 2 Hz pacing in heart failure (HF) and healthy age-matched control cells with physiological solutions (Physiol). (C–D) Frequency dependence of AP duration measured at 95% of repolarization (APD₉₅) and mid-plateau potential (V_mid-plateau). (E–F) Representative APs when cytosolic Ca²⁺ was buffered to nominally zero using 10 mM BAPTA in pipette solution (BAPTA_i) at 1 Hz and 2 Hz pacing. (G) Resting membrane potential (V_rest) is less negative in HF in line with decreased AP peak (V_peak) at 1 Hz. (H) Both maximal rate of rise (dV/dt_max) and maximal rate of phase 3 repolarization (-dV/dt_max) are significantly decreased in HF at 1 Hz. Columns and bars represent mean±SEM. n refers to cells/animals measured in each group. ANOVA with Bonferroni posttest, Ctl vs. HF, *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Increased temporal variability of APD and arrhythmogenesis in HF

(A) Representative Poincare plots of 50 consecutive APD₉₅ values at 1 Hz steady-state pacing in 4 individual myocytes from HF and age-matched control animals recorded with either preserved or buffered [Ca²⁺]_i. (B) Frequency-dependent short-term variability of APD₉₅ (STV). (C) Cumulative distribution curves of individual beat-to-beat variability values at 1 Hz pacing. (D) Percentage of total APs measured at 1 Hz pacing having beat-to-beat APD₉₅ variability of >5 ms and >10 ms in consecutive beats. Columns and bars represent mean±SEM. n refers to cells/animals measured in each group. ANOVA with Bonferroni posttest, *p<0.05, **p<0.01, ***p<0.001. (E) Arrhythmogenic diastolic activities were tested using the pacing protocol shown above. (Left) Representative records in control, HF and AIP (CaMKII inhibitor, 1 μM) treated HF cells are shown below. HF cells frequently showed delayed afterdepolarizations (DADs, enlarged in insets) and spontaneous AP (sAP, enlarged in inset). Early afterdepolarizations (EADs, enlarged in inset) were also superimposed on the sAP repolarization. (Right) Percentage of cells having DAD and sAP. 10/12 cells and 5/12 cells showed DADs and sAP in HF, respectively, whereas no DAD, sAP or EAD was observed in control and following AIP treatment in HF.

Figure 3. Ca²⁺-dependent upregulation of I_Kr in HF under AP-clamp

The rapid component of delayed rectifier K⁺ current (I_Kr) was measured as E-4031 (1 μM)-sensitive current in HF and age-matched control cells. AP-clamp using a prerecorded typical AP (shown above) was applied at 2 Hz. (A) I_Kr traces (mean±SEM) recorded under preserved [Ca²⁺]_i cycling (Physiol). I_Kr is not only increased in HF cells having [Ca²⁺]_i transients, but it activates earlier during the AP. (B) I_Kr traces (mean±SEM) recorded under buffered [Ca²⁺]_i using 10 mM BAPTA in the pipette (BAPTA_i). Buffering [Ca²⁺]_i significantly reduced I_Kr density in HF below the control level, but it had no effect in control. (C) I_Kr traces (mean±SEM) recorded in cells pretreated with the specific CaMKII inhibitor peptide AIP (1 μM). AIP had no effect on I_Kr both in control and in HF. (D) Peak I_Kr density is significantly upregulated in HF under AP by a Ca²⁺-dependent, but CaMKII-independent pathway. (E) I_Kr density measured at the mid-plateau increased in HF indicating earlier I_Kr accumulation during AP, which occurred independent of [Ca²⁺]_i level and CaMKII activity. (F) Net charges carried by I_Kr in HF and age-matched control. Columns and bars represent mean±SEM. n refers to cells/animals measured in each group. ANOVA with Bonferroni posttest, *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Ca²⁺/CaMKII-dependent upregulation of I_Ks in HF under AP-clamp

The slow component of delayed rectifier K⁺ current (I_Ks) was measured as HMR-1556 (1 μM)-sensitive current in HF and age-matched control cells. AP-clamp using a prerecorded typical AP (shown above) was applied at 2 Hz. (A) I_Ks traces (mean±SEM) recorded under preserved [Ca²⁺]_i cycling (Physiol). Basal I_Ks is a tiny current under AP, yet it is significantly upregulated in HF cells having [Ca²⁺]_i transients. (B) I_Ks traces (mean±SEM) recorded under buffered [Ca²⁺]_i using 10 mM BAPTA in the pipette (BAPTA_i). Buffering [Ca²⁺]_i reduced I_Ks in HF back to its control level. (C) I_Ks traces (mean±SEM) recorded in cells pretreated with the specific CaMKII inhibitor peptide AIP (1 μM). AIP abolished the I_Ks upregulation in HF. (D) Peak I_Ks density is significantly upregulated in HF under AP by a Ca²⁺/CaMKII-dependent pathway. (E) I_Ks density measured at the mid-plateau of the AP. (F) Net charges carried by I_Ks in HF and age-matched control. Columns and bars represent mean±SEM. n refers to cells/animals measured in each group. ANOVA with Bonferroni posttest, *p<0.05, **p<0.01.

Figure 5. Reduction of I_K1 in HF under AP-clamp

The inward rectifier K⁺ current (I_K1) was measured as Ba²⁺ (100 μM)-sensitive current in HF and age-matched control cells. AP-clamp using a prerecorded typical AP (shown above) was applied at 2 Hz. (A–C) I_K1 traces (mean±SEM) recorded under preserved [Ca²⁺]_i cycling (Physiol), [Ca²⁺]_i buffering (BAPTA_i) and CaMKII inhibition using AIP. I_K1 was significantly reduced in HF in all these conditions. (D–F) Statistics for peak I_K1 density shows that the reduction is not influenced by [Ca²⁺]_i buffering or acute CaMKII inhibition. (E) I_K1 density measured at the mid-plateau of the AP was also reduced. (F) Net charges carried by I_K1 was further reduced in HF when measured with [Ca²⁺]_i buffering. Columns and bars represent mean±SEM. n refers to cells/animals measured in each group. ANOVA with Bonferroni posttest, *p<0.05, **p<0.01, ***p<0.001.

Figure 6. Altered response of K⁺ currents to β-AR stimulation in HF

I_Ks, I_Kr and I_K1 currents were recorded following 2 min pretreatment with beta-adrenergic receptor agonist isoproterenol (ISO, 10 nM). (A) I_Ks traces (mean±SEM) under canonical AP-clamp at 2 Hz measured with preserved [Ca²⁺]_i cycling (Physiol) and [Ca²⁺]_i buffering using 10 mM BAPTA in the pipette (BAPTA_i). (B) I_Kr traces (mean±SEM) under AP-clamp following ISO pretreatment in HF and age-matched control cells. (C) I_K1 traces (mean±SEM) under AP-clamp following ISO pretreatment in physiological solutions and in BAPTA_i. (D) Robust upregulation of I_Ks peak and net charge induced by ISO, which is reduced in BAPTA_i indicating a Ca²⁺-dependent pathway in mediating the response of β-AR stimulation on I_Ks besides the classical PKA-dependent phosphorylation. HF cells showed significantly reduced I_Ks accumulation upon ISO application both with and without [Ca²⁺]_i buffering. (E) I_Kr is slightly modulated by ISO both in control and HF. (F) I_K1 peak density is not influenced by ISO, but I_K1 net charge is slightly increased in HF due to altered rectification. Symbols and bars represent mean±SEM. n refers to cells measured in each group, and the cells in each group came from three to five individual animals. ANOVA with Bonferroni posttest, *p<0.05, **p<0.01, ***p<0.001.

Figure 7. β-AR responsiveness of K⁺ currents in HF

Dose-response effect of isoproterenol (ISO) on I_Ks, I_Kr and I_K1 peak densities under AP-clamp at 2 Hz. Pipette solution contained 10 mM BAPTA. (A) I_Ks measured as HMR-sensitive current showed a robust accumulation following ISO application. I_KS sensitivity (EC₅₀) to ISO was unchanged in HF, whereas the response was significantly reduced in HF. (B) I_Kr was minimally affected by ISO treatment; however, neither the ISO-sensitivity nor the magnitude of the response were altered in HF. (C) I_K1 peak density did not change following ISO application under buffered [Ca²⁺]_i conditions and I_K1 was uniformly decreased in HF. EC₅₀ values, Hill coefficients and maximum responses were determined by fitting data to the Hill equation, indicated by solid lines. Symbols and bars represent mean±SEM. n refers to the number of cells measured in each group, and the cells in each group came from three to five individual animals. ANOVA with Bonferroni posttest, *p<0.05, **p<0.01, ***p<0.001.

Figure 8. Relative contribution of each K⁺ current to net repolarizing K⁺ current in HF

Relative contributions and magnitudes of the major repolarizing K⁺ currents (I_Kr, I_Ks, I_K1) during AP are compared in different phases of the repolarization process in HF to those in age-matched control. (A) I_Kr, I_Ks and I_K1 traces in control and HF measured under AP-clamp at 2 Hz without using any Ca²⁺ buffer or β-AR agonist. Mean traces and SEM are shown. (B) When [Ca²⁺]_i cycling is preserved, upregulation of I_Kr and I_Ks compensates the decrease of I_K1 in HF during phase 3 of AP. (C) Stimulation of β-ARs using isoproterenol (ISO, 10 nM) significantly upregulates I_Ks, thus ISO reverses I_Kr/I_Ks dominant pattern of repolarization during phase 3 of AP. However, HF cells are hyporesponsive to ISO-induced stimulation (i.e. I_Ks increases in a smaller extent than in control) thus the net repolarizing current is largely reduced in HF compared to control. Reversal in I_Kr/I_Ks dominance in repolarization following ISO treatment also fail to happen in HF. (D) CaMKII inhibition using the specific inhibitory peptide AIP abolishes the increase in I_Ks, whereas it does not affect I_Kr and I_K1. (E) When [Ca²⁺]_i is buffered (BAPTA_i), the reduction in I_K1 without the upregulation of I_Kr and I_Ks results in a significant decrease in the net repolarizing current in HF. (F) Under β-adrenergic stimulation in BAPTA_i, HF cells show decrease in all three K⁺ currents compared to control. The contributions of these K⁺ currents to total net charge are shown in the insets. Columns and bars represent mean±SEM. Statistics and n numbers are shown in Figs. 3–6.