Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels

Abstract

In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is increased. Altered Na(+) channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent (late) inward I(Na) and intracellular Na(+) concentration (as measured by the Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na(+) channels. This alters I(Na) gating to reduce availability at high heart rate, while enhancing late I(Na) (which could prolong action potential duration). In mice, enhanced CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.

Figure 1. CaMKIIδ_c enhances steady-state inactivation of rabbit myocyte I_Na (10 mM [Na⁺]_o).

(A) Mean I_Na availability (left) and I_Na during conditioning pulses (right; fit parameters in Table 1). In CaMKIIδ_c myocytes, availability was left-shifted versus β-gal (P < 0.05), and this was reversed by CaMKII inhibitors KN93 or AIP (P < 0.05). (B and C) Original I_Na traces during pre-pulses (right) and test pulse (left). I_Na amplitudes during pre-pulses were unaltered by CaMKIIδ_c (see Figure 2).

Figure 2. E_m dependence of I_Na activation in rabbit myocytes (10 mM [Na⁺]_o).

(A) Current-voltage relation (I-V) for CaMKIIδ_c versus β-gal myocytes. (B) I_Na activation curve, with relative conductance derived from maximal chord conductance and reversal potential (E_rev) for each I-V, and peak I_Na/(E_m – E_rev). The resulting conductance was normalized to the maximal chord conductance (usually between –10 and 0 mV). CaMKIIδ_C had no effect on voltage for half-activation V_1/2 or slope factor k (mV). Maximal I_Na was –4.3 ± 0.2 nA and –49.8 ± 2.4 pA/pF (C_m, 88.2 ± 5.7 pF) for CaMKIIδ_c versus –4.1 ± 0.5 nA and –42.8 ± 4.4 pA/pF (C_m, 103.8 ± 17.7 pF) for β-gal (P = NS).

Figure 3. CaMKIIδ_c increases I_IM of I_Na in rabbit myocytes (10 mM [Na⁺]_o).

(A) Increasing conditioning pulse duration (P₁) reduced peak I_Na assessed with a second pulse (P₂). CaMKIIδ_c increased the amount of accumulating I_IM (versus β-gal; P < 0.05), an effect reversed by KN93 or AIP (P < 0.05). Mean data were fitted to a single exponential (see Table 1). (B) Original traces of peak I_Na (at P₂) for different P₁ durations.

Figure 4. CaMKIIδ_c slows I_Na recovery from inactivation in rabbit myocytes (10 mM [Na⁺]_o).

(A) Increasing durations of recovery interval between conditioning pulse (P₁, causing I_Na and inactivation) and test pulse (P₂). CaMKIIδ_C significantly slowed recovery versus β-gal, an effect blocked upon CaMKII inhibition (KN93; P < 0.05). Elevation of [Ca²⁺]_i to 500 nM in myocytes overexpressing CaMKIIδ_C further slowed recovery, and KN93 completely inhibited this effect. Mean data were fit to a single exponential (fit parameters in Table 1). (B) Original traces of I_Na for different recovery intervals.

Figure 5. CaMKIIδ_c slows fast decay of I_Na.

(A) Original traces show that CaMKIIδ_c slows fast I_Na inactivation in rabbit myocytes versus β-gal. I_Na decay (first 50 ms) was fit with a double exponential: y (t) = A₁ exp (–t/τ₁) + A₂ exp (–t/τ₂) + y₀. Fits and τ₁ and τ₂ (ms) are shown in red (β-gal) and blue (CaMKIIδ_c). Deceleration of I_Na decay was significant in τ₂ but not in τ₁. KN93 could reverse the slowing of τ₂ by CaMKIIδ_C. (B) In CaMKIIδ_C-Tg mice, both τ₁ and τ₂ were slowed compared with those in WT mice. Fits to the original traces and τ₁ and τ₂ values (ms) are shown in red (WT) and blue (CaMKIIδ_C-Tg). KN93 did not reverse these effects. Average peak I_Na was –10.2 ± 0.3 nA, –79.8 ± 2.7 pA/pF, C_m 132.7 ± 4 pF for rabbit and –14.2 ± 0.6 nA, –58.9 ± 2.9 pA/pF, C_m 255.3 ± 11 pF for mouse.

Figure 6. CaMKIIδ_c enhances late I_Na and increases [Na]_i.

I_Na elicited at –20 mV (for 1,000 ms) was leak subtracted and normalized to peak I_Na. Current was integrated between 50 and 500 ms and normalized to the I_Na integral if no inactivation had occurred. (A) Original traces and mean data of normalized late I_Na. CaMKIIδ_C overexpression significantly increased late I_Na (reversed with KN93). (B) CaMKIIδ_C-Tg mice also showed late I_Na, but this was not reversible with KN93. Average peak I_Na was –10.2 ± 0.3 nA, –79.8 ± 2.7 pA/pF, C_m 132.7 ± 4 pF for rabbit and –14.3 ± 0.6 nA, –58.1 ± 2.8 pA/pF, C_m 259.7 ± 11.6 pF for mouse. (C) Mean [Na]_i at different stimulation frequencies (left) and at 1 Hz (right) in rabbit myocytes. [Na]_i was elevated in CaMKIIδ_c myocytes at all frequencies (P < 0.05) and reduced by KN93 (P < 0.05). (D) Mean data for CaMKIIδ_C-Tg mice also showing elevated [Na]_i (versus WT; P < 0.05), but this was not reversed by KN93.

Figure 7. CaMKII-dependent association with and phosphorylation of Na⁺ channels.

(A and B) Equal amounts of CaMKII were IP from mouse hearts (A, Tg versus WT) and rabbit myocytes (B, CaMKIIδ_c versus nontransfected cells) and subjected to immunoblotting. CaMKII IP showed CaMKII association with Na⁺ channels in WT and Tg hearts (n = 3 and n = 3, respectively), while IP with polyclonal rabbit anti-Ca_v1.2a did not. Similar results were seen in rabbit myocytes (CaMKIIδ_C and nontransfected cells, n = 4 and n = 2, respectively). (C) Colocalization of CaMKII and Na⁺ channel in rabbit myocytes with immunocytological staining. (D and E) Autoradiograms measuring ³²P incorporation into Na⁺ channels. Equal amounts of Na⁺ channels were immunoprecipitated from WT mouse hearts (n = 3) and directly phosphorylated with or without CaMKII, KN93 (50 μM), AIP (5 μM), or PKA/PKC inhibitor cocktail (PKA/C-I; 1 μM). (F) Endogenous CaMKII-dependent Na⁺ channel phosphorylation was activated in permeabilized rabbit myocytes by preincubating for 5 minutes in internal solutions of 500 nM [Ca²⁺] plus 2 μM CaM (versus 50 nM [Ca²⁺]). Sites not already phosphorylated were subsequently back-phosphorylated in Na⁺ channel immunoprecipitates (equal amounts) by exogenous preactivated CaMKII and ATP-γ-³²P. The intensity of ³²P is inversely related to the CaMKII-dependent phosphorylation during preincubation. (G and H) Western blots (anti-Na_v Pan) show upregulation of Na⁺ channel expression in CaMKIIδ_C-Tg versus WT mice (relative to calsequestrin [CSQ]) but unaltered expression in CaMKIIδ_C rabbit myocytes (data pooled for MOI 10 and 100).

Figure 8. Arrhythmias in CaMKIIδ_C-Tg mice.

(A) Original ECG traces during programmed electrical stimulation in vivo. In WT mice, 2 consecutive premature beats (S2-S3, coupling interval 38 ms) did not induce arrhythmias (top). In contrast, in 2 different Tg mice, the same protocol induced monomorphic (middle) or polymorphic VT (bottom). (B) Representative ECG traces before and after isoproterenol (Iso) administration. P-waves (indicated by asterisks) were apparent during normal rhythms (WT with or without isoproterenol) but were absent in Tg mice after isoproterenol administration. This apparent AV dissociation indicates arrhythmia. (C) Frequency of arrhythmia induction was significant in Tg but not WT mice for both programmed electrical stimulation (left) and isoproterenol administration (right). (D) Resting ECG parameters (RR interval, corrected QT [QT_c] interval, QRS duration, and PR interval). (E) Right-ventricular MAPs from hearts paced at basic cycle lengths (BCLs) of 100 (top) and 150 ms (bottom) for WT mouse hearts (left), Tg mouse hearts (middle), and Tg mouse hearts during infusion with KN93 (right). (F) Mean MAP durations at 90% repolarization (APD90). At slower heart rates (BCL, 150 ms), APD90 was significantly prolonged in Tg versus WT mice (but could not be reduced by KN93). At higher heart rates, there was no significant prolongation of APD90 in Tg versus WT.