Ca2+/calmodulin-dependent protein kinase II-dependent remodeling of Ca2+ current in pressure overload heart failure

Abstract

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is increased in heart failure (HF), a syndrome characterized by markedly increased risk of arrhythmia. Activation of CaMKII increases peak L-type Ca(2+) current (I(Ca)) and slows I(Ca) inactivation. Whether these events are linked mechanistically is unknown. I(Ca) was recorded in acutely dissociated subepicardial and subendocardial murine left ventricular (LV) myocytes using the whole cell patch clamp method. Pressure overload heart failure was induced by surgical constriction of the thoracic aorta. I(Ca) density was significantly larger in subepicardial myocytes than in subendocardial/myocytes. Similar patterns were observed in the cell surface expression of alpha1c, the channel pore-forming subunit. In failing LV, I(Ca) density was increased proportionately in both cell types, and the time course of I(Ca) inactivation was slowed. This typical pattern of changes suggested a role of CaMKII. Consistent with this, measurements of CaMKII activity revealed a 2-3-fold increase (p < 0.05) in failing LV. To test for a causal link, we measured frequency-dependent I(Ca) facilitation. In HF myocytes, this CaMKII-dependent process could not be induced, suggesting already maximal activation. Internal application of active CaMKII in failing myocytes did not elicit changes in I(Ca). Finally, CaMKII inhibition by internal diffusion of a specific peptide inhibitor reduced I(Ca) density and inactivation time course to similar levels in control and HF myocytes. I(Ca) density manifests a significant transmural gradient, and this gradient is preserved in heart failure. Activation of CaMKII, a known pro-arrhythmic molecule, is a major contributor to I(Ca) remodeling in load-induced heart failure.

FIGURE 1.

A and B, representative current traces (SEN myocytes) elicited by 300-ms test pulses in 20-mV increments from a holding potential of -50 mV to potentials between -30 to +50 mV at pulse intervals of 10 s. I_Ca amplitude was increased, and inactivation was slowed at all test potentials in failing ventricular myocytes. C, I_Ca-voltage relationship in ventricular myocytes from sham-operated and failing LV. Current amplitudes were normalized to cell capacitance and plotted as mean values. The vertical bars represent S.E. D, I_Ca inactivation time constants recorded in myocytes from sham-operated and failing LV. No significant differences in I_Ca inactivation kinetics were observed between SEN and SEP myocytes, and the results were pooled and fit by a biexponential function. The mean values were obtained by fitting currents recorded from each individual cell. τ1 and τ2 refer to fast and slow inactivation time constants, respectively. The vertical bars represent S.E. ^* denotes p < 0.05.

FIGURE 2.

A, surface expression of α1c protein in SEN and SEP myocytes from sham and HF LV lysates. Surface proteins were biotinylated and immobilized on avidin agarose, and equal amounts of protein were subjected to SDS-PAGE and Western blot. Densitometric analyses demonstrated statistically significant differences between SEN and SEP in sham and HF ventricle (n = 4 in each group). B, total LV lysates were immunoblotted for α1c protein, and a representative example is shown. Each lane was loaded with 25 μg of protein, and the paired bands, SEP and SEN, are from the same heart (n = 3 for each case).

FIGURE 3.

Voltage-dependent activation and inactivation of I_Ca recorded in myocytes from sham-operated and failing LV. Currents elicited by each test pulse were normalized to the maximum current. The mean values are plotted versus conditioning pulse potentials (for inactivation, inset A) or test pulse potentials (for activation, inset B). The vertical bars represent S.E.

FIGURE 4.

I_Ca recovery from inactivation in SEN myocytes from sham-operated (A) and failing LV (B). Paired 300-ms pulses at +10 mV were applied from a holding potential of -50 mV with the time interval (ΔT) varied from 50 to 1500 ms in 50-ms steps (C). The protocol was repeated every 10 s. Representative I_Ca recovery traces for myocytes from control and failing LV are shown in A and B. The mean values of the normalized peak current (+10 mV) are shown in D.

FIGURE 5.

A, current traces (SEN myocytes) showing robust frequency-dependent Ca²⁺-induced I_Ca facilitation in ventricular myocytes from control LV, which is eliminated in myocytes from failing LV. Rather, frequency-dependent I_Ca suppression was observed. B, histogram of CaMKII activities in SEN and SEP fractions from both sham and HF LV. C, representative Western blot probed for autophosphorylated, constitutively active CaMKII (pCaMKII) or total CaMKII.

FIGURE 6.

Representative I_Ca traces elicited by 300-ms test pulses in 20-mV increments from a holding potential of -50 mV to potentials between -30 to +50 mV at pulse intervals of 10 s. The I_Ca traces were recorded in ventricular myocytes from HF and sham mice following internal application of 1 μm constitutively active CaMKII.

FIGURE 7.

Upper panel, I_Ca-voltage relationships in ventricular myocytes isolated from sham-operated (n = 17) and failing (n = 12) LV following internal application of constitutively active CaMKII (1 μm). Current amplitudes were normalized to cell capacitance and plotted as mean values. The vertical bars represent S.E. Lower panel, I_Ca inactivation time constants recorded in myocytes from sham-operated and failing LV after internal application of active CaMKII (1 μm). The decay phase was fit by a biexponential function. The mean values were obtained by fitting currents recorded from each individual cell at +10 mV (peak current). τ1 and τ2 refer to fast and slow inactivation time constants, respectively. The vertical bars represent S.E.

FIGURE 8.

Representative time course of peak I_Ca recorded in an SEP myocyte isolated from failing LV. Over the course of ∼15 min, diffusion of AIP from the patch pipette into the myocyte cytoplasm leads to declines in I_Ca, followed by stabilization at a new steady state level.

FIGURE 9.

Representative current traces elicited by 300-ms test pulses in 20 mV increments from a holding potential of -50 mV to potentials between -30 to +50 mV at pulse intervals of 10 s. The I_Ca traces were recorded in an SEP myocyte from failing LV 2 min after rupture of the cell membrane (A) and again after the current reached steady state (15 min, B). Frequency-dependent Ca²⁺-induced I_Ca facilitation tested in ventricular myocytes from control LV before AIP dialysis (2 min after rupture of the cell membrane, C) and again after peak current had reached steady state (15 min, D).

FIGURE 10.

A, peak I_Ca densities (recorded at +10 mV) in control and HF myocytes (SEP and SEN) under control conditions (with normal internal solution) and after CaMKII was inhibited by internal application of AIP. The vertical bars represent S.E. ^* denotes p < 0.05. B, time constants of I_Ca inactivation measured in control and HF myocytes (SEP and SEN) under control conditions (with normal internal solution) and after CaMKII was inhibited by internal application of AIP. τ1 and τ2 refer to fast and slow inactivation time constants, respectively. The vertical bars represent S.E. ^* denotes p < 0.05.