Relationship between K+ channel down-regulation and [Ca2+]i in rat ventricular myocytes following myocardial infarction

Abstract

1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes). 2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K+ current (Ito) density (sham 19.7 +/- 1.1 pA pF-1 versus post-MI 11.0 +/- 1.3 pA pF-1; means +/- s.e.m.), inward rectifier K+ current density (sham -13.7 +/- 0.6 pA pF-1 versus post-MI -10.3 +/- 0.9 pA pF-1) and resting membrane potential (sham -84.4 +/- 1.3 mV versus post-MI -74.1 +/- 2.6 mV). Depressed Ito amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for Ito. 3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca2+]i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca2+ handling by the sarcoplasmic reticulum or in L-type Ca2+ channel density (ICa,L) were detected between the groups. 4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca2+] transient amplitude to the sham level and vice versa. 5. The net Ca2+ influx per beat via ICa,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials. 6. Our findings demonstrate that reductions in K+ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca2+ influx and [Ca2+]i transients.

Figure 1. Action potential characteristics in right ventricular myocytes following myocardial infarction

A, representative action potential traces from a sham and a post-MI myocyte. Action potentials were elicited by a brief (5 ms) suprathreshold (2 × threshold) pulse applied at 0.2 Hz. Intracellular solutions contained 5 mM EGTA. Horizontal bars indicate 0 mV. B, mean action potential duration (APD) evaluated at 50 and 90 % (APD₅₀ (□) and APD₉₀ (▪), respectively) repolarization for sham (n = 29/10) and post-MI (n = 20/6) myocytes. *P < 0.05 for APD₅₀ and APD₉₀ between sham and post-MI myocytes.

Figure 2. I_K1 in right ventricular myocytes following myocardial infarction

A, normalized traces of the inward rectifier current (I_K1) in a sham and a post-MI myocyte elicited by 500 ms voltage steps over the range -130 to -10 mV in +10 mV increments from a holding potential of -80 mV. CdCl₂ (0.3 mM) was added to avoid contamination by the calcium current. Arrows indicate 0 pA pF⁻¹. B, I_K1 current density plotted against the test potential for sham (•, n = 29/10) and post-MI (○, n = 13/6) myocytes. The current measured at the end of the test pulse in the presence of barium was subtracted from the current in the absence of barium (protocol shown in inset). Myocytes were depolarized every 5 s. *P < 0.05 between sham and post-MI myocytes at -120 mV. C, mRNA levels for IRK1 (left panel) and IRK2 (right panel) in sham (left lanes) and post-MI (right lanes) right ventricles were measured using an RNase protection assay (RPA). The upper and lower bands represent IRKx- and cyclophilin-protected mRNA fragments, respectively. The bar graphs show the mean mRNA levels in post-MI (▪) hearts expressed as a percentage of the levels observed in sham (□) hearts (i.e. % Sham).

Figure 3. I_to and I_sus in right ventricular myocytes following myocardial infarction

A, traces of the transient outward (I_to) and sustained (I_sus) current densities in a sham and a post-MI myocyte elicited by 500 ms voltage steps over the range -30 to +70 mV in +10 mV increments from a holding potential of -80 mV (protocol shown in inset, values in mV). Arrows indicate 0 pA pF⁻¹. I_to (B) and I_sus (C) were normalized to membrane capacitance and plotted against the test potential for sham-operated (•, n = 42/10) and post-MI (○, n = 21/6) hearts. Myocytes were depolarized every 5 s. *P < 0.05 between sham and post-MI myocytes at +60 mV.

Figure 4. Biophysical properties of I_to in right ventricular myocytes following myocardial infarction

A, normalized recovery from inactivation traces in a sham and a post-MI myocyte. Arrows indicate 0 pA pF⁻¹. B, plot of recovery kinetics for sham (•, n = 36) and post-MI (○, n = 14) myocytes using a two pulse protocol (inset) with two identical depolarizing pulses from -80 to +60 mV applied every 10 s at selected intervals from 10 to 10 000 ms. The recovery kinetics in the sham myocyte were best described by a monoexponential function, whereas the recovery kinetics in post-MI myocytes were best fitted by a biexponential function.

Figure 5. Representative comparison of candidate genes encoding the transient outward current in the right ventricle following myocardial infarction

A, typical mRNA measurements for Kv4.2 (left panel), Kv4.3 (middle panel) and Kv1.4 (right panel) from sham (left lanes) and post-MI (right lanes) right ventricles. The upper and lower bands represent Kvx- and cyclophilin-protected mRNA fragments, respectively. The bar graphs show mean changes in Kvx mRNA levels in post-MI (▪, n = 7/7) relative to sham (□, n = 7/7). B, Western blot analysis showing immunoreactive proteins for Kv4.2 and Kv1.4 from sham (left lanes) and post-MI (right lanes) right ventricles. The bar graphs show mean changes in Kvx mRNA levels in post-MI (▪, n = 6/6) relative to sham (□, n = 6/6).

Figure 6. Action potential and [Ca²⁺]_i characteristics recorded from sham and post-MI right ventricular myocytes

A, representative action potentials (upper traces) and [Ca²⁺]_i (lower traces) derived from a sham and a post-MI myocyte under current-clamp conditions. Arrows indicate 0 nM [Ca²⁺]_i and horizontal bars indicate 0 mV. B, mean values for diastolic and systolic [Ca²⁺]_i in sham (□, n = 13/8) and post-MI (▪, n = 13/7) myocytes. *P < 0.05 for systolic [Ca²⁺]_i between sham and post-MI myocytes.

Figure 7. Effect of action potential prolongation on [Ca²⁺]_i in right ventricular myocytes

Action potential voltage-clamp measurements in a sham and a post-MI myocyte. In A, the left panel shows record from a sham myocyte (continuous trace) stimulated with its action potential (dashed trace). The right panel shows the same myocyte stimulated with an action potential derived from a post-MI myocyte (same as that shown in B, left panel). In B, the left panel shows a post-MI myocyte stimulated with its action potential. The right panel shows the same myocyte stimulated with an action potential derived from a sham myocyte. Arrows indicate 0 nM [Ca²⁺]_i and horizontal bars indicate 0 mV.

Figure 8. Effect of short voltage-clamp pulses on the [Ca²⁺]_i transient in sham and post-MI right ventricular myocytes

A, representative [Ca²⁺]_i transients in a sham and a post-MI myocyte stimulated with short voltage-clamp pulses (100 ms) from a holding potential of -80 to +10 mV at 0.25 Hz. Arrows indicate 0 nM [Ca²⁺]_i and horizontal bars indicate 0 mV. B, mean values for systolic and diastolic [Ca²⁺]_i in sham (□, n = 10/5) and post-MI (▪, n = 10/4) myocytes. No significant difference was observed between sham and post-MI myocytes.

Figure 9. I_Ca,L in right ventricular myocytes following myocardial infarction

A, representative current traces showing cadmium-sensitive difference current densities (0.3 mM CdCl₂) from a sham and a post-MI myocyte elicited by 500 ms voltage steps to -40, 0, +10 and +30 mV from a holding potential of -80 mV. A prepulse to -40 mV (100 ms) was used to eliminate any contaminating sodium current. Arrows indicate 0 pA pF⁻¹. B, I_Ca,L density is plotted against the test potential for sham (•, n = 15/4) and post-MI (○, n = 9/3) myoctes. Myocytes were depolarized every 5 s. No significant difference was observed between sham and post-MI myocytes.

Figure 10. Effect of action potential duration on I_Ca,L

The records and graph show I_Ca,L produced under action potential voltage-clamp conditions. A, recordings from a right ventricular sham myocyte stimulated with representative sham (○) and post-MI (•) action potentials (upper traces) and corresponding cadmium-subtracted I_Ca,L traces (lower traces). Arrows indicate 0 pA and horizontal bars indicate 0 mV. B, current-voltage plot shows I_Ca,L during the action potential voltage-clamp for a sham (○) and a post-MI (•) action potential waveform. Arrows indicate increasing time.

Figure 11. Ca²⁺ content in the sarcoplasmic reticulum in right ventricular myocytes following myocardial infarction

A, original records representing I_Na-Ca tails following application of 10 mM caffeine in a sham and a post-MI myocyte. All myocytes were held at -70 mV throughout. Dotted traces show non-exchange (leakage) current flowing during the sodium-free portion of caffeine application. Bars indicate the period of caffeine application. B, sarcoplasmic reticulum Ca²⁺ content was calculated by integrating the currents and the mean data are shown. No significant difference in sarcoplasmic reticulum Ca²⁺ content was observed between sham (▪) and post-MI (□) myocytes.