Regional alterations of repolarizing K+ currents among the left ventricular free wall of rats with ascending aortic stenosis

Abstract

The effect of cardiac hypertrophy on electrocardiogram (ECG), action potential duration (APD) and repolarizing K+ currents was investigated in epicardial, midmyocardial and endocardial myocytes isolated from the rat left ventricular free wall. Cardiac hypertrophy was induced by stenosis of the ascending aorta (AS), which led to an increased pressure load (+85 +/- 10 u1v1vZ mm11Z Hg) of the left ventricle; sham-operated animals served as controls. In ECG recordings from AS rats, the QTc interval was prolonged and the main vectors of the QRS complex and the T-wave pointed in opposite directions, indicating an abnormal sequence of repolarization. APD and K+ currents were recorded using the whole-cell patch-clamp technique. In the AS group, APD90 (90 % repolarization) was significantly prolonged in epicardial and midmyocardial, but not endocardial myocytes. Corresponding to the increase in APD, the magnitude of the transient outward K+ current (Ito1) was significantly smaller (-30 %) in epicardial and midmyocardial, but not endocardial myocytes. Inactivation and steady-state inactivation of Ito1 were not affected by hypertrophy. Recovery from inactivation was slightly prolonged in endocardial myocytes from AS rats. No differences in delayed rectifier currents (IK) or inwardly rectifying K+ currents (IK1) were detected between myocytes of the three regions of sham-operated or AS animals. However, both currents were reduced by AS. The present data show that cardiac hypertrophy caused by pressure overload leads to an increase in APD and a decrease in Ito1 primarily in epicardial and midmyocardial myocytes, which implies a major role of alterations in Ito1 for the reduced gradient in APD. The effects of AS on IK1 and IK may slightly counteract the decrease in APD gradient. The observed changes in APD and underlying ionic currents could well explain the alterations in repolarization observed in the ECG induced by cardiac hypertrophy.

Figure 1. ECG recordings of sham-operated and AS rats

Representative ECG recordings 1 week after sham operation (A) or AS (B). The voltage gain was 20 mm mV⁻¹ and the paper speed was 50 mm s⁻¹. Average main vectors of the QRS complex and the T-wave are illustrated in panel C for sham-operated animals (n = 7) and in panel D for AS animals (n = 5). Continuous arrows represent recordings made before surgery; dashed arrows represent recordings 7 days after surgery. QRS and T are the main vectors before surgery; QRS’ and T’ are main vectors 7 days after surgery; AVF is the direction of lead AVF; I is the direction of lead I.

Figure 2. Effect of pressure-induced cardiac hypertrophy on regional APD

Representative APs recorded from endocardial, midmyocardial and epicardial myocytes of sham-operated (A) and AS rats (B). APs were elicited at a rate of 0.3 Hz by a depolarizing current pulse of 5 ms duration. C, average APD₉₀ obtained from myocytes of sham-operated (▪) and AS rats (□). *P < 0.05; n is the number of myocytes.

Figure 3. Selective reduction of I_to1 in epicardial and midmyocardial myocytes in pressure-induced cardiac hypertrophy

Representative outward currents recorded from endocardial, midmyocardial and epicardial myocytes of sham-operated (A) and AS rats (B). Currents were activated at a rate of 0.3 Hz by rectangular voltage pulses from a holding potential of V_pip= -90 mV to values ranging from -60 to +80 mV in steps of 20 mV. Each voltage pulse was preceded by a depolarization to V_pip= -50 mV for 20 ms to inactivate Na⁺ currents. The bath solution contained 0.3 mM Cd²⁺ to inhibit Ca²⁺ currents. Currents were normalized to C_m to correct for different cell sizes and are thus given in pA pF⁻¹. C, average current-voltage relationships of I_to1 recorded from endocardial (sham, n = 25; AS, n = 26), midmyocardial (sham, n = 21; AS, n = 29) and epicardial (sham, n = 23; AS, n = 26) myocytes of sham-operated (•) and AS (○) rats. I_to1 was quantified by subtracting the current at the end of the voltage pulse (600 ms) from the peak current. *P < 0.05, **P < 0.01, sham-operated vs. AS rats.

Figure 4. Kinetic properties of I_to1 in normal and hypertrophied hearts

Inactivation (A), steady-state inactivation (B), and recovery kinetics (C) of I_to1 recorded from endocardial, midmyocardial and epicardial myocytes of sham-operated (•) and AS (○) rats. A, inactivation time constants (τ) were estimated at holding potentials ranging from V_pip= 0 mV to V_pip=+80 mV by mono-exponential fitting of the current decay. B, steady-state inactivation was determined by a two-step pulse protocol: a conditioning pulse of 600 ms duration ranging from -80 to +10 mV in steps of 10 mV was followed by a step to +60 mV for 600 ms. The magnitude of I_to1 detected at +60 mV after the conditioning pulse was normalized to I_to1 recorded at a conditioning potential of -90 mV in each individual experiment, and is given as a function of the conditioning pulse potential. Data were fitted assuming a Boltzmann kinetic of steady-state inactivation. C, recovery from inactivation was determined by two consecutive pulses to +60 mV, each of 600 ms. During the interval between the two depolarizations, V_pip was returned to -90 mV. The interval between the voltage pulses ranged from 5 to 5000 ms and increased exponentially, with 1.5 being the exponent. Pulses were delivered at 0.1 Hz. The magnitude of I_to1 recorded during the second voltage pulse was normalized to the magnitude of the first, and plotted versus the duration of the interval between the voltage pulses. Recovery curves were fitted using a bi-exponential function.

Figure 5. Effect of pressure-induced cardiac hypertrophy on delayed rectifier K⁺ currents

Average current-voltage relationship of the steady-state current estimated at the end of a 600 ms voltage pulse and recorded from endocardial, midmyocardial and epicardial myocytes of sham-operated (•) and AS (○) rats. Current magnitude was estimated using recordings similar to those shown in Fig. 3, same n as in Fig. 3. ***P < 0.001, AS vs. sham.

Figure 6. Effect of pressure-induced cardiac hypertrophy on inwardly rectifying K⁺ currents

Recordings of Ba²⁺-sensitive currents obtained from an endocardial, midmyocardial and epicardial myocyte of sham-operated (A) and AS (B) rats. V_pip was clamped for 600 ms to a potential from -120 to +30 mV in steps of 10 mV. Using this protocol, currents recorded in the presence of 2 mM Ba²⁺ in the bath solution were subtracted from those recorded in the absence of 2 mM Ba²⁺. In the current traces recorded from midmyocardial and epicardial myocytes, a small transient outward current can be detected at positive pulse potentials. Addition of Ba²⁺ to the bath solution not only inhibits I_K1 but also shifts the activation curve of I_to1. Thus the small transient component in the Ba²⁺-sensitive currents may have resulted from the effect of Ba²⁺ on I_to1. However, since this component was only detectable at potentials positive to V_pip= -20 mV and was transient in nature, it did not interfere with the estimate of I_K1. C, average current-voltage relationship of the Ba²⁺-sensitive currents determined at the end of a 600 ms voltage pulse and recorded from endocardial (sham, n = 11; AS, n = 16), midmyocardial (sham, n = 15; AS, n = 10) and epicardial (sham, n = 10; AS, n = 8) myocytes from sham-operated (•) and AS (○) rats. *P < 0.05, **P < 0.01, sham-operated vs. AS rats.