Relationship between transient outward K+ current and Ca2+ influx in rat cardiac myocytes of endo- and epicardial origin

Abstract

1. The transient outward K+ current (Ito) is a major repolarizing ionic current in ventricular myocytes of several mammals. Recently it has been found that its magnitude depends on the origin of the myocyte and is regulated by a number of physiological and pathophysiological signals. 2. The relationship between the magnitude of Ito, action potential duration (APD) and Ca2+ influx (QCa) was studied in rat left ventricular myocytes of endo- and epicardial origin using whole-cell recordings and the action potential voltage-clamp method. 3. Under control conditions, in response to a depolarizing voltage step to +40 mV, Ito averaged 12.1 +/- 2.6 pA pF-1 in endocardial (n = 11) and 24.0 +/- 2.6 pA pF-1 in epicardial myocytes (n = 12; P < 0.01). APD90 (90 % repolarization) was twice as long in endocardial myocytes, whereas QCa inversely depended on the magnitude of Ito. L-type Ca2+ current density was similar in myocytes from both regions. 4. To determine the effects of controlled reductions of Ito on QCa, recordings were repeated in the presence of increasing concentrations of the Ito inhibitor 4-aminopyridine. 5. Inhibition of Ito by as little as 20 % more than doubled QCa in epicardial myocytes, whereas it had only a minor effect on QCa in myocytes of endocardial origin. Further inhibition of Ito led to a progressive increase in QCa in epicardial myocytes; at 90 % inhibition of Ito, QCa was four times larger than the control value. 6. We conclude that moderate changes in the magnitude of Ito strongly affect QCa primarily in epicardial regions. An alteration of Ito might therefore allow for a regional regulation of contractility during physiological and pathophysiological adaptations.

Figure 1. Magnitude of I_to in epi- and endocardial myocytes

Representative outward currents recorded from an epicardial (A) and an endocardial (B) myocyte. Current was normalized to cell capacitance to compensate for different cell sizes. Currents were activated by voltage pulses of 600 ms duration from a holding potential of -90 mV to values ranging from -60 to +60 mV in steps of 20 mV. Prior to each depolarization, V_m was clamped on -50 mV for 20 ms to inactivate Na⁺ currents. C, average I-V relation of I_to recorded from 12 epicardial (•) and 11 endocardial (○) myocytes. I_to was quantified by subtracting the current at the end of the voltage pulse (600 ms) from the peak current.

Figure 2. AP voltage-clamp recordings from epi- and endocardial myocytes

A, APs recorded in a representative epicardial (left) and endocardial (right) myocyte. Early repolarization was much stronger in the epicardial myocyte. B, Cd²⁺-sensitive current recorded with the AP voltage-clamp method using the APs depicted in A. Peak current was higher in the epicardial myocyte, but total Q_Ca (integral of the current) was much higher in the endocardial myocyte. C, I-V relation, drawn using the AP-induced Cd²⁺-sensitive current (B) and the corresponding AP (A). The peak of the inward current was recorded at a more positive potential in the endocardial myocyte (20 vs. 5 mV). D, Cd²⁺-sensitive current, activated by a rectangular depolarizing voltage pulse to 0 mV. The Na⁺ current was inactivated by a 20 ms step from a holding potential of -90 mV to -50 mV. Activated by the same command potential, Cd²⁺-sensitive currents were similar in size and inactivation kinetics in the epi- and endocardial myocyte. E, outward current, activated by a rectangular depolarizing voltage pulse to +40 mV. The Na⁺ current was inactivated by a 20 ms step from a holding potential of -90 mV to -50 mV. I_to was much more prominent in the epicardial myocyte, which explains the strong early repolarization and the smaller Q_Ca. All recordings were made in the same epi- or endocardial myocyte.

Figure 3. Dose-response relation between 4-AP and I_to

Effects of different concentrations of 4-AP on outward currents recorded in an epicardial (A) and an endocardial (B) myocyte. Cell capacitance of both myocytes was similar (133 pF in the epicardial, 137 pF in the endocardial myocyte). Currents were activated by a depolarizing rectangular voltage pulse from a holding potential of -90 mV to -50 mV for 20 ms to inactivate Na⁺ currents, and then to +60 mV. The numbers indicate the concentration of 4-AP (mM) in the bath solution. C, dose-response relation between 4-AP and I_to. I_to observed in the presence of 4-AP was normalized to I_to recorded in the absence of 4-AP in each individual myocyte. The graph contains data from endo- and epicardial myocytes. The data were fitted assuming Michaelis-Menten kinetics for the effect of 4-AP. IC₅₀ was 0.98 mM.

Figure 4. Influence of inhibition of I_to on APs and AP-induced Ca²⁺ currents in epi- and endocardial myocytes

A, APs and corresponding AP-induced Cd²⁺-sensitive currents recorded in a representative epicardial myocyte subjected to increasing inhibition of I_to with 4-AP. B, similar recordings in an endocardial myocyte. Cell capacitance was similar with 136 pF in the epicardial and 144 pF in the endocardial myocyte. The resting membrane potential was not affected by 4-AP. With increasing concentrations of 4-AP in the bath solution, APs became longer and the plateau potential increased. The increase in APD was accompanied by an increased duration, but reduced peak of the AP-induced Cd²⁺-sensitive current. These effects were more pronounced in the epicardial myocyte. With maximal inhibition of I_to, APs and AP-induced Cd²⁺-sensitive currents were similar in both myocytes. Artefacts in the current recordings prior to the activation of the Cd²⁺-sensitive current resulting from capacitive and Na⁺ currents were cut away. The spike of the AP-induced Cd²⁺-sensitive current recorded from the endocardial myocyte at 4 mM 4-AP is an artefact secondary to the slow upstroke of the corresponding AP and was not observed in similar recordings.

Figure 5. Effect of inhibition of I_to on APD_0mV, Q_Ca, I_Cd,peak and V_m,peak in epi- and endocardial myocytes

Curves were calculated from average data recorded in every experiment. Single recordings were made similar to those shown in Fig. 4. The effects of 0, 0.3, 1 and 4 mM 4-AP on APD_{0 mV} (A), Q_Ca (B), the peak AP-induced Cd²⁺-sensitive current (I_Cd,peak) (C), and the membrane voltage at which I_Cd,peak was recorded (V_m,peak) (D) are illustrated. •, data recorded in epicardial myocytes; ○, data recorded in endocardial myocytes. I_Cd,peak was estimated as the maximal inward current after the initial capacitive current. Note that differences between epicardial and endocardial myocytes disappear with increasing inhibition of I_to. Significance of difference from control values (0 mM 4-AP) was tested using Student's paired t test (*P < 0.05; **P < 0.01; ***P < 0.001).