Calcium-dependent inactivation controls cardiac L-type Ca2+ currents under β-adrenergic stimulation

Figures

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  Figure 1.. APs from isoproterenol-treated cardiomyocytes. (A) Representative AP waveforms from a cardiomyocyte before (black solid line) and after (green dashed line) 100 nM isoproterenol application. APs were elicited by 2–5 ms depolarizing current injections (100–200 pA) at 1 Hz. The horizontal line indicates zero level. (B) Phase plot of the normalized first derivative of membrane potential (dV/dt) against membrane potential (Vm) for the APs shown in A. (C) Bar graph of overshoot, threshold potentials, and mean DMPs. (D) Bar graph of maximum rate of potential change (dV/dtmax). (E) Bar graphs of APD estimated at 20%, 50%, and 90% of the repolarization phase (APD20, APD50, and APD90, respectively). Bar graphs are mean ± SEM; empty bars are control cardiomyocytes, and green bars correspond to isoproterenol-treated cardiomyocytes (n = 10; *P < 0.01, with respect to control).

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  Figure 2.. L-type calcium current during an AP upon β-adrenergic stimulus. (A and B) Representative nifedipine-sensitive current (solid line) elicited by the AP (dashed line) prerecorded from the same cell in control (A) and isoproterenol-treated (B) newborn rat cardiomyocytes. (C) Bar graph of maximal current normalized by cell capacitance (pA/pF); total time of nifedipine-sensitive current normalized by its APD (TTCnorm); and the total current integral normalized by cell capacitance (Qtotal). Data are presented as percentages with respect to the controls. The bar graph shows mean ± SEM; empty bars represent control cardiomyocytes and green bars correspond to isoproterenol-treated cardiomyocytes (n = 6; *P < 0.01 with respect to control).

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  Figure 3.. L-type channel inactivation processes during the AP. (A) State diagram of the model for L-type Ca2+ channel, the model assumes four closed states (C1, C2, C3, C4), three inactivation states (I1, I2, I3), and one open state (O). Voltage-dependent constants are α and β while γγ is calcium dependent. Kpcf and Kpcb are voltage-insensitive rate constants. (B–D) Probability of open (dark yellow line), closed (violet line), CDI (light purple line), or VDI (pink line) states of LTCCs during a simulated AP simulated in control cardiomyocytes (A) or with reduced VDI (B) or CDI (C). Normalized membrane potential (Vm, black line) for each scenario is shown for comparison.

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  Figure 4.. Physiological temperature modifies APs and L-type calcium currents in cardiomyocytes. (A) Representative AP waveforms from newborn rat cardiomyocytes at 23°–25°C (black dashed line) or 35°–37°C (brown line). (B) Representative AP waveforms at 35–37°C before (brown line) and after treatment with 1 µM SEA0400 (light blue line). APs were elicited by 2–5 ms depolarizing current injections (100–200 pA) at 1 Hz. (C) Bar graph of the APD percentage change upon isoproterenol treatment, estimated at 20% (APD20), 50% (APD50), and 90% (APD90) of the repolarization phase. (D and E) Representative nifedipine-sensitive current (solid line) elicited by the AP (dashed line) prerecorded from the same cell at 35–37°C in control cardiomyocytes (D) or treated with 1 µM SEA0400 (E). (F) Bar graph of maximal current normalized by cell capacitance (pA/pF), total time of nifedipine-sensitive current normalized by its APD (TTCnorm), and the total current integral normalized by cell capacitance (Qtotal). Data are presented as percentages with respect to control cardiomyocytes at 23–25°C. Bar graphs are mean ± SEM; empty bars represent control cardiomyocytes, brown bars correspond to cardiomyocytes at…

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  Figure 5.. APs and L-type calcium currents in CDI-impaired cardiomyocytes. (A) Representative AP waveforms from a newborn rat cardiomyocyte overexpressing CaMwt (solid black line) or CaM34 (dashed blue line). APs were elicited by 2–5 ms depolarizing current injections (100–200 pA) at 1 Hz. The horizontal line indicates zero level. (B) Bar graph of APD estimated at 20% (APD20), 50% (APD50), and 90% (APD90) of the repolarization phase. (C) Phase plot of the normalized first derivative of membrane potential (dV/dt) against membrane potential (Vm) for the APs shown in A. Empty symbols correspond to data from cardiomyocytes overexpressing CaMwt and blue symbols for cardiomyocytes overexpressing CaM34. (D) Bar graph of overshoot, threshold potentials, and mean DMPs. (E) Representative nifedipine-sensitive current (solid line) elicited by the AP (dashed line) prerecorded from the same cardiomyocyte overexpressing CaM34. (F) Bar graph of maximal current normalized by cell capacitance (pA/pF), total time of nifedipine-sensitive current normalized by its APD (TTCnorm), and the total current integral normalized by cell capacitance (Qtotal). Data are shown as percentages with respect to…

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  Figure 6.. APs and L-type calcium currents in VDI-impaired newborn rat cardiomyocytes. (A) Representative AP waveforms from a newborn rat cardiomyocyte overexpressing RFP (solid black line) or CaVβ2a (dashed plum line). APs were elicited by 2–5 ms depolarizing current injections (100–200 pA) at 1 Hz. (B) Bar graph of APD estimated at 20% (APD20), 50% (APD50), and 90% (APD90) of the repolarization phase. (C) Phase plot of the normalized first derivative of membrane potential (dV/dt) against membrane potential (Vm) for the APs shown in A. Cardiomyocytes overexpressing RFP are shown with empty symbols and cardiomyocytes overexpressing CaVβ2a with plum symbols. (D) Bar graph of overshoot, threshold potentials, and mean DMPs. (E) Representative nifedipine-sensitive current (solid line) elicited by the AP (dashed line) prerecorded from the same cardiomyocyte overexpressing CaVβ2a. (F) Bar graph of maximal current normalized by cell capacitance (pA/pF), total time of nifedipine-sensitive current normalized by its APD (TTCnorm), and the total current integral normalized by cell capacitance (Qtotal). Data are presented as percentages with respect to RFP-transduced cardiomyocytes. Bar graphs are mean…

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  Figure 7.. Role of L-type channel inactivation processes during a simulated AP. (A) Simulated L-type calcium current for the control condition (black line) or for cardiomyocytes with increased L-type channel conductance alone (light gray line) or simultaneously with an increased CDI (violet line) or VDI (pink line). (B) Experimental data showing the maximal nifedipine-sensitive current from control, CaVβ2a, and CaM34 before (empty bars) or after (green bars) isoproterenol stimulation. #, P < 0.01 with respect to control; *, P < 0.01 between values before and after isoproterenol stimulation.

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  Figure 8.. Role of VDI and CDI on APs upon β-adrenergic stimulus. (A and B) Representative AP waveforms from newborn rat cardiomyocytes overexpressing CaVβ2a (A) or CaM34 (B) before (solid lines) and after (dashed green lines) stimulus with 100 nM isoproterenol. APs were elicited by 2–5 ms depolarizing current injections (100–200 pA) at 1 Hz. (C) Bar graph of the APD percentage change upon isoproterenol treatment, estimated at 20% (APD20), 50% (APD50), and 90% (APD90) of the repolarization phase. (D and E) Phase plots of the normalized first derivative of membrane potential (dV/dt) against membrane potential (Vm) for the APs shown in A and B, respectively. Plum symbols correspond to CaVβ2a-overexpressing cardiomyocytes, blue symbols to CaM34-overexpressing cardiomyocytes, and green symbols to the isoproterenol-treated condition. (F) Bar graph of overshoot, threshold potentials, and mean DMPs from cardiomyocytes treated with isoproterenol and overexpressing CaVβ2a or CaM34. Bar graphs are mean ± SEM. *, P < 0.01, with respect to control.

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  Figure 9.. L-type channel inactivation processes upon β-adrenergic stimulus. Representative nifedipine-sensitive current (solid line) elicited by the AP (dashed line) prerecorded from the same cell in a newborn rat cardiomyocyte overexpressing RFP (A), CaVβ2a (B), CaMwt (D), or CaM34 (E). Bar graphs are mean ± SEM (n = 8) of maximal current normalized by cell capacitance (pA/pF), total time of current normalized by action potential duration (TTCnorm), and the total current integral normalized by cell capacitance (Qtotal) from a cardiomyocyte overexpressing RFP (empty bars) and CaVβ2a (plum bars). CaMwt (empty bars; B) or CaM34 (blue bars; D). Data are presented as percentages of their respective control situation [RFP for C and CaMwt for F]. *, P < 0.01 with respect to control.