Transmural Autonomic Regulation of Cardiac Contractility at the Intact Heart Level

Figures

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  FIGURE 1. β-Adrenergic stimulation increases the left ventricular developed pressure. (A) Representative traces of the left ventricular pressure before (black) and after 500 nM isoproterenol (ISO; purple). (B) Data shows the change in the normalized amplitude of the developed pressure before and after isoproterenol. Data is presented as multiple measurements (n; dot cloud) recorded on different hearts (N = 4) with mean ± SEM (solid line) *p < 0.01.

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  FIGURE 2. β-Adrenergic stimulation modulates Ca2+ transients in endocardium and epicardium. (A) Normalized fluorescent Ca2+ transients recorded, simultaneously, from endocardium and epicardium using Rhod-2 and the pulsed local field fluorescence microscopy technique. (B) Representative fluorescent traces of the endocardial Ca2+ transients before (black) and after 500 nM isoproterenol (ISO; orange). Right panel: Ca2+ transient traces normalized to their corresponding maximum fluorescence. (C) Representative Ca2+ transients recorded from the epicardium before (black) and after isoproterenol (cyan). Right panel: Ca2+ transients normalized to their maximum fluorescence. (D) Summary of the normalized amplitude of the Ca2+ transient changes, from the endocardium (circles) and epicardium (squares), before and after isoproterenol. (E) Data showing the kinetic changes in the normalized Ca2+ transients before and after isoproterenol. Means ± SEM are represented as the solid horizontal lines. *p < 0.01, N = 4 hearts.

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  FIGURE 3. mRNA differences across the ventricular wall. Endocardial and epicardial mRNA quantification of the different proteins involved in Ca2+ signaling. mRNA levels (bars) were normalized to the housekeeping gene, GAPDH and expressed as mean ± SEM. N = 5. *p < 0.05.

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  FIGURE 4. β-Adrenergic stimulation modifies AP morphology in endocardium and epicardium. (A) Normalized APs from the endocardium (gray) and epicardium (black) recorded optically using Di-8-ANEPPS and the pulsed local field fluorescence microscopy technique. The evaluated AP parameters are marked. (B) Normalized APs from the endocardium before (gray) and after perfusion with 500 nM isoproterenol (ISO; orange). Inset: Expanded view of AP phase 0 and 1 to visualize the isoproterenol effects. (C) Normalized APs recorded from the epicardium before (black) and after perfusion with 500 nM isoproterenol (cyan). Inset: Expanded view of AP phase 0 and 1 to visualize the isoproterenol effect. (D) Summary of the normalized APD30, APD half phase 2 and APD90 changes from the endocardium (circles) and epicardium (squares), before and after isoproterenol. Means ± SEM are represented as the solid horizontal lines. *p < 0.01, N = 4 hearts.

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  FIGURE 5. β-Adrenergic stimulation increases Ca2+-driven currents. (A) APs recorded from the epicardium using glass microelectrodes before (black) and after 500 nM isoproterenol (ISO; pink). Inset: Expanded view of the phase 0 and 1 showing the effects of isoproterenol on APD30. (B) Representative traces of Ca2+-driven currents recorded after the local photolysis of nifedipine using the Loose Patch Photolysis technique showing the iearly and ilate components upon β-adrenergic stimulation. (C) Data from the iearly component, representing the L-type Ca2+ currents, before (gray) and after isoproterenol (pink). The means ± SEM are represented as the solid horizontal lines. *p < 0.01, N = 4 hearts. (D) Representative traces of Ca2+-driven currents recorded consecutively at increasing UV energies (fluences) in control conditions in the presence of ryanodine (Ry, 10 μM) and thapsigargin (Tg, 2 μM). Right panel: Simultaneously recorded AP in the same conditions. (E) Ca2+-driven currents recorded consecutively at increasing UV energies (fluences) with isoproterenol in the presence of ryanodine (Ry, 10 μM) and thapsigargin (Tg, 2 μM). Right panel: Simultaneously recorded AP in the same conditions.…

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  FIGURE 6. cAMP modulates the time course of Ca2+-driven currents. (A) Ionic currents recorded during consecutive cardiac cycles before and after the photolysis of caged cAMP. (B) Ionic currents induced by cAMP taken from (A) at the indicated time points (traces black, red, and blue) are overlapped to demonstrate the effect in amplitude for both iearly and ilate components. (C) Current amplitude peaks of iearly and ilate as a function of time before and after the photolysis of caged cAMP. (D) Normalized data from (C) re-plotted as a function of time. Purple arrow indicates the time of the UV pulse (cAMP uncaging).

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  FIGURE 7. Cholinergic stimulation does not modify the left ventricular developed pressure. (A) Representative traces of the left ventricular pressure before (black) and after 5 μM carbachol (CCh; blue). (B) Data of the normalized amplitude of the developed pressure before and after carbachol. Means ± SEM are represented as the solid horizontal lines, N = 3 hearts.

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  FIGURE 8. Cholinergic stimulation has a low impact on the Ca2+ transients in endocardium and epicardium. (A) Normalized fluorescent Ca2+ transients recorded, simultaneously, from endocardium and (B) epicardium using Rhod-2 and the pulsed local field fluorescence microscopy technique before and after 5 μM carbachol (CCh) and in the presence of both carbachol and 40 μM atropine. Overlapping Ca2+ transients were normalized to their maximum fluorescence. (C) Summary of the normalized amplitude of the Ca2+ transient changes, from the endocardium (circles) and epicardium (squares), before and after carbachol. (D) Data showing the kinetic changes in the normalized Ca2+ transients before and after carbachol, and after carbachol plus atropine. Means ± SEM are represented as the solid horizontal lines. *p < 0.01, N = 5 hearts.

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  FIGURE 9. Cholinergic stimulation modifies overall AP morphology in endocardium and epicardium. (A) Normalized APs recorded optically with Di-8-ANEPPS from the endocardium and (B) epicardium before and after perfusion with 5 μM carbachol (CCh). (C) Summary of the normalized APD30, APD half phase 2 and APD90 changes from the endocardium (circles) and epicardium (squares), before and after carbachol, and after carbachol plus atropine. Means ± SEM are represented as the solid horizontal lines. *p < 0.01, N = 5 hearts.

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  FIGURE 10. Cholinergic stimulation does not modify L-type Ca2+ currents in the absence of a sympathetic drive. (A) Representative traces of Ca2+-driven currents recorded after the local photolysis of nifedipine using the Loose Patch Photolysis technique showing iearly and ilate components under control conditions and (B) in the presence of 5 μM carbachol (CCh). (C) Overlapping traces of Ca2+-driven currents before (black) and after cholinergic stimulation (brown). (D) Normalized data of the peak values of iearly, before and after carbachol.

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  FIGURE 11. Cholinergic stimulation acts via IKACh in ventricle. (A) APs recorded from the epicardium using glass microelectrodes before (black), with 100 nM tertiapin (cyan) and after tertiapin with 5 μM carbachol (CCh; yellow). (B) Epicardial APs with 5 μM carbachol (green) after 20 min washout from tertiapin + carbachol (black). (C) Effect of 40 μM atropine (dark green) on the time course of epicardial APs. (D) Data summarizing epicardial APD30, APD half of phase 2, and APD90 of the normalized APs before (gray) and after 100 nM tertiapin followed by the perfusion of 5 μM carbachol plus tertiapin. (E) APD30, APD half phase 2, and APD90 after a 20 min washout from tertiapin + carbachol (gray) and then perfusion with 5 μM carbachol (green). (F) APD30, APD half phase 2, and APD90 after perfusion of 40 μM atropine. Means ± SEM are represented as the solid horizontal lines; *p < 0.01, N = 5 hearts.