Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts cardioprotection against ischemia reperfusion injuries

Ludovit Paulis, Jeremy Fauconnier, Olivier Cazorla, Jérome Thireau, Raffaella Soleti, Bastien Vidal, Aude Ouillé, Marion Bartholome, Patrice Bideaux, François Roubille, Jean-Yves Le Guennec, Ramaroson Andriantsitohaina, M Carmen Martínez, Alain Lacampagne, Ludovit Paulis, Jeremy Fauconnier, Olivier Cazorla, Jérome Thireau, Raffaella Soleti, Bastien Vidal, Aude Ouillé, Marion Bartholome, Patrice Bideaux, François Roubille, Jean-Yves Le Guennec, Ramaroson Andriantsitohaina, M Carmen Martínez, Alain Lacampagne

Abstract

Sonic hedgehog (SHH) is a conserved protein involved in embryonic tissue patterning and development. SHH signaling has been reported as a cardio-protective pathway via muscle repair-associated angiogenesis. The goal of this study was to investigate the role of SHH signaling pathway in the adult myocardium in physiological situation and after ischemia-reperfusion. We show in a rat model of ischemia-reperfusion that stimulation of SHH pathway, either by a recombinant peptide or shed membranes microparticles harboring SHH ligand, prior to reperfusion reduces both infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. We further demonstrate in healthy animals a reduction of QTc interval mediated by NO/cGMP pathway leading to the shortening of ventricular cardiomyocytes action potential duration due to the activation of an inward rectifying potassium current sharing pharmacological and electrophysiological properties with ATP-dependent potassium current. Besides its effect on both angiogenesis and endothelial dysfunction we demonstrate here a novel cardio-protective effect of SHH acting directly on the cardiomyocytes. This emphasizes the pleotropic effect of SHH pathway as a potential cardiac therapeutic target.

Figures

Figure 1. Activation of SHH signaling shortens…
Figure 1. Activation of SHH signaling shortens QT interval in healthy animals.
(A) ECGs were acquired over 6 hours after different treatments (i.p.-injected), in control animals. Representative ECGs recorded in control, N-SHH- and MPsSHH+-treated animals indicate a shortening of QT interval after SHH signaling pathway activation. (B) Mean QTc values recorded before (t = 0, left column) and 6 hours after injection (t = 6, right column) of the vehicle (control, n = 5), N-SHH (n = 8), N-SHH+Hexamethonium (N-SHH+Hex, n = 6), MPsSHH+ (n = 7), N-SHH and cyclopamine (N-SHH+Ccl, n = 6) and cyclopamine alone (Control + Ccl, n = 6). (* p < 0.05, QTc at 6 h vs t = 0).
Figure 2. SHH mediates NO production in…
Figure 2. SHH mediates NO production in ventricular cardiomyocytes.
(A) Representative PCR gel showing the expression of Patched (Ptc) and Smoothened (Smo) and reference Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs in rat cardiomyocytes. (B) Smo and Ptc expression was revealed by Western blotting in cardiomyocytes and in testis used as reference tissue. These representative images correspond to extracted portion of membranes after transfer of the same gel and exposed respectively with anti- Smo and Ptc primary antibodies. Each band corresponds to adjacent wells of the gel. (C) Representative confocal images illustrating the increase in NO production after incubation of control cardiomyocytes with the recombinant SHH protein (N-SHH) or microparticles harboring the SHH protein (MPsSHH+) for 4 h (upper panel). DAF fluorescence level (lower panel) was quantified in control (n = 495 cells), after incubation with N-SHH (n = 365 cells), and after co-incubation with phosphoinositide-3 kinase inhibitor, LY294002 (LY, 25 μM, n = 98 cells), NOS inhibitor, NΩ-nitro-L-arginine (L-NNA, 100 μM, n = 98 cells). DAF fluorescence level was also quantified after incubation with MPsSHH+ (n = 382 cells) and after co-incubation with the SHH pathway inhibitor cyclopamine (MPsSHH+-Ccl, 30 μM, n = 137 cells). Cyclopamine alone had no effect on basal levels of NO (Ccl, 30 μM, n = 68 cells). Incubation with MPs not carrying the SHH (MPsSHH-) did not enhance NO formation (n = 23 cells). Data are mean ± SEM; *, p < 0.05 compared to control.
Figure 3. SHH reduces action potential duration.
Figure 3. SHH reduces action potential duration.
(A) Representative traces of action potential recorded in control and after SHH treatment (left panel) and in the presence the ATP-dependent potassium channel inhibitor glibenclamide (1 μM) (right panel). (B) Average values of resting membrane potential in control (n = 7), SHH (n = 8), Control+glibenclamide (n = 7), N-SHH+glibenclamide (n = 8), N-SHH+L-NNA (n = 7), N-SHH+L-NNA+glibenclamide (n = 7), N-SHH+ODQ (n = 7), N-SHH+ODQ+Glibenclamide (n = 7). (C) Action potential duration at 95% of the repolarization (APD95,) in control (n = 7), SHH (n = 8), Control+glibenclamide (n = 7), N-SHH+glibenclamide (n = 8), N-SHH+L-NNA (n = 7), N-SHH+L-NNA+glibenclamide (n = 7), N-SHH+ODQ (n = 7), N-SHH+ODQ+Glibenclamide (n = 7). (D) The effect of glibenclamide (Glib) was further evaluated in vivo on the QT interval. Representative ECGs recorded in control (top), N-SHH (middle), and N-SHH+Glib (lower panel) treated animal. (E) Summary of the mean QTc value in each condition indicating the prevention of N-SHH effect in the presence of glibenclamide. (control n = 5, N-Shh n = 8, N-Shh+Glib n = 6, *, p < 0.05 compared to vehicle).
Figure 4. SHH activates an inward rectifying…
Figure 4. SHH activates an inward rectifying potassium current glibenclamide-sensitive.
(A) Family of current difference measured before and after application of glibenclamide from -120 mV to 0 mV in the absence (black traces) or in the presence of SHH (grey traces). (B) Average steady state currents expressed as current density are represented in control (n = 8) and SHH (n = 8). Similar experiments were also performed in the presence ODQ (n = 6) and L-NNA (n = 6). (C) The average current density at -120 mV. (D) The average current density at −60 mV. * and #, p vs N-SHH.
Figure 5. SHH signal pathway inhibits excitation-contraction…
Figure 5. SHH signal pathway inhibits excitation-contraction coupling of ventricular cardiomyocytes.
(A) Representative time course of sarcomere length of control (dash line), N-SHH-treated (continuous line) and MPsSHH+-treated cardiomyocytes (dot line) for 4 h. (B) Resting sarcomere length in control, N-SHH-treated and MPsSHH+-treated cardiomyocytes. (C) Cell shortening (% of diastolic sarcomere length). (D) Representative time-course of Ca2+ transient (450/480 nm indo-1 fluorescence) of control (dashed line) cardiomyocytes and cardiomyocytes incubated with a recombinant SHH (N-SHH, continuous line) or MPsSHH+ (dotted line) for 4 h. (E) Diastolic Ca2+ levels in control, N-SHH-treated and MPsSHH+-treated cardiomyocytes. (F) Ca2+ transient amplitude. All parameters were measured in control (n = 253), N-SHH-treated (n = 82) or MPsSHH+-treated (n = 218) cardiomyocytes after incubation with phosphoinositide-3 kinase inhibitor, LY294002 (LY, 25 μM, n = 19), NOS inhibitor, Nω-nitro-L-arginine (L-NNA, 100 μM, n = 19) and SHH pathway inhibitor, cyclopamine (Ccl, 30 μM, n = 17 with N-SHH and n = 75 with MPsSHH+). Incubation with MPs lacking the SHH protein (MPsSHH-, n = 25) did not modify the shortening or Ca2+ transient. Data are mean ± SEM, *, p < 0.05 compared to control.
Figure 6. SHH signal pathway on the…
Figure 6. SHH signal pathway on the cardiac excitation-contraction coupling.
(A) Active tension of isometric permeabilized control and MPsSHH+-treated cardiomyocytes (n = 12 in each condition). (B) Relative tension of isometric permeabilized control and MPsSHH+-treated cardiomyocytes (n = 12 in each condition). (C) Effect of N-SHH on L type Ca2+ current density (ICaL in pA/pF) as a function of membrane potential recorded in voltage-clamp in control (n = 7), recombinant SHH (N-SHH)-treated (N-SHH, n = 8) cardiomyocytes for 4 h. (D) Effect of MPsSHH+ on L type Ca2+ current density (ICaL in pA/pF) as a function of membrane potential recorded in voltage-clamp in control (n = 7), Shh-harboring MPs (MPsSHH+)-treated (n = 8) cardiomyocytes for 4 h. * p < 0.05 compared to control.
Figure 7. Activation of the SHH pathway…
Figure 7. Activation of the SHH pathway decreases the infarct size in a rat model of IR.
(A) Representative sections (Upper) of TTC-stained hearts. (B) Quantification was analyzed by normalizing the infarct area (IA) to the area at risk (AAR). Treatment with N-SHH reduced infarct size after 24 h of reperfusion and this cardioprotective effect was prevented by cyclopamine. (C) No significant difference was observed in the total AAR between groups. Data are expressed as mean ± SEM (control n = 6, N-SHH n = 8, N-SHH + Ccl n = 6; * p vs control).
Figure 8. Activation of SHH signaling improves…
Figure 8. Activation of SHH signaling improves ventricular repolarization after ischemia-reperfusion.
(A) ECGs were acquired over 24 h of reperfusion following 30min ischemia, in animal treated (i.v. injected 15 min prior reperfusion) either with the vehicle, recombinant N-SHH, or N-SHH + Cyclopamine (Ccl). Typical ECGs at 6 hours post reperfusion exhibited an enlargement of QT interval corrected for heart rate (QTc). (B) Time course of QTc variation is summarized in the 3 different experimental conditions. (C) Summarizes the number of arrhythmias recorded over a period of 2 hours following reperfusion showing a reduction of ventricular arrhythmia when SHH signaling pathway is activated. (D) Three representative patterns of ventricular arrhythmias (indicated by arrows) recorded after reperfusion in animals treated with the vehicle only (control). (control n = 6, N-SHH n = 8, N-SHH + Ccl n = 6; * p
All figures (8)

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