The Modulation of Excitation-contraction Coupling in Isolated Cardiomyocytes by Microparticle-carried Sonic Hedgehog Protein

Ludovit Paulis 1,2 Jeremy Fauconnier 2 Olivier Cazorla 2 Jerome Thireau 2 Raffaella Soletti 3 Bastien Vidal 2 Aude Ouille 2 Marion Bartholome 2 Patrice Bideaux 2 Francois Roubille 2 Jean-Yves Le Guennec 2 Ramaroson Andriantsitohaina 3 M. Carmen Martinez 3 Alain Lacampagne 2
1Institute of Pathological Physiology, Faculty of Medicine, Comenius University, Bratislava, Bratislava
2UMR CNRS 9214, Inserm U1046, Universite de Montpellier, CHRU Montpellier, Montpellier
3INSERM U1063, Stress Oxydant et Pathologies Metaboliques, LUNAM Universite, Angers, Angers

Sonic hedgehog (Shh) is largely involved in embryonic tissue patterning and development. However, there is a growing evidence for a broader role of the Sonic hedgehog (Shh) signaling even in adult cardiovascular system. Recently, shed membrane microparticles (MPs) carrying the Shh protein were demonstrated to activate the PI3-K/Akt/NO pathway in endothelial cells.

We aimed to investigate the effects of Shh on excitation-contraction coupling (ECC) and the possible involvement of PI3-K/Akt/NO pathway in rat ventricular cardiomyocytes. Cardiomyocytes from adult Wistar rats were isolated by enzymatic dissociation with collagenase 4. The cells were incubated for 4 hours with recombinant Shh, MPs carrying Shh and MPs not carrying Shh; in the presence or absence of inhibitors of the Shh signaling pathway. The calcium transient and cell shortening were recorded based on Indo fluorescence and sarcomere length, NO production was determined by DAF staining in confocal microscopy and ionic currents were measured using whole-cell patch-clamp technique.

We observed that incubation of cardiomyocytes with Shh induced NO production, with subsequent activation of a glibenclamide-sensitive inward rectifier potassium channel (IKATP). Its activation resulted in reduced action potential duration and a decrease in the amplitude of calcium transient and cell contraction. These effects were prevented by cyclopamine, LY294002, L-NNA and ODQ-1 implying the activation of the Patched-Smoothened complex, PI3-kinase/Akt, NO-synthase and cGMP accumulation, respectively. While incubation with MPs not carrying the Shh protein did not reveal any effect on cardiomyocytes, Shh carried by lymphocyte-derived shed MPs mimicked the effects of the Shh protein.

We conclude that Shh pathway is functional in adult cardiomyocytes and directly modulates ECC by activating IKATP. Moreover, Shh pathway might be implied in long-range signaling in physiological and/or pathophysiological situations where MPs carrying Shh are produced. (VEGA 1-0380-14)









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