Abstract
Background/Aims: Early embryonic cardiomyocytes beat spontaneously. The hyperpolarization-activated cyclic-nucleotide-modulated current (I<sub>f</sub>) appears to be involved in its modulation as it is highly expressed at this stage. The spontaneous beating of early embryonic heart cells is slowed by acetylcholine (ACh), and our earlier studies identified a key role for nitric oxide (NO) in the regulation of the voltage dependent L-type Ca<sup>2+</sup> current (I<sub>Ca,L</sub>). The aim of the present study was to clarify whether and via which signalling pathway(s) I<sub>f</sub> is regulated upon muscarinic receptor activation in early embryonic (E9.5 to E11.5) cardiomyocytes. Methods: The whole-cell patch clamp technique in combination with pharmacology and/or knock out mouse models was used to investigate the regulation of I<sub>f</sub>. Results: We found that the ACh analogue carbachol (CCh, 10 µmol) led in the majority of cells (68%, n=50) to a significant depression of I<sub>f</sub> by 16.3±1.4% (n=34, p<0.01, voltage steps from -35 mV to –110 mV). This cholinergic inhibition was mediated by the NO/cGMP signalling pathway as it was largely reversed by superfusion with the non selective nitric oxide synthase (NOS) inhibitor N<sup>G</sup>-Methyl-L-arginine acetate salt (L-NMMA, 1 mmol), the inhibitor of the soluble guanylyl cyclase (sGC) 1H-[1, 2, 4]Oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ, 100 µmol) and a selective inhibitor of the phosphodiesterase (PDE) type 2 Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 µmol). Analysis of the muscarinic signalling in embryonic cardiomyocytes harvested from NOS2 (-/-) and NOS3 (-/-) mice revealed that the NOS3 isoform was entirely responsible for the muscarinic receptor-induced NO production. Conclusions: Muscarinic receptor stimulation depresses I<sub>f</sub> by generating NO via the NOS3 and the cGMP/PDE type 2 signalling pathway in early embryonic cardiomyocytes. This suggests that NO is a key signalling molecule involved in the regulation of chronotropy of early embryonic heart cells.
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