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Abstract
Atropine is a clinically relevant anticholinergic drug, which blocks inhibitory effects of the parasympathetic neurotransmitter acetylcholine on heart rate leading to tachycardia. However, many cardiac effects of atropine cannot be adequately explained solely by its antagonism at muscarinic receptors. In isolated mouse ventricular cardiomyocytes expressing a Förster resonance energy transfer (FRET)-based cAMP biosensor, we confirmed that atropine inhibited acetylcholine-induced decreases in cAMP. Unexpectedly, even in the absence of acetylcholine, after G-protein inactivation with pertussis toxin or in myocytes from M2- or M1/3-muscarinic receptor knockout mice, atropine increased cAMP levels that were pre-elevated with the β-adrenergic agonist isoproterenol. Using the FRET approach and in vitro phosphodiesterase (PDE) activity assays, we show that atropine acts as an allosteric PDE type 4 (PDE4) inhibitor. In human atrial myocardium and in both intact wildtype and M2 or M1/3-receptor knockout mouse Langendorff hearts, atropine led to increased contractility and heart rates, respectively. In vivo, the atropine-dependent prolongation of heart rate increase was blunted in PDE4D but not in wildtype or PDE4B knockout mice. We propose that inhibition of PDE4 by atropine accounts, at least in part, for the induction of tachycardia and the arrhythmogenic potency of this drug.
Highlights
The autonomic nervous system regulates functions of various organs via the sympathetic and parasympathetic neurotransmitters norepinephrine and acetylcholine (ACh)
We use various in vitro and in vivo techniques to test the hypothesis that atropine can inhibit the activity of cyclic adenosine monophosphate (cAMP) hydrolysing phosphodiesterases (PDEs), thereby increasing intracellular cAMP levels
Independently of its effect on muscarinic receptors, can inhibit PDE type 4 (PDE4) activity, leading to augmented cardiac contractility after β-adrenergic stimulation
Summary
Atropine increases cAMP independently of M1/2/3-muscarinic receptors. To elucidate the exact molecular mechanisms of atropine action in the heart, we studied its effects in cardiomyocytes isolated from mice expressing the Förster resonance energy transfer (FRET)-based cAMP-sensor Epac1-camps[9,10]. Sarcomere shortening measurements in isolated ventricular cardiomyocytes showed that atropine enhanced the positive inotropic effect of ISO (Fig. 3f,g) This experiments support the concept that even in absence of parasympathetic innervation in isolated cardiomyocytes or explanted hearts, atropine increases cAMP levels and cardiac contractility by inhibiting PDEs. In human atria, PDE4 accounts for only ~15% of the cAMP-specific PDE activity, whereas PDE3 represents the major PDE family[27,28]. While atropine alone or atropine treatment after ISO prestimulation did not affect force of contraction (data not shown), application of atropine to trabeculae pretreated with ISO and the PDE3 inhibitor cilostamide substantially increased contractility which was further augmented by rolipram (Fig. 3h,i) This finding confirms that the positive inotropic effect of atropine is relevant for the human heart and results from PDE4 inhibition. Since the stimulatory effect of atropine on cAMP production is only observed under catecholamine stress, it can be expected that therapeutically used β-blockers might effectively counteract atropine-induced arrhythmias associated with PDE inhibition
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