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Letters to the Editor: Cardiomyocyte‐secreted acetylcholine

I read the paper, “Cardiomyocyte-secreted acetylcholine is required for maintenance of homeostasis in the heart” (1), on acetylcholine (ACh) originating in cardiac myocytes, with great interest. The significance of this phenomenon continues to fascinate since it was first described over 40 years ago. The authors have overlooked reports and experimental limits from the earlier papers that deserve careful consideration. Edouard Coraboeuf and colleagues (2) reported in 1970 that cardiac myocytes could synthesize and release ACh. Of note, they obtained data from embryonic hearts before innervation occurred. They also observed that electrical stimulation of the embryonic chick heart caused a transient slowing of heart rate and an increased release of ACh (2). The transient slowing was opposed by atropine. So far, more recent reports (1, 3) have not asked whether stimuli can regulate ACh secretion. Divergent results on the age of animals whose heart cells can secrete ACh are another unsettled issue. You report that myocytes from neonatal mice secrete ACh. This is opposite the report by Rana et al. (3), who state that “. . . adult, but not neonatal cardiomyocytes are able to synthesize, transport and excrete acetylcholine in the rat heart.” This discrepancy calls for an explanation, inasmuch as rats and mice develop similarly after nearly equal gestation periods. The immunofluorescent detection of choline acetyltransferase (ChAT) requires validation of specificity. Some years ago (4), it was reported that the method to detect ChAT must carefully distinguish ChAT from carnitine acetyltransferase, which is found in cardiac muscle. Thus, Roskoski et al. (4) showed that embryonic chick heart cells, per se, had no ChAT activity and no neurons. Neonatal rat heart cells in culture had no ChAT, but carnitine acetyltransferase was present. Thus, in the absence of cholinergic neurons, heart cells did not synthesize ACh (4). Does the immunofluorescent method you use distinguish between ChAT and carnitine acetyltransferase? The experiments with carbachol and pyridostigmine overlook important drug properties. It is reported that addition of carbachol caused ACh release from cardiac myocytes, as indicated by increased NO production, a downstream fluorescent signal (1). There are concerns with this observation. First, cholinergic neurons have muscarinic receptors whose occupancy by ACh reduces ACh release (5). This negative-feedback mechanism, a physiological hallmark of homeostasis, seems lacking in your system. Second, “. . . pyridostigmine also augmented the fluorescent signal (Supplemental Fig. S2F).” Pyridostigmine, like neostigmine (and carbachol), has a quaternary N that not only renders it impermeable to cell membranes but also makes it an agonist acting directly at cholinoceptive sites, just like carbachol. Thus, pyridostigmine and neostigmine can activate receptors, in addition to inhibiting acetylcholinesterase. Similarly, hemicholinium-3 (HC-3) is another quaternary N compound whose actions against pyridostigmine can be explained by HC-3 binding to the same site as pyridostigmine and occluding its effect. Indeed, one should test vesamicol and HC-3 against carbachol, as was done against pyridostigmine in Supplemental Fig. 2SF. Such testing should clarify some essential elements of the hypothesis. The current state of the problem seems not to have evolved significantly from that described by earlier investigators. This is a consequence, in part, of a failure to attend to precedent.