M3 Muscarinic Receptors Mediate Positive Inotropic Responses in Mouse Atria: A Study with Muscarinic Receptor Knockout Mice

The potential functional roles of M-3 muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M-2 or M-3 receptor single knockout (M2KO, M3KO), and M-2 and M-3 muscarinic receptor double knockout mice (M-2/M3KO). Realtime quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M-2 receptor mRNA was expressed predominantly in mouse atria but that the M-1, M-3, M-4, and M-5 receptor subtypes were also expressed at low levels. Carbachol (10 nM-30 mu M) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M2KO mice confirmed that this activity is mediated by the M-2 receptor subtype. In left atria from wild-type mice, carbachol decreased the amplitude of electrical field stimulation-evoked contractions (negative inotropic action), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (positive inotropic response). In atria from M-3 KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M2KO mice, carbachol showed only positive inotropic activity. In atria from M-2/M-3 double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M-3 muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M-2 receptor activation on atrial contractility.