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

Abstract

The potential functional roles of M₃ muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M₂ or M₃ receptor single knockout (M₂KO, M₃KO), and M₂ and M₃ muscarinic receptor double knockout mice (M₂/M₃KO). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M₂ receptor mRNA was expressed predominantly in mouse atria but that the M₁, M₃, M₄, and M₅ receptor subtypes were also expressed at low levels. Carbachol (10 nM–30 μM) 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 M₂ 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 M₃KO 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 M₂/M₃ double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M₃ muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M₂ receptor activation on atrial contractility.