Regulation of Heart Contractility by M2 and M3 Muscarinic Receptors: Functional Studies Using Muscarinic Receptor Knockout Mouse

To investigate the functional roles of M2 and M3 muscarinic receptors in mouse atria, wild-type mice, muscarinic M2 or M3 single receptor knockout mice (M2KO, M3KO), and M2 and M3 muscarinic receptor double knockout mice (M2/M3KO) were used for pharmacological and molecular biological approaches. Effects of carbachol on spontaneous contraction (right atrium) or electrically-evoked contraction (left atrium) were examined in the isolated atria of the respective mice. In atria from wild-type and M3KO mice, carbachol only decreased spontaneous contraction regardless of high or low heart rate preparations. However, in M2KO mice, carbachol did not induce any chronotropic actions in the high heart rate atrium (400 beats/min), but when the heart rate was low (under 300 beats/min), carbachol caused an increase in heart rate (positive chronotropic actions). Since carbachol did not cause any chronotropic actions in either high or low heart rate atria from M2/M3KO mice, it is thought that the positive chronotropic actions were due to activation of the M3 receptor. Presence of muscarinic receptor subtype mRNAs and proteins was determined by real time RT-PCR using specific primers and immunohistochemistry using specific anti-M2 and anti-M3 receptor antibodies. Quantitative real-time RT-PCR analysis showed that M2 receptor mRNA was expressed dominantly in mouse atria but that the M1, M3, M4, and M5 receptor subtypes were also expressed at low levels. Carbachol decreased the frequency of spontaneous beating in right atria of mice through activation of the M2 receptor subtype. In left atria of wild-type mice, carbachol decreased the amplitude of electrical field stimulation (EFS)-evoked contractions (M2 receptors), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (M3 receptors). Cyclooxygenase-2 (COX-2) and prostaglandins in the endocardial endothelium were involved in the M3 receptor-mediated positive inotropic actions. In conclusion, the present studies using isolated atria of muscarinic receptor knockout mice demonstrated that myocardial M2 receptors mediate negative chronotropic/inotropic actions and that M3 muscarinic receptors mediate positive chronotropic/inotropic actions in mouse atria. Physiologically, M3 receptor-mediated excitatory cardiac effects might dampen the inhibitory effects of M2 receptor activation on cardiac contractility.