The Role of Muscarinic M2 and M3 Receptor Subtypes in Cardiopulmonary Responsiveness: Insights from In Vivo Murine Models

Abstract

Muscarinic receptors play an important role in cholinergic transmission and physiologic events. However, the study of the mechanisms by which muscarinic receptors mediate cardiopulmonary responses have been hampered by the homology of the five muscarinic receptor subtypes. We pursued three distinct methodologic approaches using murine models to dissect the fundamental roles and mechanisms of muscarinic receptor subtypes M2 (M2R) and M3 (M3R) within integrative physiology. Firstly, the role of M2R and M3R was evaluated in the development of airway hyperresponsiveness (AHR) in ovalbumin sensitized murine models lacking functional M2Rs and M3Rs. We provide novel evidence that the M3R, but not M2R, was critical for allergen-induced AHR, based on respiratory mechanics during airway challenge. The lack of the M3R also resulted in an unexpected bronchoconstrictor hyperresponsive phenotype independent of OVA sensitization and inflammation. Next, we examined the cardiopulmonary impact of skeletal M3R (activation of Gq G-protein pathway) on thermoregulation and cardiopulmonary function in a muscarinic (M3Dq) Designer Receptor Exclusively Activated by Designer Drug (DREADD) model. Based on telemetry from freely behaving mice, skeletal M3Dq expression had no impact on the daily cardiopulmonary phenotype, whereas, activation of skeletal M3Dq by Clozapine N-Oxide elicited a phenotype characterized by reduced ventilation, heart rate and body temperature, consistent with a presumptive reduction in metabolism. Lastly, we tested the ability of two novel structure-based designed muscarinic antagonists, ABH 423 and JHH 378, to inhibit differentially, the M3R over the M2R in a C57BL/6 murine model. Both compounds demonstrated a 4,000 x or greater -fold in vivo selectivity (p<0.001) of the M3R over the M2R, which was reflected in the inhibition of methacholine-induced bronchoconstriction and preservation of methacholine-induced bradycardia. Our findings revealed: 1. the key role of muscarinic M3 receptors in the development of airway responsiveness, 2. the integrated cardiopulmonary response due to skeletal muscle M3Dq pathway activation, possibly linked to torpor and, 3. the first pre-clinical data for structure-based highly selective M3R antagonists with therapeutic potential for obstructive lung disease.