The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice

We hypothesized that muscarinic receptors (MRs) in the heart have a role in stress responses and thus investigated changes in MR signaling (gene expression, number of receptors, adenylyl cyclase (AC), phospholipase C (PLC), protein kinase A and C (PKA and PKC) and nitric oxide synthase [NOS]) in the left ventricle, together with telemetric measurement of heart rate (HR) in mice (wild type [WT] and M-2 knockout [KO]) during and after one (1R) or seven sessions (7R) of restraint stress (seven mice per group). Stress decreased M-2 MR mRNA and cell surface MR in the left ventricle in WT mice. In KO mice, 1R, but not 7R, decreased surface MR. Similarly, AC activity was decreased in WT mice after 1R and 7R, whereas in KO mice, there was no change. PLC activity was also decreased after 1R in WT and KO mice. This is in accord with the concept that cAMP is a key player in HR regulation. No change was found with stress in NOS activity. Amount of AC and PKA protein was not changed, but was altered for PKC isoenzymes (PKC alpha, beta, gamma, eta and epsilon (increased) in KO mice, and PKCi (increased) in WT mice). KO mice were more susceptible to stress as shown by inability to compensate HR during 120 min following repeated stress. The results imply that not only M-2 but also M-3 are involved in stress signaling and in allostasis. We conclude that for a normal stress response, the expression of M-2 MR to mediate vagal responses is essential.