Muscarinic acetylcholine receptor mediated stimulation of cardiac excitation and contraction: Signal transduction and physiological implication

Vagus nerve and muscarinic acetylcholine receptor (mAChR) stimulation are largely associated with inhibition of cardiac function. Stimulatory effects on heart have also been observed with Ach but have not been thoroughly characterized. In this study, the muscarinic agonist carbachol (CCh) produced an increase in spontaneous beat rate of primary cultured neonatal rat ventricular myocytes (cnrvms). Positive inotropy was also observed in electrically paced cnrvms where CCh produced an increase in cytosolic calcium concentrations [Ca 2+]c. In paced ventricular tissue, CCh produced a rebound increase in force of contraction. Pharmacological studies demonstrated that the stimulatory effects of CCh were atropine, pirenzepine and neomycin sensitive but pertussis toxin and methoctramine insensitive, suggesting that a mAChR subtype other than m2 is responsible for mediating stimulation. Single-cell rt-pcr and immunocytochemistry revealed the existence of m1 mAChR subtype mRNA and functional protein in cnrvms. These results support the hypothesis that the stimulatory effects of muscarinic agonists/vagal nerve stimulation occur through activation of m1 mAChRs. M1 mAChR activation produced an increase in phosphotyrosine content of several cellular proteins, including a protein immunologically related to the cellular tyrosine kinase PYK2. Furthermore, pretreatment of cnrvms with the tyrosine kinase inhibitor genistein attenuated the ability of CCh to produce an increase in [Ca2+]c, suggesting that CCh mediated positive inotropy involves activation of cellular tyrosine kinases.; The physiological importance of m1 mAChR signaling on cardiac function was investigated in wild-type and m1 mAChR knockout mice. Heart-rate measurements performed on anesthetized mice in response to ACh injection revealed that wild-type mice experienced a rebound increase in heart rate following an initial drop in rate. Knockout m1(--/--) mice did not display rebound increase in rate with Ach, and interestingly, several animals were unable to restore their heart rates and died following the first or subsequent exposure to ACh. These results suggest that m1 mAChR activation in heart prevents fatal cardiac depression following chronic exposure to muscarinic agonists. Contractility studies on paced papillary muscle from neonatal, adult male and adult female ventricles demonstrated that the CCh mediated increase in contractility exhibits the following pattern, neonate > adult male > adult female. These findings suggest developmental and hormonal regulation of m1 mAChR expression and signaling in cardiac tissue. Western blot analysis of m1 mAChR levels in ventricular myocytes detected lower levels of m1 mAChR in female ventricles compared to males.