Loss of M2 muscarinic receptor function inhibits development of hypoxic bradycardia and alters cardiac β-adrenergic sensitivity in larval zebrafish (Danio rerio)

Steele SL, Lo KH, Li VW, Cheng SH, Ekker M, Perry SF. of M-2 muscarinic receptor function inhibits development of hypoxic bradycardia and alters cardiac beta-adrenergic sensitivity in larval zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 297: R412-R420, 2009. First published June 10, 2009; doi:10.1152/ajpregu.00036.2009.-Fish exposed to hypoxia develop decreased heart rate, or bradycardia, the physiological significance of which remains unknown. The general muscarinic receptor antagonist atropine abolishes the development of this hypoxic bradycardia, suggesting the involvement of muscarinic receptors. In this study, we tested the hypothesis that the hypoxic bradycardia is mediated specifically by stimulation of the M-2 muscarinic receptor, the most abundant subtype in the vertebrate heart. Zebrafish (Danio rerio) were reared at two levels of hypoxia (30 and 40 Torr PO2) from the point of fertilization. In hypoxic fish, the heart rate was significantly lower than in normoxic controls from 2 to 10 days postfertilization (dpf). At the more severe level of hypoxia (30 Torr PO2), there were significant increases in the relative mRNA expression of M-2 and the cardiac type beta-adrenergic receptors (beta 1AR, beta 2aAR, and beta 2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Torr PO2) or significantly attenuated (at 30 Torr PO2) in larvae experiencing M-2 receptor knockdown (using morpholino antisense oligonucleotides). Sham-injected larvae exhibited typical hypoxic bradycardia in both hypoxic regimens. The expression of beta 1AR, beta 2aAR, beta 2bAR, and M-2 mRNA was altered at various stages between 1 and 4 dpf in larvae experiencing M-2 receptor knockdown. Interestingly, M-2 receptor knockdown revealed a cardioinhibitory role for the beta(2)-adrenergic receptor. This is the first study to demonstrate a specific role of the M-2 muscarinic receptor in the initiation of hypoxic bradycardia in fish.