Loss of M₂muscarinic receptor function inhibits development of hypoxic bradycardia and alters cardiac β-adrenergic sensitivity in larval zebrafish (Danio rerio)

Abstract

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 PO(2)) 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 PO(2)), there were significant increases in the relative mRNA expression of M(2) and the cardiac type beta-adrenergic receptors (beta1AR, beta2aAR, and beta2bAR) at 4 dpf. The hypoxic bradycardia was abolished (at 40 Torr PO(2)) or significantly attenuated (at 30 Torr PO(2)) 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 beta1AR, beta2aAR, beta2bAR, 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.

Evidence for a role of heme oxygenase-1 in the control of cardiac function in zebrafish (Danio rerio) larvae exposed to hypoxia

“…The dose of 0.05 mg ml −1 of MS-222 was chosen because it was the lowest dose able to keep the larvae immobilized. Other studies have shown that high doses of MS-222 decrease f H in zebrafish larvae; however, the concentration used in this study is lower than that used in those studies (Steele et al, 2009(Steele et al, , 2011. Furthermore, in our own experiments only a dose of MS-222 above 0.12 mg ml −1 caused a decrease in f H after 10-15 min exposure of 4 dpf larvae.…”

“…In larval zebrafish, the onset of cardiac control via the parasympathetic and sympathetic nervous systems (or circulating catecholamines) is evident as early as 4 dpf (Steele et al, 2009(Steele et al, , 2011. The development of bradycardia in the face of chronic hypoxia in zebrafish larvae is mediated by cardiac muscarinic receptors (Bagatto, 2005) of the M 2 sub-type (Steele et al, 2009), while the tachycardia response to acute hypoxia is probably controlled by a release of inhibitory vagal tone or stimulation of adrenergic receptors by elevated circulating catecholamine levels or catecholamines released from sympathetic neurons (Steele et al, 2009(Steele et al, , 2011.…”

Cardiac responses to hypercapnia in larval zebrafish (Danio rerio): The links between CO2 chemoreception, catecholamines and carbonic anhydrase

“…For example, although hypoxic bradycardia was observed in 4 d.p.f. larvae in at least two studies (Bagatto, 2005;Steele et al, 2009), there are also reports of the zebrafish heart being unresponsive to hypoxia until 30 d.p.f. at which time bradycardia develops (Barrionuevo and Burggren, 1999), or actually exhibiting tachycardia (Jacob et al, 2002).…”

“…Previous studies have also reported a lack of an effect of atropine on resting heart rate until about 12 d.p.f. (Schwerte et al, 2006;Steele et al, 2009), suggesting that parasympathetic cholinergic tone is absent until ~12 d.p.f. However, Hsieh and Liao (Hsieh and Liao, 2002) reported a significant reduction in heart rate in 3 d.p.f.…”

Vitreoscilla Hemoglobin (VHb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio)

“…Because early life stage (ELS) is one of the most sensitive life stages to toxicity during the life cycle, ELS tests with fish species are commonly used as routine toxicity tests in risk assessment (OECD 1998;Roex et al 2002). D. rerio was recommended as a model fish species for short-term embryo-larval toxicity testing (OECD 1998), and there are many studies investigating the effects of hypoxia on zebrafish embryo or larvae (Padilla and Roth 2001;Ton et al 2003;Rombough and Drader 2009;Steele et al 2009;Van Rooijen et al 2009;Yaqoob et al 2009). We found that the 7-day-old F 3 vhb transgenic larvae may be suitable for study of the hypoxia tolerance traits.…”