In vivo and in vitro assessment of cardiac β-adrenergic receptors in larval zebrafish (Danio rerio)

Abstract

SUMMARY β-Adrenergic receptors (βARs) are crucial for maintaining the rate and force of cardiac muscle contraction in vertebrates. Zebrafish (Danio rerio) have one β1AR gene and two β2AR genes (β2aAR and β2bAR). We examined the roles of these receptors in larval zebrafish in vivo by assessing the impact of translational gene knockdown on cardiac function. Zebrafish larvae lacking β1AR expression by morpholino knockdown displayed lower heart rates than control fish, whereas larvae deficient in both β2aAR and β2bAR expression exhibited significantly higher heart rates than controls. These results suggested a potential inhibitory role for one or both β2AR genes. By using cultured HEK293 cells transfected with zebrafish βARs, we demonstrated that stimulation with adrenaline or procaterol (a β2AR agonist) resulted in an increase in intracellular cAMP levels in cells expressing any of the three zebrafish βARs. In comparison with its human βAR counterpart, zebrafish β2aAR expressed in HEK293 cells appeared to exhibit a unique binding affinity profile for adrenergic ligands. Specifically, zebrafish β2aAR had a high binding affinity for phenylephrine, a classical α-adrenergic receptor agonist. The zebrafish receptors also had distinct ligand binding affinities for adrenergic agonists when compared with human βARs in culture, with zebrafish β2aAR being distinct from human β2AR and zebrafish β2bAR. Overall, this study provides insight into the function and evolution of both fish and mammalian β-adrenergic receptors.

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

“…To resolve potential problems associated with non-specific effects of pharmacological blockade of β-receptors, a gene knockdown approach was employed to specifically target the β 1 receptor (Steele et al, 2011). In contrast to the results of Steele et al (Steele et al, 2011), β 1 -AR knockdown did not lower resting heart rate. However, similar to β-AR receptor pharmacological blockade, the tachycardia induced by hypercapnia was prevented by β 1 -AR knockdown.…”

“…(Bagatto, 2005;Schwerte et al, 2006;Wang et al, 2009;Steele et al, 2011), it is not straightforward to distinguish between tone arising from a functional sympathetic innervation of the heart and tone associated with circulating catecholamines. Control of catecholamine secretion, although well characterized in adults (Reid et al, 1998;Perry and Bernier, 1999;Perry and Capaldo, 2011), has not been studied in larval fish.…”

“…Morpholinos (tagged with 3′-carboxyfluorescein) for zebrafish cytosolic CA (zCAc; NM_199215, also referred to as CA2-like a) (Gilmour et al, 2009), the β 1 -adrenergic receptor (β 1 -AR; AB294717) (Steele et al, 2011) and a control morpholino were purchased from Gene Tools (Philomath, OR, USA) with the following sequences, respectively: 5′-AGTGGTCAGCCAT -TCCGCCAGCTGT-3′, 5′-ACGGTAGCCCGTCTCCCATTG-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3′. All morpholinos were prepared to a final concentration of 4 ng nl −1 in Danieau buffer (58 mmol l −1 NaCl, 0.7 mmol l −1 KCl, 0.4 mmol l −1 MgSO 4 , 0.6 mmol l −1 Ca(NO 3 ) 2 , and 5 mmol l −1 Hepes with a final pH of 7.6), and Phenol Red was used as an indicator of positive injection.…”

“…Injections into one-or two-cell embryos were performed using a Narishige IM300 microinjector (Narishige International USA Inc., Long Island, NY, USA) with needles made from 1.0 mm borosilicate glass (Sutter Instrument, Novato, CA, USA). Dosage and sequence of zCA isoforms are based on Gilmour et al (Gilmour et al, 2009) and β 1 -AR from Steele et al (Steele et al, 2011). Neither study reported adverse effects when using a dosage of 4 ng per embryo.…”

β-Adrenergic regulation of Na+uptake by larval zebrafishDanio rerioin acidic and ion-poor environments

“…For the dose of injected morpholino, see Ref. 36. Control groups were injected with a standard control morpholino (5=-CCTCTTACCTCAGTTACAATTTATA-3=) that was prepared and injected as the other morpholinos.…”