Aim Although zebrafish are gaining popularity as biomedical models of cardiovascular disease, our understanding of their cardiac control mechanisms is fragmentary. Our goal was to clarify the controversial role of the ß1‐adrenergic receptor (AR) in the regulation of heart rate in zebrafish. Methods CRISPR‐Cas9 was used to delete the adrb1 gene in zebrafish allowing us to generate a stable adrb1−/− line. Larval heart rates were measured during pharmacological protocols and with exposure to hypercapnia. Expression of the five zebrafish adrb genes were measured in larval zebrafish hearts using qPCR. Results Compared with genetically matched wild‐types (adrb1+/+), adrb1−/− larvae exhibited ~20 beats min−1 lower heart rate, measured from 2 to 21 days post‐fertilization (dpf). Nevertheless, adrb1−/− larvae exhibited preserved positive chronotropic responses to pharmacological treatment with AR agonists (adrenaline, noradrenaline, isoproterenol), which were blocked by propranolol (general ß‐AR antagonist). Regardless of genotype, larvae exhibited similar increases in heart rate in response to hypercapnia (1% CO2) at 5 dpf, but tachycardia was blunted in adrb1−/− larvae at 6 dpf. adrb1 gene expression was abolished in the hearts of adrb1−/− larvae, confirming successful knockout. While gene expression of adrb2a and adrb3a was unchanged, adrb2b and adrb3b mRNA levels increased in adrb1−/− larval hearts. Conclusion Despite adrb1 contributing to the setting of resting heart rate in larvae, it is not strictly essential for zebrafish, as we generated a viable and breeding adrb1−/− line. The chronotropic effects of adrenergic stimulation persist in adrb1−/− zebrafish, likely due to the upregulation of other ß‐AR subtypes.
The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent β-adrenoreceptor (AR)-mediated increase in fV that does not require expression of β1-ARs, as the hyperventilatory response to β-AR stimulation was unaltered in adrb1−/- mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min−1 without propranolol to 68±17 breaths min−1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild-types and adrb1−/- mutants. Thus β1-AR is not required for the HVR, and other β-ARs, though not required for the initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system.
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