Regulation of heart rate following genetic deletion of the ß1 adrenergic receptor in larval zebrafish.

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 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% CO ) 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.