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

Abstract

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.

Cardiovascular Response to Exercise in Vertebrates: Update

“…In the studied vertebrates, HR and its adjustment to physical exercise show notable differences and could be influenced by several intrinsic factors, such as age (as has already been evidenced in humans) [65,[84][85][86][87][88][89]. As illustrated in Table 1, fish, amphibians, and reptiles show a limited range of HR.…”

“…HR in vertebrates is determined by the activity of the heart pacemaker and various factors, such us temperature, acetylcholine, adrenaline, or calcium, that modulate its electrical activity via nervous and hormonal pathways [64][65][66][67][68]. SV is determined by contractility and the characteristics of the ventricle, mainly regarding its filling capacity.…”

Evolutionary loss of the ß1-adrenergic receptor in salmonids

“…However, it appears plausible that loss of adrb1 might not have been sustainable without the expansion of adrb2 and adrb3 repertoires. By analogy, knockout of adrb1 in zebrafish leads to increased cardiac expression of adrb2b and adrb3b genes (Joyce et al, 2022). As such, this study contributes to the growing appreciation of the redundancy between adrb paralogs, particularly in teleosts.…”

“…In mice, deletion of Adrb1 results in high prenatal mortality and renders the heart insensitive to ß-adrenergic stimulation (Rohrer et al, 1996). In zebrafish, by contrast, adrb1 knockout has no obvious adverse effects on viability and fertility, at least when raised in optimized aquaculture conditions (Joyce et al, 2022), although it is not known if the knockout fish would survive under natural conditions. Routine heart rate is suppressed in adrb1 knockout larval zebrafish, although they are still able to respond to ß-adrenergic stimulation with tachycardia, presumably mediated by other ß-ARs (Joyce et al, 2022).…”

Acquirement of the autonomic nervous system modulation evaluated by heart rate variability in medaka (Oryzias latipes)

“…Zebrafish and medaka have recently been established as model animals of human diseases [24][25][26][27]. Several pharmacological studies have revealed that larval-stage zebrafish express receptors for sympathetic and parasympathetic neural transmission are expressed in zebrafish larvae and that heart rates in zebrafish larvae change in response to both sympathetic and parasympathetic input with or without anesthesia [17,21,[28][29][30], although one of these studies further demonstrated that the autonomic components of the reflex are poorly developed in 5-days-old larvae of zebrafish, suggesting that ANS function is incompletely developed at this stage [21]. Moreover, to evaluate long-term changes in ANS activity such as those induced by environmental changes, it is necessary to study adult animals to avoid major alterations in ANS activity during embryonic development, growth or sexual maturation.…”