Hypoxia inducible factor 1-α is minimally involved in determining the time domains of the hypoxic ventilatory response in adult zebrafish (Danio rerio)

https://doi.org/10.1016/j.resp.2021.103774Get rights and content

Highlights

  • Discrete time domains of the hypoxic ventilatory response in adult zebrafish.

  • Dominant time domain is hypoxic ventilatory decline during sustained hypoxia.

  • Pronounced hypoventilation during recovery following sustained hypoxia.

  • Hif-1α plays only minor role in regulating hypoxic ventilatory patterns.

Abstract

In the current study, adult zebrafish (Danio rerio) were exposed to 72 h hypoxia (90 mmHg) to assess the time domains of the hypoxia ventilatory response (HVR) and the consequence on a subsequent more severe (40 mmHg) bout of acute hypoxia. Experiments were performed on wild-type fish and mutants in which one or both paralogs of hypoxia inducible factor-1α (hif-1α) were knocked out. Although there were subtle differences among the wild-type and knockout fish, resting fV was reestablished after 2–8 h of continuous hypoxia in both groups, a striking example of hypoxic ventilatory decline (HVD). When fish were subsequently exposed to more severe hypoxia, a rapid increase in fV was observed, the magnitude of which was independent of genotype or prior exposure history. During recovery, fish that had been exposed to 72 h of 90 mmHg hypoxia exhibited a pronounced undershoot in fV, which was absent in the hif-1α double knockouts. Overall, the results revealed distinct time domains of the HVR in zebrafish that were largely Hif-1α-independent.

Introduction

When exposed to sufficiently severe hypoxia, fish increase the volume of water flowing over the gills, a phenomenon that is termed the hypoxic ventilatory response (HVR). The HVR helps to maintain normal rates of O2 uptake across the gill despite the lower inspired O2 tensions by maximizing water-to-blood O2 diffusion gradients (Davis and Cameron, 1971). The HVR is characterised by increased ventilation frequency (fV) and/or amplitude [reviewed by Perry et al. (2009)]. The relative importance of fV versus amplitude adjustments to the overall increase in ventilation volume during hypoxia varies greatly among species (Gilmour, 2001; Perry et al., 2009). In adult zebrafish (Danio rerio), the HVR is driven exclusively by an increase in fV with no obvious adjustment of amplitude (Porteus et al., 2021; Vulesevic et al., 2006).

In mammals, the HVR is associated with discrete time domains consisting of specific phases of ventilatory adjustments during sustained hypoxia that occur acutely (seconds-to-minutes) or with long-term (hour-to-days-to months) exposures (Powell et al., 1998). Acute time domains beginning within 1–2 breaths of hypoxia exposure include phases of short-term depression and short-term potentiation (Powell et al., 1998). Little is known about the acute time domains in fishes (Porteus et al., 2011). With prolonged bouts of hypoxia, specific time domain phases include hypoxic ventilatory decline (HVD; a decrease in ventilation during sustained hypoxia relative to the acute response), ventilatory acclimation to hypoxia (VAH; an increase in hypoxic sensitivity) and hypoxic desensitization (HD; a decrease in hypoxic sensitivity usually associated with chronic or lifetime exposure to hypoxia) (Porteus et al., 2011; Powell et al., 1998). Because CO2 is such a potent driver of ventilation in mammals, the VAH is physiologically significant in offsetting the hypoventilatory influence of respiratory alkalosis under conditions when ventilation needs to be maximized (e.g. during exposure to high altitude). In fish, where CO2 plays a less important role in regulating ventilation, HVD may be the preferred response during sustained hypoxia because decreasing ventilation will lower the ventilatory convection requirement thereby reducing the energetic costs of breathing, which are higher in water- versus air-breathers. (Dejours, 1976, 1981; Maina, 2000).

Few studies have sought to define time domains of the HVR in fish. Typically, experiments examining the ventilatory responses of fish to hypoxia are acute and last for less than a few hours. These studies, while informative, were not designed to evaluate time domains of the HVR, especially those potentially occurring with sustained long-term hypoxia. With a few notable exceptions in a single species (see below), the results of previous studies that have monitored ventilation for 90 min to 24 h of hypoxia suggest that hyperventilatory responses are more-or-less independent of duration of exposure and thus provide no support for time domains during this period (Borch et al., 1993; Florindo et al., 2006; Forgue et al., 1989; Glass et al., 1990; Thomas and Hughes, 1982a, b). We are aware of a single study (Porteus et al., 2014b) that was designed specifically to investigate time domains in fish exposed to hypoxia. In that study, fV and breathing amplitude were monitored in bowfin (Amia calva) exposed for 3 days to sustained hypoxia (26 mm Hg at 8 °C or 45 mm Hg at 22 °C) while being denied access to air. A distinct pattern emerged, consisting of an acute hyperventilatory phase followed by a period of diminished hyperventilation that preceded a secondary increase in ventilation (Porteus et al., 2014b). It was suggested (Porteus et al., 2014b) that the decrease in ventilation is analogous to the HVD in mammals and likewise serves to conserve energy.

Although not specifically designed to assess time domains, the results of two other studies (Moyson et al., 2015; Stecyk and Farrell, 2002) using common carp (Cyprinus carpio) demonstrated an attenuation of the HVR with sustained hypoxia. In the more recent study (Moyson et al., 2015), exposure of carp to 7 days of continuous hypoxia (1.3–1.5 mg O2 L−1; estimated by us to be ∼35 mmHg) was accompanied by a maximal increase in fV by 5 h, which declined gradually to reach baseline (normoxic) values after 119 h (Moyson et al., 2015), a response that is indicative of HVD. It is unclear, however, whether the decrease in fV observed by Stecyk and Farrell (2002) truly reflected HVD given that the extremely severe levels of hypoxia employed (<0.3 mg O2 L−1) presumably were accompanied by metabolic depression.

The precise causes of the HVD in bowfin and carp are unclear although it is possible that they reflect a decrease in chemosensory activity (Porteus et al., 2014b). Alternatively, the ventilatory decline could be related to a gradual improvement of O2 transfer and transport owing to non-ventilatory compensatory adjustments. With long term exposure to hypoxia, a variety of physiological responses are initiated to enhance O2 uptake and delivery beyond that achieved by hyperventilation alone. These responses include an elevation in O2 carrying capacity of the blood (Nikinmaa and Tervonen, 2004), cardiovascular adjustments (Sandblom and Axelsson, 2011), modifications of carbohydrate transport and metabolism (Weber et al., 2016; Zhu et al., 2013), mitochondrial changes (Chen et al., 2020) and an increase in haemoglobin–O2 binding affinity (Cadiz et al., 2019; Wood and Johansen, 1972). All of these changes potentially could contribute to HVD although the increase in haemoglobin-O2 binding affinity arguably would be the most significant. By allowing equivalent levels of haemoglobin-O2 saturation to be achieved at lower arterial PO2 values (see reviews by (Nikinmaa, 2001; Perry and Wood, 1989), ventilation can be reduced appropriately. Many, if not all of the compensatory responses listed above are thought to be initiated by one or more isoforms of hypoxia inducible factor (Pelster and Egg, 2018), a master transcription factor that increases with hypoxia in fish (Borowiec et al., 2018; Chen et al., 2012; Geng et al., 2014; Köblitz et al., 2015; Kopp et al., 2011; Levesque et al., 2019; Mohindra et al., 2013; O’Brien et al., 2020; Rissanen et al., 2006; Robertson et al., 2014; Sollid et al., 2006; Zhang et al., 2017). In mice, HIF-1α was found to play a role in fine-tuning the VAH time domain of the HVR during long-term hypoxia (Kline et al., 2002; Peng et al., 2006; Yuan et al., 2013). Therefore, it is possible that Hif-1α also plays a role in regulating the time domains during the HVR in fishes exposed to long-term hypoxia, although to date this idea has not been tested.

With this background, the current study was designed to monitor fV continuously in adult zebrafish during 96 h of exposure to moderate hypoxia (90 mmHg) to test the hypothesis that long-term hypoxia is associated with hypoxia inducible factor-dependent HVD. Thus, it was predicted that fV would decline in wild-type fish but not in fish experiencing hif-1α knockout. Further experiments were designed to assess the impact of prolonged hypoxia on ventilatory sensitivity to hypoxia by measuring fV during acute severe hypoxia after prior exposure to milder hypoxia. It was predicted that zebrafish previously exposed to hypoxia would exhibit a decreased response to acute severe hypoxia, consistent with continuing HVD or a manifestation of HD owing to Hif-1α-dependent compensatory responses occurring during the previous bout of hypoxia.

Section snippets

Animals

Adult wild-type (WT) zebrafish, Danio rerio (Hamilton 1822) were reared in house (stocks of adult fish were obtained initially from commercial supplier Big Al’s Aquatics, Ottawa). Additionally, three Hif-1α knockout lines were used. The hif1aa−/− and hif1aa−/−ab−/− mutants were generated and validated by Gerri et al. (2017) and kindly provided to us as embryos. A separate line of hif1ab−/− fish were generously provided by Joanna Yeh (Harvard Medical School, MA, USA). These lines were maintained

Responses of wild-type fish

Control fish maintained for 72 h under normoxic conditions exhibited stable fV at 120 ± 14 breaths min−1 (Fig. 1A). Acute imposition of hypoxia (40 mmHg) at 72 h caused a marked increase of fV of 47 ± 8 breaths min−1 (Fig. 1A, Table 1). Upon return to normoxia, resting fV was reestablished (Fig. 1A).

In fish exposed to sustained 90 mmHg hypoxia, fV peaked between 1–3 h depending on the particular fish (peak ΔfV = 47 ± 12 breaths min−1; Fig. 1B; Table 1). Ventilation frequency had returned to

A critique of the methods

A major goal of the current study was to assess the time domains of the HVR in zebrafish over a 3-day exposure to continuous hypoxia. Thus, it was essential that the technique being used to monitor ventilation could adequately detect potentially subtle changes in fV as well as differentiate between ventilatory adjustments associated with time spent in the experimental chamber versus the specific effects of acclimation to hypoxia. Typically, previous experiments that have monitored fV in

Conclusions

Similar to mammals, there are discrete time domains of the HVR in adult zebrafish, but the patterns of these ventilatory responses differ. The dominant time domain during prolonged hypoxia in zebrafish is HVD, an energetically favourable response, with no evidence of HD or VAH. The difference in ventilation patterns between mammals and zebrafish are not unexpected given the differences in energetic costs of breathing and the role of CO2 in regulating ventilation between water- and

Funding

This work was supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery grant (RGPIN 2017-05545) to Dr. Steve Perry.

Acknowledgements

We thank Dr. Velislava Tzaneva for her advice on experimental methodology. We also thank Christine Archer and the University of Ottawa aquatic care facility for their help and knowledge of animal husbandry.

References (70)

  • S.F. Perry et al.

    Mechanisms and consequences of carbon dioxide sensing in fish

    Respir. Physiol. Neurobiol.

    (2012)
  • C.S. Porteus et al.

    Time domains of the hypoxic cardio-respiratory response in bowfin (Amia calva)

    Respir. Physiol. Neurobiol.

    (2014)
  • C. Porteus et al.

    Respiratory responses to external ammonia in zebrafish (Danio rerio) Comp

    Biochem. Physiol. A, Mol. Integr. Physiol.

    (2021)
  • F.L. Powell et al.

    Time domains of the hypoxic ventilatory response

    Respir. Physiol.

    (1998)
  • E. Sandblom et al.

    Autonomic control of circulation in fish: a comparative view

    Auton. Neurosci.

    (2011)
  • S. Thomas et al.

    A study of the effects of hypoxia on acid-base status of rainbow trout blood using an extracorporeal blood circulation

    Respir. Physiol.

    (1982)
  • B. Vulesevic et al.

    Developmental plasticity of ventilatory control in zebrafish, Danio rerio

    Respir. Physiol. Neurobiol.

    (2006)
  • J. Altimiras et al.

    Non-invasive recording of heart rate and ventilation rate in rainbow trout during rest and swimming. Fish go wireless!

    J. Fish Biol.

    (2000)
  • K. Borch et al.

    Cardiac activity, ventilation rate and acid-base regulation in rainbow trout exposed to hypoxia and combined hypoxia and hypercapnia

    Fish Physiol. Biochem.

    (1993)
  • B.G. Borowiec et al.

    Distinct metabolic adjustments arise from acclimation to constant hypoxia and intermittent hypoxia in estuarine killifish (Fundulus heteroclitus)

    J. Exp. Biol.

    (2018)
  • M.L. Burleson et al.

    Afferent inputs associated with cardioventilatory control in fish

  • J. Chen et al.

    Specific cyprinid HIF isoforms contribute to cellular mitochondrial regulation

    Sci. Rep.

    (2020)
  • J.C. Davis et al.

    Water flow and gas exchange at the gills of rainbow trout, Salmo gairdneri

    J. Exp. Biol.

    (1971)
  • P. Dejours

    Water versus air as the respiratory medium

  • P. Dejours

    Principles of Comparative Respiratory Physiology

    (1981)
  • L.H. Florindo et al.

    The role of branchial and orobranchial O2 chemoreceptors in the control of aquatic surface respiration in the neotropical fish tambaqui (Colossoma macropomum): progressive responses to prolonged hypoxia

    J. Exp. Biol.

    (2006)
  • J. Forgue et al.

    Maintenance of oxygen consumption in resting Silurus glanis at different levels of ambient oxygenation

    J. Exp. Biol.

    (1989)
  • J. Fox et al.

    An R Companion to Applied Regression

    (2011)
  • C. Gerri et al.

    Hif-1α regulates macrophage-endothelial interactions during blood vessel development in zebrafish

    Nat. Commun.

    (2017)
  • M.L. Glass et al.

    Combined effects of environmental PO2 and temperature on ventilation and blood gases in the carp Cyprinus Carpio L

    J. Exp. Biol.

    (1990)
  • J.D. Hadfield

    MCMC methods for multi-response generalized linear mixed models: the MCMCglmm for R

    J. Stat. Softw.

    (2010)
  • W. Joyce et al.

    Hypoxia inducible factor-1α knockout does not impair acute thermal tolerance or heat hardening in zebrafish

    Biol. Lett.

    (2020)
  • A. Kerstens et al.

    The ventilation, extraction and uptake of oxygen in undisturbed flounders, Platichthys flesus: responses to hypoxia acclimation

    J. Exp. Biol.

    (1979)
  • R. Kinkead et al.

    The role of circulating catecholamines in the ventilatory and hypertensive responses to hypoxia in the Atlantic cod (Gadus morhua)

    Physiol. Zool.

    (1991)
  • D.D. Kline et al.

    Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α

    Proc. Natl. Acad. Sci. U. S. A.

    (2002)
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