Hypoxia/Reoxygenation Cardiac Injury and Regeneration in Zebrafish Adult Heart

Abstract

Aimsthe adult zebrafish heart regenerates spontaneously after injury and has been used to study the mechanisms of cardiac repair. However, no zebrafish model is available that mimics ischemic injury in mammalian heart. We developed and characterized zebrafish cardiac injury induced by hypoxia/reoxygenation (H/R) and the regeneration that followed it.Methods and Resultsadult zebrafish were kept either in hypoxic (H) or normoxic control (C) water for 15 min; thereafter fishes were returned to C water. Within 2–6 hours (h) after reoxygenation there was evidence of cardiac oxidative stress by dihydroethidium fluorescence and protein nitrosylation, as well as of inflammation. We used Tg(cmlc2:nucDsRed) transgenic zebrafish to identify myocardial cell nuclei. Cardiomyocyte apoptosis and necrosis were evidenced by TUNEL and Acridine Orange (AO) staining, respectively; 18 h after H/R, 9.9±2.6% of myocardial cell nuclei were TUNEL+ and 15.0±2.5% were AO+. At the 30-day (d) time point myocardial cell death was back to baseline (n = 3 at each time point). We evaluated cardiomyocyte proliferation by Phospho Histone H3 (pHH3) or Proliferating Cell Nuclear Antigen (PCNA) expression. Cardiomyocyte proliferation was apparent 18–24 h after H/R, it achieved its peak 3–7d later, and was back to baseline at 30d. 7d after H/R 17.4±2.3% of all cardiomyocytes were pHH3+ and 7.4±0.6% were PCNA+ (n = 3 at each time point). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic and Systolic Areas were used to compute Fractional Area Change (FAC). FAC decreased from 29.3±2.0% in normoxia to 16.4±1.8% at 18 h after H/R; one month later ventricular function was back to baseline (n = 12 at each time point).Conclusionszebrafish exposed to H/R exhibit evidence of cardiac oxidative stress and inflammation, myocardial cell death and proliferation. The initial decrease in ventricular function is followed by full recovery. This model more closely mimics reperfusion injury in mammals than other cardiac injury models.

Evidence for a role of heme oxygenase-1 in the control of cardiac function in zebrafish (Danio rerio) larvae exposed to hypoxia

“…Other CO targets may include ion channels such as voltage-gated Ca 2+ (T-type Ca 2+ channels; reviewed in Capel and Terrar, 2015) and K + channels (HCN and ERG K + channels) important for establishing the robust T-type Ca 2+ current coupled with a 'funny' current (I f ) and a rapid delayed rectifier K + current (I Kr ) that are important for the diastolic depolarization rate of the developing zebrafish heart (Nemtsas et al, 2010;Alday et al, 2014). Studies from mammals have shown that endogenous CO inhibits all isoforms of T-type Ca 2+ channels as well as ERG K + channels (responsible for I Kr ) under normoxic conditions (reviewed in Peers et al, 2015) but there is no information on the effect of endogenous CO on HCN channels (responsible for I f ). The effects of endogenous CO on the cardiac pacemaker currents has not been studied in teleost fish thus far; therefore, the following interpretation of the mechanisms of action is an extrapolation from studies on mammals.…”

“…HO-1 mRNA was detected in the red blood cells of wild-type zebrafish larvae as early as 34 h post-fertilization (Craven et al, 2005) and expression in whole larvae was shown to increase with exposure to the toxicant 3,4-dichloroaniline (Voelker et al, 2008). More recently, a significant increase in HO-1 mRNA was reported in the hearts of adult zebrafish exposed to 9 h of hypoxia (Parente et al, 2013). Although HO-1 is known to increase in zebrafish under toxic (e.g.…”

Regeneration across Metazoan Phylogeny: Lessons from Model Organisms

“…Many important signaling pathways, including FGF, RA, PDGF and Jak1/Stat3, regulate heart regeneration. genetic ablation , cryocauterization (Gonzalez-Rosa et al, 2011) and hypoxia/reoxygenation (Parente et al, 2013). In these models, zebrafish can fully regenerate the heart after 30, 130 and 30 days, respectively.…”

Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration

“…Unlike mammals, adult zebrafish completely regenerate their hearts following amputation, cryoinjury, hypoxia/reoxygenation injury, or genetic ablation of cardiomyocytes (3)(4)(5)(6)(7)(8)(9)(10)(11). Lineage tracing studies have demonstrated that new cardiomyocytes arise by proliferation of partially dedifferentiated cardiomyocytes spared from injury (12,13).…”