Hypoxia/reoxygenation cardiac injury and regeneration in zebrafish adult heart.
OBJECTIVE
the 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.


RESULTS
adult 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).


CONCLUSIONS
zebrafish 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.
Laboratorio di Biologia Vascolare e Medicina Rigenerativa, Centro Cardiologico Monzino, Istituto di Ricovero e Cura a Carattere Scientifico, Milan, Italy.
Valeria
V
Parente
Serena
S
Balasso
Giulio
G
Pompilio
Lorena
L
Verduci
Gualtiero I
GI
Colombo
Giuseppina
G
Milano
Uliano
U
Guerrini
Lidia
L
Squadroni
Franco
F
Cotelli
Ombretta
O
Pozzoli
Maurizio C
MC
Capogrossi
D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D006321:Heart / Q000503:physiopathology*; D006335:Heart Injuries / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D006352:Heart Ventricles / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D020127:Recovery of Function; D012038:Regeneration; D015027:Zebrafish
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010100:Oxygen