P573A novel model of hypoxia/reoxygenation injury in zebrafish adult heart

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Abstract

The adult zebrafish heart, unlike the mammalian heart, exhibits the capacity to regenerate after ventricular resection; for this reason it has been used to investigate the mechanisms of cardiac repair. We developed a novel system to induce hypoxia/reoxygenation (H/R) injury in the adult zebrafish heart, a model that more closely mimics ischemia/reperfusion injury in humans and other mammals than ventricular resection.

The aim of the present study was to establish the effect of H/R on myocardial cell death, cell proliferation and cardiac function.

Transgenic Tg (cmlc2:nucDsRed) adult zebrafish were kept either in 5% (hypoxic, H) or in 80% (normoxic control, C) O2 fish water for 15 minutes. Thereafter fishes were returned to C water and hearts were explanted for analysis at different time points.

At 18 hours post hypoxia we observed a significant increase of apoptotic myocardial cells in H/R vs C fishes (TUNEL assay; C=1.3 ± 0.01%, H/R=10 ± 0.045%; n=3, p < 0.05) and a higher number of necrotic myocardial cells in H/R than C fishes (acridine orange staining; C=3%, H/R=14%; n=2). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic Area (VDA) and Systolic Area (SA) were measured to compute Shortening Fraction (SF); fishes exhibited a marked decrease in SF at 18h after H/R (C=29.3 ± 1.9%, H/R=16.4 ± 1.7%; n= 12, p < 0.001), and one month later ventricular function was back to baseline. At 7 days post hypoxia we observed an increase of pHH3 + myocardial cells (C=2.29 ± 0.65%, H/R=17.35 ± 1.43%; n=3, p < 0,05) and PCNA + in H/R vs C fishes, a result indicative of cell cycling and suggestive of cell proliferation. One month after H/R myocardial pHH3 and PCNA expression was back to baseline.

In conclusion, the present study shows that zebrafish exposed to the H/R injury exhibit significant cardiac damage with a marked and transient decrease in ventricular contractility. Future studies will address whether myocardial regeneration occurs in zebrafish heart exposed to H/R and microRNAs role in cardiac response to injury.

* These authors contributed equally to this work.

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