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.
Highlights
Recent studies have shown that the adult zebrafish heart, unlike the mammalian heart, exhibits the ability to fully regenerate within weeks after surgical removal of the ventricular apex [1], [2], cryoinjury [3,4,5] and genetic cardiomyocyte ablation [6]
Mechanical cardiac injury, cryoinjury, and genetic cardiomyocyte ablation lack some of the key elements that cause and are associated with heart damage in mammals following coronary occlusion [3,4,5], e.g. there is no oxygen deprivation, it is unknown whether free radicals are produced, the cardiac apex is removed and this prevents the development of extensive cell death in the injured area as it occurs in the severely ischemic heart
There is a need to develop a zebrafish model of cardiac injury that more closely mimics what occurs in mammals following coronary artery occlusion; such model would be expected to be more suitable to study the mechanisms of damage and regeneration in zebrafish than surgical amputation of the apex, cryoinjury, or genetic cardiomyocyte ablation
Summary
Recent studies have shown that the adult zebrafish heart, unlike the mammalian heart, exhibits the ability to fully regenerate within weeks after surgical removal of the ventricular apex [1], [2], cryoinjury [3,4,5] and genetic cardiomyocyte ablation [6]. About three months after amputation, the regeneration process is complete and, at a morphological level, previously damaged hearts are indistinguishable from uninjured hearts [1] This species peculiarity has made the zebrafish an attractive model to study the mechanisms for heart regeneration following injury. Mechanical cardiac injury, cryoinjury, and genetic cardiomyocyte ablation lack some of the key elements that cause and are associated with heart damage in mammals following coronary occlusion [3,4,5], e.g. there is no oxygen deprivation, it is unknown whether free radicals are produced, the cardiac apex is removed and this prevents the development of extensive cell death in the injured area as it occurs in the severely ischemic heart. We developed a hypoxia/reoxygenation (H/R) injury model that affects the zebrafish heart and exhibits characteristics of reperfusion injury in the mammalian heart; acute cardiac damage is followed by spontaneous regeneration and functional recovery
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