Hypoxia/Reoxygenation Cardiac Injury and Regeneration in Zebrafish Adult Heart

<div><p>Aims</p><p>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.</p><p>Methods and Results</p><p>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).</p><p>Conclusions</p><p>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.</p></div

Oxidative stress detection after H/R <i>in vivo.</i>

<p>DHE staining (a-d) and N-Tyr immunofluorescence (e-f) of hearts under control conditions (C) and exposed to H/R. (a) Representative confocal microscopy images of DHE staining in C and 2 h after H/R. (b) Merge of DHE and Hoechst nuclear staining. Calibration bar = 20 µm. White arrow-heads indicate DHE⁺ nuclei. (c) 3D representation of DHE fluorescence intensity distribution in the analyzed area: the <i>z-axis</i> shows the fluorescence intensity in cardiac nuclei, the <i>y-axis</i> and <i>x-axis</i> show the spatial distribution of nuclei on a plane. (d) Graph shows Mean Fluorescence Intensity (MFI) in C and 2 h to 14 h after H/R (n = 4 at each time point; ** <i>p</i><0.01 <i>vs.</i> C). Time course analysis revealed a peak of oxidative stress at 2 h in zebrafish adult heart sections, detected by DHE staining. (e) Representative confocal microscopy images of N-Tyr immunofluorescence, where green fluorescence indicates anti-N-Tyr and Hoechst nuclei staining: control (C, left panel) and 2 h after H/R (right panel). Calibration bar = 10µm. (f) Graph shows Mean Fluorescence Intensity (MFI) in C and 2 h to 14 h after H/R (n = 4 at each time point; ** <i>p</i><0.01 <i>vs.</i> C). H/R induced protein nitrosylation with a peak effect at the 2 h time point.</p

Necrotic myocyte cell death induced by H/R <i>in vivo.</i>

<p>Necrotic myocyte cell death was assessed under baseline conditions, and 18 h and 30d after H/R in the Tg(cmlc2:nucDsRed) zebrafish line. At 18 h after H/R it was found a marked increase in necrotic myocardial cell number, which was back to control value at the 30d time point. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R showing colocalization of DAPI, DsRED and AO stainings. Arrows indicate cardiomyocyte AO⁺ nuclei. (b) AO⁺ cardiomyocytes nuclei in control (C) animals, and 18 h and 30d after H/R (n = 3 at each time point; ** <i>p</i><0.01 <i>vs.</i> C).</p

Apoptotic myocyte cell death induced by H/R <i>in vivo.</i>

<p>Apoptotic myocyte cell death was assessed under baseline conditions, and 18 h and 30d after H/R in the Tg(cmlc2:nucDsRed) zebrafish line. At 18 h after H/R it was found a marked increase in apoptotic myocardial cell number, which was back to control value at the 30d time point. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R, showing colocalization of DAPI, DsRED and TUNEL stainings. Arrows indicate cardiomyocyte TUNEL⁺ nuclei, whereas arrow-head indicates non-cardiomyocyte TUNEL⁺ nuclei. (b) TUNEL⁺ cardiomyocytes nuclei in control (C) animals, and 18 h and 30d after H/R (n = 3 at each time point; * <i>p</i><0.05 <i>vs.</i> C).</p

Detection of HIF-1α-dependent genes expression in whole hearts after H/R <i>in vivo.</i>

<p>Graphs show selected HIF-1α-dependent genes expression in whole hearts in control (C) and at different time points (3 h, 6 h, and 9 h) after H/R. (a) <i>Hmox1</i> mRNA expression exhibited a progressive increase and, at the 9 h time point, <i>hmox1</i> was ∼8-fold higher than in C. (b) <i>Vegfaa</i> mRNA increased and achieved its peak 6 h after H/R. (c) <i>Epo</i> mRNA exhibited a peak increase at 3 h which was ∼1.7-fold higher than in C but failed to achieve statistical significance. (n = 6; * <i>p</i><0.05 and *** <i>p</i><0.001 <i>vs.</i> C).</p

Inflammatory response induced by H/R <i>in vivo.</i>

<p>Representative confocal microscopy images (a-h) showing neutrophils (yellow fluorescence or green fluorescence) and macrophages (red fluorescence) infiltration in double transgenic line Tg(MPO:EGFP)×Tg(LysC:DsRed) in control (C) and at different time points (4 h, 6 h, and 14 h) after H/R. Neutrophils are either yellow (LysC⁺/MPO⁺) or green (predominantly, MPO⁺) cells (arrows in the 6 h image); red macrophages are LysC⁺ cells (arrow in the 4 h image). Hoechst stains cell nuclei; (a-d) calibration bar = 100 µm, (e-h) calibration bar = 20 µm.The peak inflammatory response occurred at the 6 h time point after H/R This experiment was performed three times with similar results.</p

Myocardial cells positive for PCNA induced by H/R <i>in vivo.</i>

<p>Cardiomyocytes proliferation was assessed under baseline conditions and 18 h to 30d after H/R in Tg(cmlc2:nucDsRed) zebrafish line. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R showing colocalization of DAPI, DsRed and PCNA stainings. Arrows indicate cardiomyocyte PCNA⁺ nuclei. (b) Following H/R, there was a progressive increase in PCNA⁺ cardiomyocytes nuclei; the peak increase was achieved at the 3d time point, and at 30d the number of PCNA⁺ myocardial cells was back to control value (n = 3 at each time point; ** <i>p</i><0.01 and *** <i>p</i><0.001 <i>vs.</i> C).</p

Myocardial cells positive for pHH3 induced by H/R <i>in vivo.</i>

<p>Cardiomyocytes proliferation was assessed under baseline conditions and 18 h to 30d after H/R in Tg(cmlc2:nucDsRed) zebrafish line. (a) Representative image of a zebrafish heart ventricular section 3d after H/R showing colocalization of DAPI, DsRED and pHH3 stainings. Arrows indicate cardiomyocyte pHH3⁺ nuclei. (b) The increase in pHH3⁺ cardiomyocytes was apparent 18 h after H/R, achieved its peak at the 7d time point and was back to baseline at the 30d time point (n = 3 at each time point; ** <i>p</i><0.01 and *** <i>p</i><0.001 <i>vs.</i> C).</p