Physiological Responses to Swimming-Induced Exercise in the Adult Zebrafish Regenerating Heart

Figures

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  Figure 1. Evaluation of cardiomyocyte proliferation and cardiac function by 2D-echochardiography in response to swimming-induced exercise. (A) Schematic representation of the experimental design. Fish were induced to swim for a total of 20 days and injected with EdU (green arrows). (B–F) Representative section stained by EdU (green), Mef2c (red) and nuclei staining with Hoechst (blue). (B) Image of a whole ventricle section (dotted lines) labeled with EdU. Scale bar represents 100 μm. Higher magnification views of the framed area in (B): (C) merged image from (D–F). White arrowheads indicate EdU+-MEF2c+ cardiomyocyte nuclei. Yellow arrowheads indicate EdU+-MEF2c− non-cardiomyocyte nuclei, as shown by the absence of MEF2c staining in (E) (open yellow arrowheads). Scale bar represents 10 μm. (G) There is a significant increase in the percentage of proliferative cardiomyocytes after exercise (P = 0.026). (H) Percentage of non-cardiomyocyte proliferative nuclei. Exercised (E, n = 6) and Control (C, n = 6) groups. (I) Representative SDS-PAGE of p38 MAPK as a cell differentiation/growth marker. (J) Quantification by densitometry of p38 MAPK activity as the ratio between phosphorylated p38 MAPK…

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  Figure 2. Effects of swimming-induced exercise prior to ventricular cryoinjury on heart regeneration. (A) Schematic representation of the experimental design. CI, cryoinjury; IA, injured area; BrdU injection (green arrow). (B) Evaluation of cardiac function after a cardiac injury in previously exercised (E) and non-exercised (C) fish. The relative change of ejection fraction shows a significant decrease at 7 days post injury (dpi; E, P = 0.0023; C, P = 0.0005) in comparison with basal conditions (non-injured ventricles) that is subsequently recovered after 28 dpi, with previously exercised fish (P = 0.0835) showing a better recovery (i.e., EF values at 28 dpi closer to basal values) than non-exercised fish (P = 0.0526). (E, n = 7; C, n = 7; *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey's post-hoc test). (C,D) AFOG staining of representative ventricle sections from C and E fish at 28 dpi. Scale bar represents 100 μm. (E,F) Higher magnification views of serial sections corresponding to the framed areas in (C,D). Immunofluorescence with BrdU (5-bromo-2′-deoxyuridine) combined with MEF2c. White arrowheads indicate BrdU+-MEF2c+ nuclei (cardiomyocytes). Yellow arrowheads…

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  Figure 3. Effects of swimming-induced exercise after ventricular cryoinjury on heart regeneration. (A) Schematic representation of the experimental design. CI, cryoinjury; IA, injured area; EdU injection (green arrow). (B–E) After 13 days of swimming, lesioned hearts were examined for proliferation at 15 dpi. (B,D) AFOG staining. Boxed area shows the injured area (IA). Scale bars represent 100 μm. (C,E) Higher magnification views of the framed area in (B,D). EdU labeling (green), MEF2 (red) and nuclei staining with Hoechst (blue). White arrowheads indicate EdU+-MEF2+ nuclei (cardiomyocytes). Yellow arrowheads indicate EdU+-MEF2− nuclei (non-cardiomyocytes). Scale bar represents 10 μm. (F–G) Percentage of proliferative cardiomyocytes is increased both in the IA and distant to IA (P = 0.029 and P = 0.029, respectively; *P < 0.05, Mann Whitney test) (F), whereas no differences were found for non-cardiomyocyte nuclei (G). (H,I) Fibrotic area differences at 7 and 15 dpi after AFOG staining. (H) Total lesioned area. (I) Fibrin and collagen deposition quantification from the same images analyzed in I. 7 dpi: E, n = 5; C, n = 5 and 15 dpi: E, n = 4; C, n = 4. Bars represent the mean ± SEM. dpi,…

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  Figure 4. Transcriptomic analysis of adult zebrafish regenerating ventricles in response to swimming-induced exercise after ventricular cryoinjury. (A) Schematic representation of the experimental design. CI, cryoinjury; RNA-seq, RNA-sequencing. (B) Heat map for the 512 significant differential expressed genes (Padj < 0.1) from exercised (E) and control or non-exercised (C) zebrafish at two time points analyzed, 7 and 14 dpi. After hierarchical clustering, four clusters were obtained (see legend). (C) Expression trend of the genes included in each cluster. Gene counts (log2 scale) were plotted to show differential time expression between exercised (blue lines) and non-exercised (red lines) at 7 and 14 dpi.

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  Figure 5. Functional analysis of differentially expressed genes in adult zebrafish regenerating ventricles in response to swimming-induced exercise. (A) Overview chart of the most significant GO terms (Biological Process) from all the significant DEGs (Padj < 0.1). **Indicates significant association of the genes represented in the GO terms (P < 0.05). (B) Selection of genes (human gene symbol) that were associated with GO terms in (A). Large nodes show the GO term as shown in (A). Small nodes represent selected genes associated with the GO terms (edge color) that correspond to the four clusters shown in Figure 4 (see legend).