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Fig 2. Long-term monitoring reveals persisting fibrotic scar in adult frog hearts after cardiac amputation.
Top: Schematic timing of heart collection following endoscopic cardiac biopsy in adult frogs from 1 dpa up to 11 mpa. (A-D) Histology of adult heart after picrosirius (PSR) staining to label cytoplasm (i.e. cardiomyocytes) in blue and fibrous matrix (i.e. pericardium, epicardium and endocardium as well as fibrotic scar tissue) in red, for a control non-operated heart (A, CTRL), compared to heart sections at one day (B, 1 dpa), one month (C, 1 mpa) and six months (D, 6 mpa) post amputation. Black arrows indicate amputation sites; faint blue staining formed a clot around the ventricle at 1 dpa; note the presence of a low to intense red-stained scar at the site of amputation for 1 mpa and 6 mpa respectively. (EE’-LL’) Magnifications of sections stained with PSR and adjacent sections immuno-labelled for tropomyosin (CH1, red), fibronectin (fn, green) and counterstained with DAPI (nuclei, blue), for control (E-E’), and operated hearts at 1 dpa (F-F’), 7 dpa (G-G’), 1 mpa (H-H’), 2 mpa (I-I’), 3 mpa (J-J’), 6 mpa (K-K’), and 11 mpa (L-L’). PRS labelling allowed the observation of scars on each amputated section, the red labelling displaying an increase of intensity with time (E to L): a low and diffuse red staining was detectable at 1 dpa and 7 dpa around the site of amputation (*) and intensifies from 1 mpa to 11 mpa. Likewise, fibronectin immunolabelling shares a similar pattern as PSR with an increasing accumulation at the site of amputation from 1 mpa to 11 mpa (E’ to L’). White arrows: pe, pericardium; ep, epicardium. Animals: CTRL, n = 2; 1dpa, n = 1; 7dpa, n = 2; 1mpa, n = 3; 2mpa, n = 3; 3mpa, n = 2; 6mpa, n = 2, 11mpa, n = 2. Scale bars, 1 mm (A–D, E-L and E’-L’). |
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Fig 5. Long lasting cardiac hypertrophy occurs in amputated frog heart.
Cardiomyocytes were observed at the site of amputation (A, D, G, J, M, P) and in a remote zone of the amputated ventricle (B, E, H, K, N, Q), using immuno-detection of natriuretic peptide A as an indicator of cardiac hypertrophy. Sections were labelled for tropomyosin (CH1, red), natriuretic peptide A (NPPA, green) and DAPI-counterstained nuclei (blue) at different times-points after amputation (7 dpa and 1, 3, 6 & 11 mpa) and compared with a control non-amputated heart (CTRL). In the vicinity of the amputation site, a weak NPPA signal was detected in CTRL (A), slightly increased at 7 dpa (D), whereas a clear increase of NPPA labelling in the myocardium was evident from 1 mpa to 11 mpa (G, J, M, P). In the remote zone, the NPPA labelling seemed appeared to slightly increase over time compared to the CTRL (compare B, E, H, K, N, Q). As a labelling control, all samples displayed an intense NPPA signal in atrium (C, F, I, L, O, R), since nppa is highly expressed in this tissue. Animals, CTRL, n = 1; 7dpa, n = 1; 1mpa, n = 1; 3mpa, n = 1; 6mpa, n = 1, 11mpa, n = 1. Scale bars, 200 μm (A–R). |
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Fig 6. Sarcomeric organisation of cardiomyocytes is deteriorated near the amputation site.
(A-L) Immuno-labelled sections for tropomyosin (CH1, red), fibronectin (fn, green) and DAPI-counterstained nuclei (blue), for a control non-amputated heart (CTRL) compared to 1 & 7 dpa, and 1, 6, and 11 mpa). Sarcomere organisation is observed at the amputation site (A, C, E, G, I, K, and their respective magnification) and in a remote zone of the amputated ventricle (B, D, F, H, J, L, and their respective magnification). The tropomyosin signal revealed a thin and well-organised striated structure of the cardiomyocytes for the CTRL heart. In contrast, at the site of amputation, the sarcomere organisation was completely disorganised at 1 dpa, lost at 7 dpa, partially recovered but not completely at 6 mpa and 11 mpa respectively (compare left magnifications). No evident change was seen between CTRL and AMP hearts in the remote zone (compare right magnifications). Note the general increase of the fibronectin staining around the cardiomyocytes for 1 mpa, 6 mpa and 11 mpa both at the site of amputation and in the remote zone. An asterix (*) marks the amputation site for time-points where fibrotic scar is not obvious. Animals: CTRL, n = 2; 1dpa, n = 1; 7dpa, n = 2; 1mpa, n = 3; 6mpa, n = 2, 11mpa, n = 2. Scale bars, 200 μm (A–L), 20 μm (magnification). |
