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Fig 1. Ventricular resection endoscopic biopsy and animal survival in adult Xenopus laevis.
(A-C) Key steps of the heart ventricular biopsy procedure using an endoscope. After an abdominal incision, the endoscope is inserted into the pleuroperitoneal cavity (A), using a xenon light source and light cable (A’) to search for the heart and observe the procedure internally. Biopsy forceps are used to break the falciform ligament, open the pericardial sac (B), then collect a calibrated piece of cardiac tissue from the apical region of the ventricle (see also S1 File). The surgery is ended (C) by performing a single suture to close the incision. (D-E) Comparison of a control non-operated heart (D) with an operated heart, displaying a large blood clot 1 day post-amputation (E), the size of biopsy tissue (E’) collected corresponds to approximately 4% of the ventricle. (F) Graphical representation of survival after the procedure, detailing total number of animals used for the full amputation procedure (n = 34), and animals ethically sacrificed (n = 2) or experimentally sacrificed at the end of the planned protocol (n = 32). Scale bars, 2 mm (D, E and E’).
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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 3. Cardiomyocytes show evidence of cell hypertrophy at the amputation site.
Cardiomyocytes were observed at the site of amputation (*; see A, C, E, G, I, K and their respective magnifications A’, C’, E’, G’, I’, K’) and in a remote zone of the amputated ventricle (see B, D, F, H, J, L and their respective magnifications B’, D’, F’, H’, J’, L’), using WGA labelling as an indicator of hypertrophy. (A-L) Sections were labelled with WGA (cell membranes, red) and DAPI (nuclei, blue) for different times after amputation (1 & 7 dpa and 1, 6 & 11 mpa) and compared with a control non-amputated heart (CTRL). On the right of each picture, a post treatment of the red/WGA images allows better visualisation of the signal intensity. Note that in the control heart the myocardium showed a comparable level of labelling in the apex or in the remote zone of the ventricle (A, B), with a stronger signal in the epicardium (A). An increase of WGA labelling in the myocardium was evident from 1 mpa to 11 mpa in the vicinity of the amputation site (compare the green colour for G, I, K with C, E), whereas no differences were seen in the remote zone with the CTRL (compare the green colour for D, F, H, J, L with B). (A’-L’) Magnification of a longitudinal view of WGA-labelled cardiomyocytes: control heart showed a thin and regular WGA labelling (A’ and B’) whereas for the amputated heart, an increase of the thickness and the irregularity of the WGA signal was observed from 1 dpa up to 11 mpa compared to the control (A’) or with the WGA signal in the remote zone (see B’, D’, F’, H’, J’, L’). 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 (A’–L’).
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Fig 4. Increased cardiomyocyte size persists eleven months post amputation.
(A) Using the WGA labelled heart pictures, the cell-membrane labelled boundaries of orthogonal view cardiomyocytes (left panel) was delineated in red (middle panel) and filled with blue colour (right panel) for non-amputated control hearts (CTRL) and different time-points post-amputation (7 dpa, 1, 3, 6 and 11 mpa). (B) The cross-sectional area of each cell was automatically calculated using ImageJ software. Quantification was performed on 2 or 3 independent heart sections, corresponding to minima of 300 cell areas counted for each group, and samples were compared to the CTRL. Animals: CTRL, n = 1; 7dpa, n = 1; 1mpa, n = 1; 3mpa, n = 1; 6mpa, n = 1, 11mpa, n = 1. An unpaired non-parametric t-test (Mann Whitney) was performed: ****, p<0.0001.
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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 7. Short-term transcriptional responses in adult frog heart after cardiac injury display evidence of a scarring process.
(A) Heart ventricles from different experimental conditions were collected for RT-qPCR analysis: control non-operated hearts (CTRL), SHAM-operated hearts where only the pericardium was opened (scissor 1, SHAM), and amputated hearts where a ventricle biopsy was performed following pericardium opening (scissor 1 & 2, AMP). (B) RNA extraction was performed on whole heart ventricles, collected at days 1, 3 and 7 post-amputation followed by RT qPCR to quantify gene expression. Gene markers of fibrosis (fn1, col1a1, ctgf), hypertrophy (odc, nppa, nppb), cellular structure (actn3, tnnt2), proliferation (pcna, ccnd1, tert), inflammation (cebpb, il1b, cxcl8), actl6a and akt1 were monitored. Aligned dot plot with median and interquartile range, n≥4 for each group with exact n displayed above the x-axis. Normalisation was performed using the geometric mean of two reference genes (smarcd1/smn2), fold-change is shown in log2 scale, respective to SHAM for each gene at each individual time-point. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.
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Fig 8. Endoscopic ventricle biopsy procedure in adult frogs induces a fibrous scar with absence of heart regeneration.
Endoscopic procedure in anesthetised frog allows “in situ” live visualisation of the operating field and collection ventricle tissue in the apical region using biopsy forceps. The resulting outcome for the frog heart is the induction of scar related gene expression during the first week post-amputation (SHORT TERM) and the establishment of a persistent fibrous scar, cardiac hypertrophy and sarcomere disorganisation at the amputation site, without the capacity for the cardiac tissue to regenerate, even after almost one year (LONG TERM)
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