Marshall L et al. (2017), Persistent fibrosis, hypertrophy and sarcomere ...

XB-ART-53154

PLoS One 2017 Jan 01;123:e0173418. doi: 10.1371/journal.pone.0173418.

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Persistent fibrosis, hypertrophy and sarcomere disorganisation after endoscopy-guided heart resection in adult Xenopus.

Marshall L , Vivien C , Girardot F , Péricard L , Demeneix BA , Coen L , Chai N .

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Models of cardiac repair are needed to understand mechanisms underlying failure to regenerate in human cardiac tissue. Such studies are currently dominated by the use of zebrafish and mice. Remarkably, it is between these two evolutionary separated species that the adult cardiac regenerative capacity is thought to be lost, but causes of this difference remain largely unknown. Amphibians, evolutionary positioned between these two models, are of particular interest to help fill this lack of knowledge. We thus developed an endoscopy-based resection method to explore the consequences of cardiac injury in adult Xenopus laevis. This method allowed in situ live heart observation, standardised tissue amputation size and reproducibility. During the first week following amputation, gene expression of cell proliferation markers remained unchanged, whereas those relating to sarcomere organisation decreased and markers of inflammation, fibrosis and hypertrophy increased. One-month post-amputation, fibrosis and hypertrophy were evident at the injury site, persisting through 11 months. Moreover, cardiomyocyte sarcomere organisation deteriorated early following amputation, and was not completely recovered as far as 11 months later. We conclude that the adult Xenopus heart is unable to regenerate, displaying cellular and molecular marks of scarring. Our work suggests that, contrary to urodeles and teleosts, with the exception of medaka, adult anurans share a cardiac injury outcome similar to adult mammals. This observation is at odds with current hypotheses that link loss of cardiac regenerative capacity with acquisition of homeothermy.

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Species referenced: Xenopus laevis
Genes referenced: actl6a akt1 ccnd1 col1a1 ctrl fn1 il1b jmjd6 mef2a nppa tpm1
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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’).
	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’).

	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.



	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)

References [+] :

Andersen, Persistent scarring and dilated cardiomyopathy suggest incomplete regeneration of the apex resected neonatal mouse myocardium--A 180 days follow up study. 2016, Pubmed