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. 2018 Mar 13;10(3):860-874.
doi: 10.1016/j.stemcr.2018.01.042. Epub 2018 Mar 1.

Neonatal Apex Resection Triggers Cardiomyocyte Proliferation, Neovascularization and Functional Recovery Despite Local Fibrosis

Vasco Sampaio-Pinto  1 , Sílvia C Rodrigues  2 , Tiago L Laundos  1 , Elsa D Silva  2 , Francisco Vasques-Nóvoa  3 , Ana C Silva  4 , Rui J Cerqueira  5 , Tatiana P Resende  2 , Nicola Pianca  6 , Adelino Leite-Moreira  5 , Gabriele D'Uva  6 , Sólveig Thorsteinsdóttir  7 , Perpétua Pinto-do-Ó  1 , Diana S Nascimento  8
Affiliations

Neonatal Apex Resection Triggers Cardiomyocyte Proliferation, Neovascularization and Functional Recovery Despite Local Fibrosis

Vasco Sampaio-Pinto et al. Stem Cell Reports. .

Abstract

So far, opposing outcomes have been reported following neonatal apex resection in mice, questioning the validity of this injury model to investigate regenerative mechanisms. We performed a systematic evaluation, up to 180 days after surgery, of the pathophysiological events activated upon apex resection. In response to cardiac injury, we observed increased cardiomyocyte proliferation in remote and apex regions, neovascularization, and local fibrosis. In adulthood, resected hearts remain consistently shorter and display permanent fibrotic tissue deposition in the center of the resection plane, indicating limited apex regrowth. However, thickening of the left ventricle wall, explained by an upsurge in cardiomyocyte proliferation during the initial response to injury, compensated cardiomyocyte loss and supported normal systolic function. Thus, apex resection triggers both regenerative and reparative mechanisms, endorsing this injury model for studies aimed at promoting cardiomyocyte proliferation and/or downplaying fibrosis.

Keywords: cardiac fibroblasts; cardiac injury response; cardiac regeneration; cardiomyocyte proliferation; extracellular matrix; fibrosis; neonatal apex resection; neovascularization; stereology.

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Figures

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Graphical abstract
Figure 1
Figure 1
Hearts Do Not Fully Regenerate the Resected Apex (A) Representative MT-stained sections of apex-resected and sham controls following surgery. Sections exhibiting myocardial disruption and/or cardiac fibrosis are highlighted by a dashed line. Scale bars, 2 mm. High-magnification images of the injury site (A1, 0 day; A2, 21 days; A3, 60 days; and A4, 180 days) show collagen (blue staining) from 21 days onward. Scale bars, 250 μm. (B and C) Ventricular surface area (from left to right, n = 8, 10, 8, 14, 3, 7, 4, 4) (B) and heart length/ body weight (BW) (from left to right, n = 11, 11, 8, 10, 4, 7, 4, 4) (C) of resected and sham-operated hearts were determined in MT-stained paraffin sections at 0, 7, 14, 21, 60, and 180 days post-surgery. (D and E) Injury extension (from left to right, n = 8, 12, 7, 4) (D) and ventricular and scar volume (from left to right, n = 19, 14, 7, 4) (E) were calculated on MT-stained paraffin sections. All values are presented as means ± SD. See also Figure S1 for representative MT sections at 7 and 14 days after surgery, data on heart weight, heart to BW ratio, and percentage of fibrosis in the apex.
Figure 2
Figure 2
Injured Hearts Display Morphological Alterations but Exhibit Normal Systolic Function (A) Representative echocardiographic parasternal long-axis view 180 days post-surgery. (B–G) End-diastolic left ventricle anterior wall thickness (LVAW), left ventricle posterior wall thickness (LVPW), left ventricle internal diameter (LVID), heart length, eccentricity index, and heart volume. (H–J) Ejection fraction (EF), stroke volume and cardiac output determined by Simpson's method. (K and L) Ratio between left ventricular early (E) to late (A) filling velocities and myocardial performance index (also known as Tei index) (from left to right, n = 7, 9, 22, 24, 18, 15, 18, 14). (M) Representative ECG signal traces at 180 days post-surgery (n = 10, 6; 180 days sham, 180 days injury). See also Table S1 for detailed quantification of ECG parameters. All values are presented as means ± SD.
Figure 3
Figure 3
Apex Resection Triggers Local ECM Remodeling and Fibroblast Activation (A) Representative confocal images of CD45/TN-C/FN, α-SMA/TN-C/FN, and s-α-Actinin/TN-C/FN immunolabelling at the injury site 48 hr (n = 4) and 7 days post-surgery (n = 4). Arrowhead, CD45+ hematopoietic cells; arrows, α-SMA+ myofibroblasts; dashed segment, resection line. Scale bars, 30 μm. See also Figure S2 for detailed analysis of cellular and extracellular matrix (ECM) remodeling following injury. (B) Cardiac fibroblasts were isolated and prospectively identified by CD140a, CD90, and SCA-1 expression throughout ontogeny (see Figure S4), 7 days post-resection (n = 3) and sham surgery (n = 6). CD140a+CD90+SCA-1− (purple/SP1), CD140a+CD90−SCA-1− (blue/SP2), and CD140a+CD90+SCA-1+ (gray/SP3) fibroblasts were FACS-sorted (see Figure S5 for sorting strategy). (C) Transcriptomic profiling of SP1 to SP3 subpopulations isolated from injured (n = 5) and sham hearts (n = 4) was performed by real-time qPCR. Expression levels were normalized against Gapdh. All values are presented as means ± SEM. See also Figures S4 and S5 for detailed phenotypic characterization of cardiac fibroblasts throughout ontogeny and sorting strategy, respectively.
Figure 4
Figure 4
Activation of CM Proliferation and Not Binucleation Generates a Heart with Increased CM Number upon Apex Resection (A and B) Quantification of PH3 expression by CMs in the area between papillary muscles and the apex (A) (n = 10, 10; 7 days sham, 7 days injury) and of isolated CMs from the same region (B) (n = 12, 11; 7 days sham, 7 days injury). (C) Representative confocal image and respective orthogonal views of a CM expressing PH3 and clearly displaying sarcomere disassembly. Scale bar, 20 μm. See also Movies S1, S2, and S3. (D) Quantification of Aurora B expression in CMs in regions close to the apex and remote myocardium (n = 3, 3; 7 days sham, 7 days injury). (E) Representative image of Aurora B expression in proliferating CMs at 7 days post-surgery (n = 3). Scale bar, 20 μm. (F) Assessment of CM nucleation in the apex region at 7 days post-surgery and representative images (n = 5, 4; 7 days sham, 7 days injury). (G) Ratio between round-shaped (immature) and rod-shaped (mature) CMs in mononucleated and multinucleated subpopulations of CMs isolated from the apex (n = 6, 5; 7 days sham, 7 days injury). (H) Three EdU pulses (days 1, 6, and 8) were given to trace proliferating CMs. Hearts were harvested 14 days post-surgery. (I) Representative confocal images of PCM-1+EdU+ CM 14 days post-surgery. Scale bars, 40 μm. (J) Quantification of EdU incorporation in PCM-1+ CMs in the apical and remote myocardium (n = 4, 7; 14 days sham, 14 days injury). (K) CM nucleus density at 14 days post-surgery (n = 4, 7; 14 days sham, 14 days injury). (L) Representative confocal image of COLL IV expression in the myocardium, which allowed the delineation of CM cell boundaries and nucleation evaluation (n = 4, 7; 14 days sham, 14 days injury). Scale bar, 30 μm. (M) Stereological estimate of the total number of CMs in the myocardium (n = 4, 7; 14 days sham, 14 days injury). All values are presented as means ± SD.
Figure 5
Figure 5
Resected Hearts Display Increased Coronary Vasculature but Not Hypertrophy or Edema (A and B) Capillary density at 60 days post-surgery (i.e., number of CD31+ endothelial cells per unit area) (n = 6, 7; 60 days sham, 60 days injury). Scale bars, 10 μm. (C and D) CM cross-sectional area at 60 and 180 days post-surgery (n = 6, 6, 4, 4; 60 days sham, 60 days injury, 180 days sham, 180 days injury). Scale bars, 25 μm. (E and F) Area of isolated CMs at 60 and 180 days post-surgery (n = 3, 5, 4, 4; 60 days sham, 60 days injury, 180 days sham, 180 days injury). Scale bar, 100 μm. (G) Volume of isolated CMs at 60 days post-surgery (n = 4, 6 sham, injury). (H) Representative images of 3D segmentation of CMs. Scale bars, 50 μm. (I) Myocardial edema was assessed by myocardial water content quantification (i.e., percentage of myocardial weight that is lost after desiccation) 180 days post-surgery (n = 10, 6; 180 days sham, 180 days injury). All values are presented as means ± SD.
Figure 6
Figure 6
Proposed Model of the Biological Response Elicited by Neonatal Apex Resection Apex resection promotes local infiltration of inflammatory cells in the first 48 hr, which leads to the deposition of a transient FN and TN-C-rich ECM. At 7 days post-injury, rates of CM proliferation are increased throughout the left ventricular myocardium and cardiac fibroblasts are activated at the injury site. These cellular dynamics result in a thickening of left ventricle walls, de novo vessel formation and deposition of a permanent fibrotic scar at the midpoint of the injured area. Long-term evaluation showed preserved systolic function, shortened long-axis and thicker left ventricle, without hypertrophy and edema.
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