Targeting chronic cardiac remodeling with cardiac progenitor cells in a murine model of ischemia/reperfusion injury

Figures

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  Fig 1. Study protocol. A) Timeline diagram of procedures and (B) flowchart of mouse experiment. Mice without significant cardiac damage (LVESV< 31), outliers or unsuccessful CPC injections (BLI signal after 2 days <20.000U) were excluded from primary analysis. For additional analysis, the CPC group was divided based on injury extent with the median LVEDV prior to cell treatment as cut-off point. ‘CPC small’ refers to animals with a LVEDV <median and ‘CPC large’ to animals with a LVEDV >median.

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  Fig 2. Echo-guided intramyocardial injection resulted in successful delivery of CPC. A) Representative B-mode image of intra-myocardial injection (29 Gy needle) at 4 weeks after the onset of I/R injury. The needle tip (marked by white arrowhead) is located in the anterior wall of the left ventricle. B) BLI images 2 days after intramyocardial injection with CPC demonstrated that 18/23 injections were successful (marked with *). C-D) CPC retained in the tissue up to 28 days after injection, as visualized by immunofluorescent staining for human lamin A/C (green), troponin I (red) and nuclei (blue). CPC were predominantly located along the needle track in the anterior and septal wall, at the level of the papillary muscles. (E) Squares mark H&E slides at the level of traced CPC. The left sided H&E slide corresponds to the CPC tracing slide in ‘C and D’.

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  Fig 3. Functional measurements by echocardiography of day 28 (pre-treatment). Upon I/R injury the LVEDV (A) and LVESV (B) are increased, and the LVEF (C) is decreased with no differences between CPC treated or vehicle treated mice. CPC group: n = 19, Vehicle group; n = 16. * p< 0.001.

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  Fig 4. Functional outcome measurements by echocardiography at 1 month follow-up demonstrated preservation of the end-diastolic volume. A) LVEDV in the CPC group differed from the control group at experimental day 56 (post-treatment). No significant difference between the groups was observed for (B) LVESV and (C) LVEF. Black lines indicate vehicle treated mice (n = 16) and grey lines indicate mice treated with CPC (n = 13). * p< 0.05.

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  Fig 5. Histological analysis of matrix composition in the injured myocardium. Representative images of cryosections stained with picrosirius red used for quantification of the collagen density of the infarcted area (A). The left and middle panels show a brightfield and polarized light image of the CPC group, repectively. The right image represents the vehicle treated group. The collagen composition was assessed by immunofluorescent imaging of (B) collagen type I (red) and (C) collagen type III (green). (D) Immunofluorescent staining showed an increase of CD31+ (red) and a-SMA+ (green) vessels in the infarcted area compared to the remote area. No difference was observed between CPC or vehicle treated mice. Nuclei were stained with Hoechst (blue) and the myocardial structure is shown in grey. In B-D the upper panel represents the CPC group and the lower panel the vehicle group. (E) Table with quantification of the stainings.

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  Fig 6. Myocardial deformation. (A) Schematic overview of 6 myocardial segments and deformation vectors; Basal-anterior (BA), mid-anterior (MA), apical-anterior (AA), apical-posterior (AP), mid-posterior (MP), basal-posterior (BP). Red line indicates infarct region. (B) Graphic representation of radial and longitudinal deformation. (C) Representative echocardiographic image of myocardial deformation (radial SR) after I/R injury, with concomitant vector lines, (D) for individual tracking points, and (E) combined with electrocardiogram and short axis view.