An Integrated Evaluation Framework for Adult Heart Regeneration

Figures

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  FIGURE 1. Evaluation of cardiac function and fibrosis. (A) The schematic diagram of assessment methods of cardiac systolic function by echocardiography, including the preparation for the detection (A(i)), the detection of cardiac systolic function (A(ii)), animal recovery (A(iii)) and data analysis (A(iv)). (B) A representative B‐mode echocardiographic image of the cardiac short‐axis view during systolic function assessment. (C, D) Representative M‐mode echocardiographic images and analysis of the same mouse under initial anaesthesia (C) and near‐awakening conditions (D). (E) The schematic diagram of assessment methods of cardiac fibrosis area by Masson's trichrome staining, including hearts harvesting and embedding (E(i)), paraffin‐embedded tissue section (E(ii)), Masson's trichrome staining (E(iii)) and quantification of cardiac fibrosis area (E(iv)). (F) A representative image illustrating the ligation site and section plane of cardiac tissue collected 56 days after ischemia/reperfusion (I/R) injury. (G) Representative Masson's trichrome staining images of paraffin‐embedded heart sections at different layers, collected 56 days after I/R injury.

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  FIGURE 2. Cardiomyocyte proliferation detection and quantitative counting. (A) The schematic diagram of cycling cardiomyocytes by immunofluorescence staining, including hearts harvesting and embedding (A(i)), paraffin‐embedded tissue section (A(ii)), immunofluorescence staining (A(iii)) and quantification of cycling cardiomyocytes (A(iv)). (B) The schematic diagram of the cell cycle and proliferation markers. (C–E) Representative immunofluorescence staining images of Ki67+ (B), EdU+ (C), pH3+ (D) and Aurora B+ (E) cardiomyocytes. (F) Schematic of the MADM lineage tracing principle for assessing cardiomyocyte proliferation. (G) Representative fluorescence images of single‐labelled (red or green; arrowheads), double‐labelled (yellow), and unlabeled (without fluorescence) cardiomyocytes in MADM mice. (H) The schematic diagram of enzymatic dissociation of cardiomyocytes and total cardiomyocyte counting, including hearts harvesting and digesting (H(i)), dilution and slide preparation of cardiomyocytes (H(ii)) and images acquisition and cardiomyocytes counting (H(iii)). (I) Complete dissociation of cardiomyocytes yields a pellet of digested cardiomyocyte. (J) Representative light field images of…

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  FIGURE 3. The integrated framework for assessing heart regeneration.