Clearance of p16‐positive cardiac cells improves age‐related cardiac remodeling in mice

Figures

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  FIGURE 1. Study design and treatment‐associated changes in structural indicators of hypertrophy in aged INK‐ATTAC mice treated with vehicle or AP. (a) Study design: INK‐ATTAC mice were treated twice‐weekly with vehicle or AP, from 12 months until 18 months of age. (b) Changes in left ventricular (LV) mass/end‐diastole dimension (LVDd) and absolute values at 12 and 18 months. (c) Changes in LV anterior wall thickness (LVAWT) and absolute values at 12 and 18 months. (d) Changes in LV mass and absolute values at 12 and 18 months. (e) Heart weight. (f) Body weight. (g) Heart weight normalized to body weight (For all analyses in b–g, Young mice: n = 20; Aged mice: Vehicle‐treated: n = 23; AP‐treated: n = 25). Statistical analysis for changes in echocardiogram parameters: unpaired t‐test; for the rest of the analyses: one‐way ANOVA followed by Tukey's test. Echocardiographic results are reported as absolute values and changes occurring from 12 to 18 months during treatment with AP (Aged‐AP) or Vehicle (Aged‐Vehicle). Each point represents results from one mouse; bars and horizontal lines are means and SD.

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  FIGURE 2. Echocardiographic assessment of cardiac structure and function in aged INK‐ATTAC mice treated with vehicle or AP. The echocardiographic data are reported as changes (Δ) from 12 to 18 months in each group (Aged‐Vehicle, Aged‐AP). (a) Left ventricular (LV) structural parameters: LV dimension at end systole (LVDs); LV dimension at end diastole (LVDd); LV mass/body weight (BW). (b) Systolic function parameters: fractional shortening % (FS%), ejection fraction % (EF%), cardiac output (CO). (c) LV diastolic function parameters: transmitral flow atrial filling (a), transmitral flow early filling (e). Statistics: Unpaired t‐test for parametric or Mann‐Whitney test for non‐parametric variables. Each point represents results from one mouse; bars and horizontal lines are means and SDs.

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  FIGURE 3. Hemodynamic parameters in Young, Aged‐Vehicle and Aged‐AP INK‐ATTAC. (a) Isovolumic relaxation time. (b) End‐diastolic left ventricular (LV) pressure. (c) Maximum rate of decrease in pressure during relaxation (Min dP/dt). (d) Mean arterial pressure. (e) Mean systolic pressure. (f) Maximum rate of increase in pressure (Max dP/dt). (g) Minimum pressure (Pmin). (h) Maximum pressure (Pmax). (i) Systolic ejection period. (j) End‐systolic pressure. (k) Developed pressure. (l) Diastolic filling period. (m) Mean diastolic pressure. (n) Contraction time. (o) Contraction index (Young mice: n = 9; Aged mice: Vehicle‐treated: n = 12; AP‐treated: n = 10). Statistical analysis for all panels: one‐way ANOVA followed by Tukey's test. Each point represents results from one mouse; bars and horizontal lines are means and SDs.

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  FIGURE 4. Fluorescence‐activated cell sorting (FACS) for non‐cardiomyocyte cardiac cells in Young, Aged‐Vehicle and Aged‐AP groups. a–c: (a) Young group (b) Aged‐Vehicle group (c) Aged‐AP group. First column: CD31− CD45− (fibroblast) population. Second column: CD31+ (endothelial cell) population. Third and fourth columns: CD45+ (immune cell) population. (d) Quantification of GFP‐positive (p16‐expressing senescent) cells as a percentage of the total population in each cell type marker for Aged‐Vehicle and Aged‐AP mouse samples. Statistical analysis was performed using an unpaired t‐test with a significance level of P < 0.05. SSC: Side scatter, indicating granularity of the cells (Aged mice: Vehicle‐treated: n = 10; AP‐treated: n = 9). (e) Validation of sorted cell population post FACS with qPCR. Fibroblast markers (Tcf21 and Ddr2), immune‐cell marker of (CD45), endothelial‐cell marker (CD31). Statistical analysis was performed using one‐way ANOVA followed by Tukey's test. Ddr2: discoidin domain receptor tyrosine kinase2; Tcf21: transcription factor 21. Each point represents results from one mouse; bars and horizontal lines are means and SD.

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  FIGURE 5. Double immunofluorescence for p16 and cardiac non‐cardiomyocyte cell type markers in left ventricle of Young, Aged‐Vehicle and Aged‐AP groups. (a) Young (b) Aged‐Vehicle (c) Aged‐AP results. For all groups, left column: double staining of p16 and Vimentin (marker of fibroblasts), middle column: double staining of p16 and aSMA (marker of myofibroblasts), right column: double staining of p16 and CD31 (marker of endothelial cells). Each column contains two panels, one (left) at lower magnification and another (to the right) showing magnified views of the area in the yellow box to the left. All panels show cell‐marker staining (green), staining for p16 (red) and 4,6‐diamino‐2‐phenylindole (DAPI, Blue), wheat germ agglutinin (WGA, purple).Yellow arrows point to double‐stained cells (p16 and cardiac cell type marker). The horizontal white lines in the left panels are 100 µm scale markers (Young mice: n = 5; Aged mice: Vehicle‐treated: n = 5; AP‐treated: n = 5). (d) Quantification of the co‐immunostaining data corresponding to the experiments shown above. Statistical analysis for all panels: one‐way ANOVA followed by Tukey's test. Each point represents results from one mouse; bars and…

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  FIGURE 6. Double immunofluorescence for p16 and cardiomyocyte marker proteins. (a) Heart‐tissue sections stained with senescent‐cell marker p16 (red), nuclear stain 4,6‐diamino‐2‐phenylindole (DAPI; blue), and cardiomyocyte marker, troponin I (green) (Young mice: n = 5; Aged mice: Vehicle‐treated: n = 5; AP‐treated: n = 5). (b) Data are expressed as the percentage of p16+ cardiomyocyte nuclei relative to the total number of cardiomyocyte nuclei detected per section.(c) Myosin heavy chain 7 (Myh7) gene‐expression. Statistical analysis for all panels: one‐way ANOVA followed by Tukey's test. Each point represents results from one mouse; bars and horizontal lines are means and SD.

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  FIGURE 7. Left‐ventricular fibrosis quantification, profibrotic marker gene expression and optical mapping. (a) Collagen content analyzed with Masson's Trichrome immunostaining (Young mice: n = 5; Vehicle‐treated mice: n = 11; AP‐treated mice: n = 9). (b) Hydroxyproline quantification. (c) Profibrotic marker gene expression. (d) Activation map. (e) Conduction velocity, APD 50, APD 70, APD 90 (Young mice: n = 9; Aged mice: Vehicle‐treated: n = 10; AP‐treated: n = 10). Statistical analysis for all panels: one‐way ANOVA followed by Tukey's test. Each point represents results from one mouse; bars and horizontal lines are means and SD.

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  FIGURE 8. In vitro model of cardiomyocyte exposure to senescent cardiac fibroblast secretome. (a) Study design (b) Senescence and SASP marker gene expression in control fibroblasts and those treated with 200 µM H2O2 (n = 8). (c) Hypertrophy marker gene expression in cardiomyocytes co‐cultured with control versus senescent fibroblasts. Statistical analysis for all panels: unpaired t‐test. Each point represents results from one mouse; bars and horizontal lines are means and SD.

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  FIGURE A1. Hypertrophy markers, ion channels and calcium handling proteins gene expression. Statistical analysis for all panels: one‐way ANOVA followed by Tukey's test, significance level P < 0.05. (Young mice: n = 6; Aged mice:Vehicle‐treated: n = 6; AP‐treated: n = 6). Each point represents results from one mouse; bars and horizontal lines are means and SD.