TLR2 mediates cell cycle re-entry and survival of neonatal cardiomyocytes in response to injury

Figures

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  Fig. 1. Pressure overload maintains the overexpression of pro-regenerative genes that are normally transiently activated after MI. A Venn diagram showing similarities in overexpressed genes at 1 dps P1 MI with those at 14 dps P1 nTAC. B Gene Ontology (GO) analysis of overlapping genes at 1 dps P1 MI compared to 14 dps P1 nTAC. C Heatmap showing gene expression of Ccl4, S100a8, and C1qa at 1.5 dps and 3 dps P1 MI and sham hearts, obtained from database [7]. D Heatmap showing gene expression of Ccl4, S100a8, and C1qa in 14 dps P1 nTAC and sham hearts [5]

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  Fig. 2. Pressure overload increases the number of CCL4, C1QA, and S100A8-expressing cells in the heart. A-F Immunostaining of heart sections at P14 in non-injured (n.i.) control and 14 dps P0 nTAC for CD45, PDGFRα, DAPI, and S100A8 (A), C1QA (C), CCL4 (E), and quantification of S100A8+ cells (B), C1QA+ cells (D), and CCL4+ cells (F) per area. Scale bar = 20 μm, (*p < 0.05; **p < 0.01; unpaired t-test)

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  Fig. 3. Pool3 enhances cell cycle activity and reduces the apoptosis rate of nCM and EC in vitro. A Schematic image of the experiment showing co-isolation and co-culture of nCM and EC and treatment with the proteins. B-D Immunofluorescence images and quantification of Ki67+ nCM (C, red arrowheads in B) and EC (D, white arrowheads in B) in control (Ctrl) and Pool3-treated nCM. E-G Immunofluorescence images and quantification of cCasp3+ nCM (F) and EC (G). Scale bar = 100 μm, (*p < 0.05; **p < 0.01; ns: not significant; one-way ANOVA with Šídák’s multiple comparisons test)

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  Fig. 4. Pool3 enhances nCM cell cycle activity and survival in vitro. A Schematic image of the experiment showing isolation of primary mouse nCM from P0-P1 hearts and in vitro treatment with CCL4, S100A8, C1QA, or Pool3. B-D Immunofluorescence images of Ki67 and PHH3 staining in nCM in Ctrl and Pool3-treated nCM (B) and quantification of Ki67+ (C) and PHH3+ nCM (D). E Quantification of Aurora B+ nCM. F-G Anillin+ mid-body shown by arrowhead and enlarged inside the insert, and its quantification (G). H-J Immunofluorescence images of cCasp3+ and TUNEL+ nCM (H) and quantification of cCasp3+ (I) and TUNEL+ (J) nCM. Scale bar = 50 μm, (*p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant; one-way ANOVA with Šídák’s multiple comparisons test)

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  Fig. 5. TLR2 activation as a central signaling hub mediating pro-survival and cell cycle re-entry in nCM in vitro and in vivo. A Schematic images of the experiment. B Volcano plot of regulated genes in nCM upon treatment with Pool3. n = 5 per condition. C-D Selected GO analysis terms, their corresponding genes, and p-value of upregulated (C) and downregulated (D) genes upon treatment of nCM with Pool3. E Venn diagram showing the overlap of upregulated genes at 14 dps P1 nTAC compared to Pool3-treated nCM. F Selected GO terms, their p-value, and gene names related to the overlapping genes in 14 dps P1 nTAC and Pool3-treated nCM. G-H STRING analysis showing protein-protein interaction and k-means clustering of overlapping genes in 14 dps P1 nTAC and Pool3-treated nCM with gene names in each cluster (H). I-J Western blot and quantification of BCL2 in the LV of P14 n.i. and 14 dps P0 nTAC hearts. (*p < 0.05, unpaired t-test)

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  Fig. 6. Pool3 does not affect cell cycle activity and apoptosis rate of TLR2 KO nCM in vitro. A Schematic image showing the experiment. B-D Immunostaining images, single channel, and quantification of Ki67 (B-C), PHH3 (D-E), and Aurora B (F-G) of TLR2 KO nCM in Ctrl and Pool3-treated nCM. E-F Immunostaining images, single channel and quantification of cCasp3+ (H-I), and TUNEL+ (J-K) TLR2 KO nCM. Scale bar = 50 μm (ns: not significant; unpaired t-test)

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  Fig. 7. Blocking of TLR2 abolishes Pool3-induced nCM cell cycle re-entry in vitro. A Schematic images of the experiment showing the presence of TLR2 in wild-type nCM and inhibition of TLR2 using a specific inhibitor (C29). B-G Immunostaining images and quantification of Ki67+ (B-C), PHH3+ (D-E), and Aurora B+ (F-G) nCM. Scale bar = 50 μm, (*p < 0.05; **p < 0.01; ns: not significant, one-way ANOVA with Šídák’s multiple comparisons test)

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  Fig. 8. Blocking of TLR2 abolishes the pro-survival effect of Pool3. A-F Immunostaining images and quantification of cCasp3+ and TUNEL+ nCM in Ctrl and Pool3-treated nCM in the presence or absence of TLR2 inhibitor (C29). G Quantification of the binucleation rate of nCM in Pool3 and Ctrl-treated nCM in the presence or absence of TLR2 inhibitor (C29). Scale bar = 50 μm, (*p < 0.05; **p < 0.01; ****p < 0.0001; ns: not significant, one-way ANOVA with Šídák’s multiple comparisons test)

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  Fig. 9. Activation of TLR2 by its agonist (zymosan) enhances the survival and cell cycle re-entry of nCM in vitro. A Schematic image of the experiment and an image of unstained nCM treated with zymosan conjugated with Alexa Fluor 488 (Z-488). B Immunofluorescence images of nCM stained for cTnT and TLR2 and Z-488 showing the interaction of zymosan with TLR2 on nCM. Insert showing the enlarged image of the zymozan interaction with nCM via TLR2. C-L Immunostaining images of Ctrl and Z-488-treated nCM and quantification of Ki67+ (C-D), PHH3+ (E-F), Aurora B+ (G-H), cCasp3+ (I-J), and TUNEL+ (K-L) nCM. Scale bar in B = 20 μm and C-K = 50 μm (*p < 0.05; **p < 0.01, unpaired t-test)

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  Fig. 10. Zymosan reduces the nCM apoptosis rate under hypoxia conditions in vitro. A Schematic representation of the experiment culturing and timeline of nCM under hypoxia and treatment with Z-488. B-E Immunostaining images and quantification of cCasp3+ nCM (B-C) and TUNEL+ nCM (D-E) in Ctrl and Z-488-treated nCM. Scale bar = 50 μm (*p < 0.05; **p < 0.01, unpaired t-test)

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  Fig. 11. TLR2 KO mice are unable to adapt to pressure overload. A Schematic image of the experimental timeline using TLR2 KO mice. B Kaplan-Meier survival curve of TLR2 KO sham and P0 nTAC. C-D Echocardiographic images showing long (C) and short (D) axis views of the heart in sham and nTAC TLR2 KO mice at 7 dps. E-I Quantification of ejection fraction (EF) (E), fractional shortening (FS) (F), LV wall thickness (G), LV end diastolic area (LVEDA) (H), and LV end systolic area (LVESA) (I) of sham and nTAC TLR2 KO mice at 7 dps. J Macroscopic images of the hearts in TLR2 KO sham and P0 nTAC at 7 dps. K Heart weight/body weight (HW/BW) in sham and P0 nTAC TLR2 KO mice at 7 dps. Scale bar in C-D and J = 1 mm (*p < 0.05; **p < 0.01, ***p < 0.001, unpaired t-test). Blue dots indicate tissues collected postmortem, and black dots are those sacrificed