Malat1 deficiency prevents neonatal heart regeneration by inducing cardiomyocyte binucleation

Figures

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  Figure 1. Malat1 deficiency prevents neonatal heart regeneration. (A) Expression level of Malat1 (counts per million mapped reads, CPM) in regenerative (P1) versus mature nonregenerative cardiomyocytes (CMs) (P56). n = 4 for both groups. Available RNA-seq data were used for analysis (66). (B) Scheme of MI experiment in neonatal mice. (C and D) Sirius red staining of heart sections from Malat1+/+ and Malat1–/– mice 7 days after MI. (C) Representative histology images of Sirius red staining. Panels show different cross sections from apex to base. Arrowheads indicate persistent scarring. (D) Quantification of scarring after MI. n = 9 for Malat1+/+ and n = 8 for Malat1–/–. (E and F) Sirius red staining of heart sections from Malat1+/+ and Malat1–/– mice 21 days after MI. (E) Representative histology images of Sirius red staining. Arrowheads indicate persistent scarring. (F) Quantification of scarring. n = 9 for Malat1+/+ and n = 8 for Malat1–/–. (G and H) Echocardiographic analysis 21 days after MI. (G) Representative images of hearts in parasternal long-axis. (H) Quantification of the left ventricular ejection fraction. n = 7 for Malat1+/+ and n = 8 for Malat1–/–. (I and J) Apoptosis analysis…

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  Figure 2. Malat1 deficiency impairs regenerative response and inhibits cardiomyocyte proliferation. (A) Schematic of MI experiment in neonatal mice. (B and C) CD45+ cell infiltration in infarct zone 7 days after MI. (B) Representative immunofluorescence images showing CD45+ cell infiltration in the infarct zone. Arrowheads indicate CD45+ cells. (C) Quantification of infiltrated CD45+ cells. n = 6 for Malat1+/+ and n = 8 for Malat1–/–. (D and E) Vascular density and endothelial cell proliferation analysis 7 days after MI. (D) Representative immunofluorescence images of CD31+PH3+ cells from border (top) and remote (bottom) zones. Arrowheads indicate PH3+ endothelial cells. (E) Top: Quantification of CD31+ cell density (left) in the border zone. n = 5 for Malat1+/+ and n = 8 for Malat1–/–. Quantification of CD31+PH3+ cells (right) in border zone. n = 7 for Malat1+/+ and n = 11 for Malat1–/–. Bottom: Quantification of CD31+ cells density (left) in the remote zone. n = 7 for Malat1+/+ and n = 7 for Malat1–/–. Quantification of CD31+PH3+ cells (right) in the remote zone. n = 8 for Malat1+/+ and n = 11 for Malat1–/–. (F and G) Cardiomyocyte mitosis 7 days after MI. (F) Representative…

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  Figure 3. Malat1–/– mice show increased number of binucleated cardiomyocytes on P3. (A) Macroscopic analysis of hearts from Malat1+/+ and Malat1–/– mice on P3. (B) Heart weight (HW) to body weight (BW) ratio, body weight, and heart weight comparisons. n = 4 for Malat1+/+ and n = 7 for Malat1–/–. (C and D) Vascular density analysis of Malat1+/+ and Malat1–/– hearts on P3. (C) Representative immunofluorescence images of CD31+ cells. (D) Quantification of CD31+ cell density. n = 5 for Malat1+/+ and n = 5 for Malat1–/–. (E and F) Analysis of cardiomyocyte (CM) binucleation on P1, P3, and P7. (E) Representative immunofluorescence images of mono- and binucleated cardiomyocytes on P3. (F) Quantification of binucleated cardiomyocytes on P1, P3, and P7. n = 4 for Malat1+/+ and n = 7 for Malat1–/– (P1), n = 5 for Malat1+/+ and n = 4 for Malat1–/– (P3), n = 7 for Malat1+/+ and n = 3 for Malat1–/– (P7). Scale bars: 20 μm (C) and 50 μm (E). Data are shown as mean ± SEM. P values were calculated by unpaired, 2-tailed Student’s t test between each group for each time point.

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  Figure 4. Cardiomyocyte-specific deletion of Malat1 abolishes cardiac regeneration and induces cardiomyocyte binucleation. (A) Representative images of RNAscope in situ hybridization. Arrowheads indicate Malat1 transcripts in neonatal cardiomyocytes (α-Actinin+). (B) RT-qPCR–based detection of Malat1 levels in control and Malat1ΔCM cardiomyocytes (CMs) on P3. (C) Representative immunofluorescence images of isolated cardiomyocytes (cTnT+ cells) from control and Malat1ΔCM mice on P3. Arrowheads indicate binucleated cardiomyocytes. (D) Quantification of binucleated cardiomyocytes on P3. n = 3 for control and n = 5 for Malat1ΔCM. (E) Schematic of MI experiment in neonatal mice. (F) Representative histology images of Sirius red staining 21 days after MI. Arrowheads indicate persistent scarring. (G) Quantification of scarring. n = 5 for control and n = 7 for Malat1ΔCM. (H) Representative images of echocardiography analysis of the hearts in parasternal long axis. (I) Quantification of the left ventricular ejection fraction. n = 5 for control and n = 6 for Malat1ΔCM. Scale bars: 5 μm (A), 50 μm (C), and 2 mm (H). Data are shown as mean ± SEM. P values were calculated by unpaired, 2-tailed…

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  Figure 5. Cardiomyocyte-specific deletion of Malat1 reduced cardiomyocyte proliferation 21 days after MI. (A and B) CD45+ cell infiltration in infarct zone 21 days after MI. (A) Representative immunofluorescence images showing CD45+ cell infiltration in the infarct zone. Arrowheads indicate CD45+ cells. (B) Quantification of infiltrated CD45+ cells. n = 5 for Control and n = 7 for Malat1ΔCM. (C and D) Cardiomyocyte mitosis 21 days after MI. (C) Representative immunofluorescence images showing PH3+cTnT+ cells (mitotic cardiomyocytes) in border (top) and remote (bottom) zones. Arrowheads indicate PH3+cTnT+ cells. (D) Top: Quantification of PH3+cTnT+ cells in border zones. Bottom: Quantification of PCM1+PH3+ cells in remote zones. n = 5 for Control and n = 7 for Malat1ΔCM. (E and F) Cardiomyocyte cytokinesis 7 days after MI. (E) Representative immunofluorescence images showing Aurora B+cTnT+ cells (dividing cardiomyocytes) in the border zone. (F) Quantification of Aurora B+cTnT+ cells in the border zone. n = 5 for Control and n = 7 for Malat1ΔCM. Scale bars: 20 μm. Data are shown as mean ± SEM. P values were calculated by unpaired, 2-tailed Student’s t test (B and D) or Mann-Whitney U test (F).

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  Figure 6. LNA-mediated silencing of Malat1 induces binucleation in mouse HL-1 cells and regulates cardiac maturation and mitosis-associated gene expression. (A) RT-qPCR–based confirmation of Malat1 silencing in mouse HL-1 cells. n = 3. (B–D) Proliferation and binucleation analysis after Malat1 silencing in mouse HL-1 cells. (B) Representative immunofluorescence images of binucleated (upper figures) or proliferating cells (lower figures, Aurora B+ cells). Scale bars: 20 μm. (C) Quantification of Aurora B+ mouse HL-1 cells (proliferating cells) 48 hours after Malat1 silencing. n = 3. (D) Quantification of binucleated mouse HL-1 cells 72 hours after Malat1 silencing. n = 8. (E) Volcano plot of down- and upregulated genes upon Malat1 silencing. P < 0.05, log2(fold change) < –0.585 or > 0.585, FDR ≤ 0.5. (F) GO enrichment analysis of differentially expressed genes. Numbers in brackets indicate the number of genes regulated in the GO terms. (G) Representation of gene expression analysis of selected genes by RNA sequencing (upper row) and confirmation by RT-qPCR analysis (bottom row). n = 3. Data are shown as mean ± SEM. P values were calculated by unpaired, 2-tailed Student’s t test with Welch’s…

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  Figure 7. Malat1 interacts with hnRNP U and hnRNP U deficiency induces binucleation in HL-1 cells. (A) Enrichment of Malat1 in anti–hnRNP U immunoprecipitations. n =3. (B and C) RT-qPCR– and Western blot–based confirmation of hnRNP U silencing in HL-1 cells. Representative image of Western blot is shown. Lanes were run on the same gel but were noncontiguous; the complete Western blot is shown in Supplemental Figure 3F. n = 3. (D and E) Analysis of HL-1 cell proliferation 48 hours after hnRNP U knockdown. (D) Representative immunofluorescence images of PH3+ (mitotic) cells. Arrowheads indicate mitotic HL-1 cells. (E) Quantification of PH3+ HL-1 cells. n = 3. (F and G) Cytokinesis of HL-1 cells 48 hours after hnRNP U knockdown. (F) Representative immunofluorescence images of Aurora B+ (proliferating) HL-1 cells. Arrowheads show proliferating HL-1 cells. (G) Quantification of Aurora B+ HL-1 cells n = 3. (H and I) Binucleation analysis of HL-1 cells 72 hours after hnRNP U knockdown. (H) Representative immunofluorescence images of binucleated HL-1 cells. Arrowheads indicate binucleated cells. (I) Quantification of binucleated HL-1 cells. n = 6. (J) Quantification of PH3+ HL-1 cells after Malat1…