Research ArticleCardiology

Control of cytokinesis by β-adrenergic receptors indicates an approach for regulating cardiomyocyte endowment

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Science Translational Medicine  09 Oct 2019:
Vol. 11, Issue 513, eaaw6419
DOI: 10.1126/scitranslmed.aaw6419
  • Fig. 1 Cardiomyocytes in infants with ToF/PS fail to divide.

    (A) Immunostaining and (B and C) quantification of cardiomyocytes in heart tissue from patients with tetralogy of Fallot and pulmonary stenosis (ToF/PS). Multinucleated (≥2) cardiomyocytes are indicated by filled symbols and solid lines in (B). Each symbol in (B) and bar in (C) represents one human heart (ToF/PS: n = 12; no heart disease: n = 5). (D) Immunostaining and (E) quantification of ploidy of nuclei in mononucleated cardiomyocytes determined with microscopy (ToF/PS: n = 5). Quantification in (E) includes age-matched published results for no heart disease (n = 5) (2). (F to J) Myocardium analyzed from a 4-week-old human infant with ToF/PS labeled with 15N-thymidine (given orally). Myocardium was analyzed by multiple-isotope imaging mass spectrometry (MIMS) at 7 months. 31P staining in nuclei and 32S morphologic detail, including striated sarcomeres, in (F) mononucleated and (G) binucleated myocytes. Nuclei are dark in the 32S image. The 15N/14N ratio image reveals 15N-thymidine incorporation. The blue end of the scale is set to natural abundance (no label uptake), and the upper bound of the rainbow scale is set to 100% (F) or 50% (G) above natural abundance. The white arrows in (F) indicate the boundaries of a labeled mononucleated cardiomyocyte, and in (G) the nuclei of a binucleated cardiomyocyte. (H) Quantification of labeled binucleated and mononucleated cardiomyocytes [mononucleated cardiomyocytes analyzed: n = 282; 15N+ mononucleated cardiomyocytes: n = 25; binucleated cardiomyocytes analyzed: n = 104 (208 total nuclei); 15N+ binucleated cardiomyocytes: n = 20 (40 total nuclei)]. (I) Ploidy of 15N-thymidine–positive mononucleated cardiomyocytes analyzed by microscopy of Hoechst staining of adjacent sections. (J) Quantification of diploid and polyploid cardiomyocytes. Statistical significance was tested with Student’s t test (E). Scale bars, 20 μm (A and D) and 10 μm (F, G, and I).

  • Fig. 2 Ect2 regulates cardiomyocyte cytokinesis and binucleation.

    (A) Live cell imaging of neonatal rat cardiomyocytes (NRVM, P2-P3, n = 52 cardiomyocytes), corresponding to movie S1. Cleavage furrow (white arrows) ingression is between 300 and 335 min, regression at 355 min, and formation of a binucleated cardiomyocyte at 510 min. (B) Transcriptional profiling of single cycling (+) and not cycling (−) cardiomyocytes at embryonic day 14.5 (E14.5) and 5 days after birth (P5) for 61 Dbl homology family Rho-GEFs. Ect2 is significantly repressed in cycling P5 cardiomyocytes (P < 0.05). The color code is given below, and the frequency of genes with corresponding expression is indicated with a black line. (C) Immunostaining and quantification of RhoA-GTP at the cleavage furrow (E14.5: n = 3 cell isolations; P2: n = 4 cell isolations) in binucleating cardiomyocytes. (D to H) NRVM transduced with Adv-GFP-Ect2 or Adv-GFP. (D) Live cell imaging of GFP-ECT2 in cycling NRVM, corresponding to movie S4. (E) Images and quantification of binucleated cardiomyocytes (n = 3 cell isolations). (F) Quantification of cardiomyocytes in the S phase (n = 3 cell isolations). (G) Images and quantification of cardiomyocytes in the M phase (n = 3 cell isolations). (H) Analysis of ploidy of nuclei (GFP: n = 3 cell isolations; GFP-Ect2: n = 2 cell isolations). Statistical significance was tested with Student’s t test. Scale bars, 20 μm (C) and 50 μm (E and G).

  • Fig. 3 Ect2 gene inactivation lowers cardiomyocyte endowment and is lethal in mice.

    (A to H) Ect2flox gene inactivation with αMHC-Cre in mice. (A) Immunostaining and quantification of binucleated cardiomyocytes at P1 (Ect2F/Wt n = 6, Ect2F/F n = 6 hearts). (B) DNA content per nucleus (Ect2F/Wt n = 642 cardiomyocytes, Ect2F/F n = 647 cardiomyocytes). (C) Cardiomyocyte endowment, quantified by counting of fixation-digested hearts (Ect2F/Wt n = 12, Ect2F/F n = 5 hearts). (D) Immunostaining and quantification of hypertrophy (cardiomyocyte size). (E) Quantification of mono- and binucleated cardiomyocyte size. (D and E) (Ect2F/Wt n = 1138 cardiomyocytes, 1101 mono, 37 bi, from 6 hearts; Ect2F/F n = 1015 cardiomyocytes, 892 mono, 123 bi, from 6 hearts) and (F) heart weight (Ect2F/Wt n = 14, Ect2F/F n = 6 hearts). (G) Echocardiographic analysis of myocardial dysfunction at P0 (LV endocardium outlined in yellow, Ect2Wt/Wt n = 4, Ect2F/F n = 3 mice; movies S5 and S6). (H) Pup survival (fig. S9B and movie S7). (I) Immunostaining and quantification of cardiomyocyte binucleation after Ect2 rescue (n = 3 cell isolations). (J) Immunostaining and quantification of cell cycle entry in cardiomyocytes after Ect2flox gene inactivation with αMHC-MerCreMer, tamoxifen P0, P1, and P2, followed by 3 days of culture in the presence of BrdU (Ect2Wt/Wt n = 3, Ect2F/F n = 2 cell isolations). (K) Immunostaining and quantification of cell cycle progression to the M phase in vivo after αMHC-Cre inactivation of Ect2flox (P1, Ect2F/Wt n = 6, Ect2F/F n = 6 hearts). Statistical significance tested with Student’s t test (A to D, F, G, and I to K), one-way ANOVA with Bonferroni’s multiple comparisons (E), and Fisher’s exact test (H). Scale bars, 20 μm (A and I), 30 μm (J and K), and 100 μm (D).

  • Fig. 4 Hippo signaling regulates cardiomyocyte abscission and binucleation.

    (A) Luciferase assay in human embryonic kidney 293 cells analyzing Ect2 promoter activity after removal of the five TEAD1/2-binding sites. WT, wild-type Ect2 promoter; ∆1–5, All five putative TEAD-binding sites removed; ∆2kB, the continuous 2 kB DNA sequence containing all five TEAD-binding sites removed; vector, empty vector that did not contain Ect2 promoter (n = 3 cultures) (see also fig. S11). (B) Quantification of Ect2 expression after knockdown of TEAD1 and TEAD2 by siRNA (n = 4 cell isolations). (C) Immunostaining and quantification of the proportion of binucleated NRVMs after knockdown of TEAD1 and TEAD2 (P2, n = 3 cell isolations). (D) Immunostaining and quantification of binucleated cardiomyocytes generated in NRVM after increasing expression of TEAD1 (n = 3 cell isolations). (E) Quantification of Ect2 and (F) immunostaining and quantification of binucleated cardiomyocytes after adenoviral overexpression of YAP1-WT and a nonphosphorylatable version containing a S127A mutation (YAP1-S127A) in NRVMs (P2) (n = 4 cardiomyocyte isolations). Scale bars, 20 μm. Statistical significance was tested with one-way ANOVA with Bonferroni’s multiple comparisons (A to C, E, and F) and Student’s t test (D).

  • Fig. 5 β-AR signaling regulates cardiomyocyte abscission and binucleation.

    (A) Cardiac expression of YAP target genes Cyr61 and CTGF and of (B) Ect2 (n = 3 hearts per group) after inactivation of β1- and β2-AR genes (DKO) in mice (P4). (C) Immunostaining and quantification of multinucleated (P4: n = 4 hearts per group; P10: n = 6 hearts for WT, n = 3 hearts for DKO) and (D) total cardiomyocytes in DKO mice (quantified by counting fixation-digested hearts, P4: n = 7 hearts for WT, n = 5 hearts for DKO; P10: n = 6 hearts for WT, n = 3 hearts for DKO). (E) Immunostaining and quantification of M phase cardiomyocytes (n = 4 hearts per group) in vivo at P4. (F) Immunostaining and quantification of binucleated cardiomyocytes generated by knockdown of Ect2 in cultured neonatal cardiomyocytes from β-AR DKO mice (P2, n = 3 cell isolations). Sc, scrambled siRNA (see fig. S12) for Ect2 siRNA validations. Scale bars, 20 μm. Statistical significance was tested with two-way ANOVA with Bonferroni’s multiple comparisons (C and D) and Student’s t test (A, B, E, and F).

  • Fig. 6 Pharmacologic alterations of β-AR signaling regulate cardiomyocyte abscission and endowment.

    (A) Ect2 mRNA expression in cultured NRVMs treated with Fsk (n = 5 cardiomyocyte isolations). (B) Immunostaining and quantification of multinucleated cardiomyocytes after Fsk administration in vivo (1 μg/g body, one intraperitoneal injection per day in newborn mice, n = 6 hearts per group). (C) Immunostaining and quantification of multinucleated cardiomyocytes (PBS: n = 4, prop: n = 3 hearts for P4; n = 4 hearts per group for P8) and (D) total number of cardiomyocytes (quantified by counting fixation-digested hearts; PBS: n = 7, prop: n = 6 hearts for P4; n = 4 hearts per group for P8) after propranolol administration (prop, 10 μg/g body, two intraperitoneal injections per day in newborn mice). (E) Immunostaining and quantification of cell cycle entry (n = 5 hearts per group for P8) and (F) M phase activity (n = 4 hearts per group for P8). (G) Immunostaining and quantification of multinucleated cardiomyocytes and (H) total number of cardiomyocytes (quantified by counting fixation-digested hearts) after alprenolol administration (alp, 10 μg/g body, two intraperitoneal injections per day in newborn mice, n = 6 hearts per group). Scale bars, 20 μm. Statistical significance was tested with Student’s t test (A, E, and F) and two-way ANOVA with Bonferroni’s multiple comparisons (B to D, G, and H).

  • Fig. 7 Propranolol-induced increase in the cardiomyocyte endowment in the neonatal period improves adult cardiac function and remodeling after MI.

    Mice received propranolol (prop, 10 μg/g body, two intraperitoneal injections per day, P1 to P12) or PBS. (A) Quantification of cardiomyocytes in adult hearts at baseline (fixation-digested hearts, n = 6 hearts per group for P42). (B) Ejection fraction of adult hearts at baseline (P60, n = 11 hearts for PBS, n = 6 hearts for prop). (C) Diagram of experimental design. MI was induced by permanent ligation of the left anterior descending coronary artery between 6 weeks (P44) and 2 months after birth (P60). MRI was performed in the acute phase [1 to 3 days post injury (dpi)] and recovery phase (10 to 12 dpi). (D) MRI of late gadolinium enhancement (LGE) in both groups in the acute and recovery phases. MI size is indicated in purple and quantified (n = 6 mice per group for the acute phase; PBS: n = 7, prop: n = 6 mice for the recovery phase). (E) MRI images and quantification of EF in the acute and recover phases in propranolol- or PBS-treated mice (PBS: n = 4, prop: n = 5 mice for the acute phase; PBS: n = 7, prop: n = 6 mice for the recovery phase). (F) MRI images and analysis of stretched myocardial wall (PBS: n = 5, prop: n = 4 mice for the recovery phase). (G to I) MRI images and analysis of systolic myocardial thickening in the acute phase (G and I; PBS: n = 4, prop: n = 5 mice) and the recovery phase (H and I; PBS: n = 7, prop: n = 6 mice). (J) Scar size quantified by AFOG staining (PBS: n = 5, prop: n = 4 hearts for the recovery phase) at 12 dpi. (K) Number of cardiomyocytes (determined by stereology, n = 5 hearts per group) and (L) heart weight–to–body weight ratio (PBS: n = 5, prop: n = 6 hearts). Statistical significance was tested with Student’s t test (A, B, F, and J to L) and one-way ANOVA with Bonferroni’s multiple comparisons (D, E, and I).

  • Fig. 8 β-Adrenergic signaling regulates cytokinesis in cardiomyocytes from patients with ToF/PS.

    (A) Expression of cell cycle and cytokinesis-related genes in cycling cardiomyocytes from patients with ToF/PS and non-ToF/PS fetuses. Each symbol represents one cycling cardiomyocyte (fetal: n = 66 cardiomyocytes from four hearts, ToF/PS: n = 14 cardiomyocytes from three hearts). (B) Ect2-positive cycling cardiomyocytes in hearts (fetal: n = 4 hearts, ToF/PS: n = 3 hearts). (C) Immunostaining and quantification of cytokinesis failure in cultured human fetal cardiomyocytes (Ctrl, control; Fsk, forskolin; prop, propranolol; and dobu, dobutamine: 10 μM), measured by formation of binucleated daughter cells (n = isolations from four hearts). (D) Analysis of Ect2-positive midbodies (n = isolations from two hearts). (E) Analysis of cytokinesis in cultured myocardium (n = cultures from three patients with ToF/PS) treated with Fsk, Dobu, or Dobu + prop. The interrupted red line indicates maximal cytokinesis failure induced by Fsk (positive control). (F) Proposed model connecting cytokinesis failure to endowment changes. Scale bars, 40 μm (C) and 20 μm (D and E). Statistical significance was tested with Student’s t test (B) and one-way ANOVA with Bonferroni’s multiple comparisons test (C to E).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/513/eaaw6419/DC1

    Methods

    Fig. S1. Approach for determining the ploidy of 15N-thymidine–labeled nuclei.

    Fig. S2. The two nuclei in binucleated cardiomyocytes have the same 15N-thymidine intensity, demonstrating that they share the same cell cycle history.

    Fig. S3. A single-cell transcriptional profiling strategy identifies molecular mechanisms of cardiomyocyte binucleation.

    Fig. S4. Strategy for isolation and characterization of cycling cardiac cells.

    Fig. S5. Strategy for identifying single mouse cardiomyocytes for single-cell transcriptional analysis.

    Fig. S6. The expression of Ect2 gene decreases in cycling neonatal mouse cardiomyocytes.

    Fig. S7. Adenoviral-mediated transduction efficiency of Ect2 in NRVMs is >90%.

    Fig. S8. Adenoviral-mediated transduction of GFP-Ect2 does not induce apoptosis in cultured NRVMs.

    Fig. S9. Survival analysis shows that Ect2 gene inactivation in development induces decreased pup viability.

    Fig. S10. Inactivation of Ect2 gene in development does not induce apoptosis.

    Fig. S11. The WT Ect2 promoter was modified to test the effect of the putative TEAD1/2-binding sites on the Ect2 promoter activity.

    Fig. S12. Knockdown of Ect2 using siRNA reduces Ect2 mRNA and protein and induces cytokinesis failure and binucleation in cardiomyocytes.

    Fig. S13. Approaches for quantification of BrdU+ cardiomyocytes in vivo.

    Fig. S14. Altering β-AR signaling does not affect the heart weight.

    Fig. S15. The optical dissector operates optimally at 3-μm distance between lookup and counting frames.

    Fig. S16. Validation of BrdU assay specificity in cultured neonatal cardiomyocytes.

    Fig. S17. Representative immunostained cardiomyocytes isolated from cultured human myocardium.

    Table S1. List of differentially expressed genes encoding 61 different Dbl homology family Rho-GEFs between E14.5 and P5 cycling and noncycling mouse cardiomyocytes.

    Table S2. Cell cycle genes for Fig. 8A.

    Table S3. Quantification of numeric data.

    Table S4. Antibody manufacturers and dilutions.

    Table S5. Image acquisition hardware and settings.

    Table S6. PCR primers and 5′-3′ oligonucleotides.

    Table S7. Animal strains used in this study.

    Table S8. Clinical information corresponding to human samples.

    Movie S1. Live cell imaging in NRVMs shows that cleavage furrow regression precedes formation of binucleation.

    Movie S2. Live cell imaging of a neonatal cardiomyocyte undergoing division.

    Movie S3. Live cell imaging of a neonatal cardiomyocyte undergoing cytokinesis failure.

    Movie S4. Live cell imaging shows appropriate and dynamic localization of GFP-Ect2 during the cell cycle.

    Movie S5. Heart function of newborn (P0) WT mice.

    Movie S6. Heart function of newborn (P0) mouse pup with Ect2 knockout.

    Movie S7. Ect2 inactivation induces heart failure and death of newborn mice.

    Movie S8. Heart function of the left anterior descending coronary artery (LAD) ligation–injured adult mouse.

    References (7075)

  • The PDF file includes:

    • Methods
    • Fig. S1. Approach for determining the ploidy of 15N-thymidine–labeled nuclei.
    • Fig. S2. The two nuclei in binucleated cardiomyocytes have the same 15N-thymidine intensity, demonstrating that they share the same cell cycle history.
    • Fig. S3. A single-cell transcriptional profiling strategy identifies molecular mechanisms of cardiomyocyte binucleation.
    • Fig. S4. Strategy for isolation and characterization of cycling cardiac cells.
    • Fig. S5. Strategy for identifying single mouse cardiomyocytes for single-cell transcriptional analysis.
    • Fig. S6. The expression of Ect2 gene decreases in cycling neonatal mouse cardiomyocytes.
    • Fig. S7. Adenoviral-mediated transduction efficiency of Ect2 in NRVMs is >90%.
    • Fig. S8. Adenoviral-mediated transduction of GFP-Ect2 does not induce apoptosis in cultured NRVMs.
    • Fig. S9. Survival analysis shows that Ect2 gene inactivation in development induces decreased pup viability.
    • Fig. S10. Inactivation of Ect2 gene in development does not induce apoptosis.
    • Fig. S11. The WT Ect2 promoter was modified to test the effect of the putative TEAD1/2-binding sites on the Ect2 promoter activity.
    • Fig. S12. Knockdown of Ect2 using siRNA reduces Ect2 mRNA and protein and induces cytokinesis failure and binucleation in cardiomyocytes.
    • Fig. S13. Approaches for quantification of BrdU+ cardiomyocytes in vivo.
    • Fig. S14. Altering β-AR signaling does not affect the heart weight.
    • Fig. S15. The optical dissector operates optimally at 3-μm distance between lookup and counting frames.
    • Fig. S16. Validation of BrdU assay specificity in cultured neonatal cardiomyocytes.
    • Fig. S17. Representative immunostained cardiomyocytes isolated from cultured human myocardium.
    • Legend for table S1
    • Table S2. Cell cycle genes for Fig. 8A.
    • Table S3. Quantification of numeric data.
    • Table S4. Antibody manufacturers and dilutions.
    • Table S5. Image acquisition hardware and settings.
    • Table S6. PCR primers and 5′-3′ oligonucleotides.
    • Table S7. Animal strains used in this study.
    • Table S8. Clinical information corresponding to human samples.
    • Legends for movies S1 to S8
    • References (7075)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of differentially expressed genes encoding 61 different Dbl homology family Rho-GEFs between E14.5 and P5 cycling and noncycling mouse cardiomyocytes.
    • Movie S1 (.mov format). Live cell imaging in NRVMs shows that cleavage furrow regression precedes formation of binucleation.
    • Movie S2 (.mov format). Live cell imaging of a neonatal cardiomyocyte undergoing division.
    • Movie S3 (.mov format). Live cell imaging of a neonatal cardiomyocyte undergoing cytokinesis failure.
    • Movie S4 (.mov format). Live cell imaging shows appropriate and dynamic localization of GFP-Ect2 during the cell cycle.
    • Movie S5 (.mov format). Heart function of newborn (P0) WT mice.
    • Movie S6 (.mov format). Heart function of newborn (P0) mouse pup with Ect2 knockout.
    • Movie S7 (.mov format). Ect2 inactivation induces heart failure and death of newborn mice.
    • Movie S8 (.mov format). Heart function of the left anterior descending coronary artery (LAD) ligation–injured adult mouse.

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