Research ArticleRegenerative Medicine

A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice

See allHide authors and affiliations

Science Translational Medicine  18 Mar 2015:
Vol. 7, Issue 279, pp. 279ra38
DOI: 10.1126/scitranslmed.3010841
  • Fig. 1. miR302-367 is expressed in the early heart and is important for cardiomyocyte proliferation.

    (A) Relative expression of miR302-367 cluster members during heart development as determined by qRT-PCR. (B) Hematoxylin and eosin (H&E)–stained and immunostained sections of control E14.5 hearts showing thinning of ventricular wall, hypoplastic ventricular septum, and reduced cardiomyocyte proliferation in Nkx2.5cre:miR302-367flox/flox mutants compared with miR302-367flox/flox mice. RV, right ventricle; LV, left ventricle; VS, ventricular septum; RA, right atrium. (C) Quantification of Ki67 (proliferation) from images in (B). (D) Gene expression changes associated with cardiomyocyte proliferation and differentiation in Nkx2.5cre:miR302-367flox/flox null mutants versus controls at E14.5. (C and D) Data are means ± SEM (n = 3). P values determined by Student’s t test.

  • Fig. 2. Overexpression of miR302-367 cluster in the developing heart results in increased cardiomyocyte proliferation and cardiomegaly.

    (A) At E18.5, Nkx2.5cre:R26R-miR302-367Tg/+ mutants have an enlarged heart with thickened ventricular myocardium and ventricular septal defects compared to Nkx2.5cre controls. (B) Cardiomegaly and increased cardiomyocyte proliferation in Nkx2.5cre:R26R-miR302-367Tg/+ mutant hearts at P20. (A and B) Immunostainings for PH3 and α-actinin and wheat germ agglutinin (WGA) show the number of mitotic cardiomyocytes. Scale bars, 100 μm. High-magnification reveals PH3+ cardiomyocytes. LA, left atrium. Data are means ± SEM (n = 3). P values determined by Student’s t test.

  • Fig. 3. miR302-367 overexpression leads to increased cell proliferation as well as altered differentiation and metabolism in cardiomyocytes.

    (A) Wheat germ agglutinin and α-actinin staining of hearts at P20. Cell surface area was quantified at E18.5 and P20. Quantitative analyses represent counting of five fields from three independent samples per group. (B) Heatmap and pathways profile of microarray analysis of Nkx2.5cre and Nkx2.5cre:R26R-miR302-367Tg/+ mutant hearts at P14. (C and D) Gene expression changes related to cell proliferation (C) and programmed cell death (D) at P14. (E and F) Gene expression changes related to differentiation (E) and fatty acid metabolism (F) at P14 and P23. Data are means ± SEM (n = 3 per group). *P < 0.05 versus Nkx2.5cre control animals (Student’s t test).

  • Fig. 4. miR302-367 promotes cardiomyocyte proliferation through regulation of Hippo pathway kinases.

    (A) Expression of Mob1b, Lats2, and Mst1 in Nkx2.5cre:R26R-miR302-367Tg/+ mutant hearts at E18.5 and Nkx2.5cre:R26R-miR302-367flox/flox null mutant hearts at E12.5 by qRT-PCR. (B) Luciferase reporter assays showing that miR302-367 can repress Mst1, Lats2, and Mob1b expression through their respective 3′UTRs. This repression can be reversed by mutations of the miR302-367 binding sites. (C) Confocal fluorescence microscopy of phospho-Yap and nuclear staining of Yap, with or without DAPI, in ventricular cardiomyocytes of Nkx2.5cre:R26R-miR302-367Tg/+ mouse hearts at E18.5. Cytoplasmic and nuclear ratio for total Yap protein was quantified using Fiji software. (A to C) *P < 0.05, **P < 0.01 versus Nkx2.5cre control hearts (Student’s t test). (D) Overexpression of miR302-367 in primary mouse neonatal cardiomyocytes. Cardiomyocyte proliferation was quantified using Ki67 immunostaining. **P < 0.01 versus Yap shRNA control. Scale bars, 100 μm. (E) Proposed model of miR302-367 promoting cardiomyocyte proliferation through regulation of Hippo pathway kinases. (A to D) Data are means ± SEM (n = 3 for A, C, and D; n = 5 for B).

  • Fig. 5. miR302-367 can promote cardiomyocyte proliferation in the adult heart.

    (A) Schematic of tamoxifen-inducible miR302-367 overexpression in the hearts of adult Myh6mercremer mice. (B to D) Confocal images with z-stacking and quantification showing the number of cells reentering the cell cycle [5-bromo-2′-deoxyuridine (BrdU)+] (B), undergoing mitosis (PH3+) (C), or undergoing cytokinesis [Aurora B (AuB)+] (D) 7 days after induction of miR302-367 expression in the adult heart. (E) Number of cardiomyocytes in Myh6mercremer and Myh6mercremer:R26R-miR302-367Tg/+animals. Inset: Fluorescence-activated cell sorting (FACS) plot shows that 98.6% of the isolated cells counted in the adult hearts are cTnT+ cardiomyocytes. (F) Number of nuclei in control and Myh6mercremer:R26R-miR302-367Tg/+ cardiomyocytes. Scale bars, 100 μm. mono, mononucleated; bi, binucleated; multi, multinucleated About 1 × 103 cardiomyocytes were counted per sample. (G) Cell sizes of the isolated cardiomyocytes. Data are means ± SEM (n = 3). P values determined with Student’s t test. n.s., no significant change.

  • Fig. 6. Prolonged miR302-367 overexpression in the adult heart reduces fibrotic scar size but compromises cardiac function after MI.

    (A) Study design of miR302-367 overexpression by tamoxifen intraperitoneal injection after MI by ligation of the left anterior descending (LAD) coronary artery. (B) Masson’s trichrome–stained heart sections from the site of ligation toward the apex of control and Myh6mercremer:R26R-miR302-367Tg/+ mice at 21 days after MI. Serial sections were cut at 500-μm intervals from the site of the ligature toward the apex. One representative Myh6mercremer and two Myh6mercremer:R26R-miR302-367Tg/+ hearts are shown (n = 6 per group). (C) Quantification of the fibrotic regions in heart sections in (B). (D) Immunostaining and quantification of PH3+/α-actinin+ cells in Myh6mercremer and Myh6mercremer:R26R-miR302-367Tg/+ hearts at 21 days after MI. (E) Cardiac function in mice subjected to LAD ligation, evaluated by echocardiography (n = 7 per group). EF, ejection fraction; FS, fractional shortening; EDV, end-diastolic volume; ESV, end-systolic volume. Data are means ± SEM (n = 6 to 7). (C and D) P values determined with Student’s t test. (E) *P < 0.05 versus sham, by one-way analysis of variance (ANOVA).

  • Fig. 7. Transient miR302 mimic therapy promotes cardiac regeneration and improves function of injured hearts.

    (A) Schematic of 7-day miR302 mimic treatment after MI (n = 3 sham; n = 8 control; n = 18 miR302). (B) Masson’s trichrome staining of heart sections 50 days after MI and 42 days after final treatment with control or miR302 mimic. Serial sections were cut at 500-μm intervals from the site of the ligature toward the apex. Two representative control and three miR302 mimic–treated hearts are shown. (C) Quantification of the fibrotic areas in heart sections. Data are means ± SEM. (D and E) Cardiac function of mice subjected to LAD ligation was evaluated by echocardiography. Data are means ± SEM. P values determined by one-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/279/279ra38/DC1

    Materials and Methods

    Fig. S1. Generation of mice with a conditional deletion of the miR302-367 cluster.

    Fig. S2. Generation of the mice with conditional overexpression of the miR302-367 cluster.

    Fig. S3. miR302-367 regulates cardiomyocyte proliferation through the Hippo pathway.

    Fig. S4. Gene expression profiles in adult hearts after inducible overexpression of miR302-367.

    Fig. S5. Hippo signaling activity and myocardial features in the adult heart after inducible overexpression of miR302-367.

    Fig. S6. Half-life of miR302-367 mimic treatment and the effects on cardiomyocyte proliferation, apoptosis, and vascular perfusion.

    Fig. S7. Expression of miR302 mimics in the lung and organ histology after systemic treatment with mimics.

    Table S1. miRNAs identified from HITS-CLIP.

    Table S2. Overlapping genes between HITS-CLIP and predicted targets of miR302.

    Table S3. miR302 targets identified from HITS-CLIP.

    Table S4. miRNA qRT-PCR analysis in the adult mouse heart.

    Table S5. Primers for genotyping, qRT-PCR, and luciferase reporter analyses.

    References (41, 42)

  • Supplementary Material for:

    A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice

    Ying Tian,* Ying Liu, Tao Wang, Ning Zhou, Jun Kong, Li Chen, Melinda Snitow, Michael Morley, Deqiang Li, Nataliya Petrenko, Su Zhou, Minmin Lu, Erhe Gao, Walter J. Koch, Kathleen M. Stewart, Edward E. Morrisey*

    *Corresponding author. E-mail: ying.tian{at}temple.edu (Y.T.); emorrise{at}mail.med.upenn.edu (E.E.M.)

    Published 18 March 2015, Sci. Transl. Med. 7, 279ra38 (2015)
    DOI: 10.1126/scitranslmed.3010841

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Generation of mice with a conditional deletion of the miR302-367 cluster.
    • Fig. S2. Generation of the mice with conditional overexpression of the miR302-367 cluster.
    • Fig. S3. miR302-367 regulates cardiomyocyte proliferation through the Hippo pathway.
    • Fig. S4. Gene expression profiles in adult hearts after inducible overexpression of miR302-367.
    • Fig. S5. Hippo signaling activity and myocardial features in the adult heart after inducible overexpression of miR302-367.
    • Fig. S6. Half-life of miR302-367 mimic treatment and the effects on cardiomyocyte proliferation, apoptosis, and vascular perfusion.
    • Fig. S7. Expression of miR302 mimics in the lung and organ histology after systemic treatment with mimics.
    • Table S1. miRNAs identified from HITS-CLIP.
    • Table S2. Overlapping genes between HITS-CLIP and predicted targets of miR302.
    • Table S3. miR302 targets identified from HITS-CLIP.
    • Table S4. miRNA qRT-PCR analysis in the adult mouse heart.
    • Table S5. Primers for genotyping, qRT-PCR, and luciferase reporter analyses.
    • References (41, 42)

    [Download PDF]

Navigate This Article