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A calcineurin–Hoxb13 axis regulates growth mode of mammalian cardiomyocytes

Abstract

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1–Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

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Fig. 1: Identification of Hoxb13 as a Meis1-interacting transcription factor in postnatal cardiomyocytes.
Fig. 2: Loss of Meis1 and Hoxb13 induces cardiomyocyte proliferation and heart regeneration.
Fig. 3: Calcineurin regulates Hoxb13 expression in the postnatal hearts.
Fig. 4: Increased calcineurin activity suppresses postnatal cardiomyocyte proliferation.

Data availability

The data that support the findings of this study are available within the paper and its Supplementary Information. Source Data for Figs. 14 and Extended Data Figs. 16, 8, 9 are provided with the online version of the paper. ChIP–seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE141769. Data or other materials are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank S. Zhang for MRI studies at the Mouse MRI Core, Advanced Imaging Research Center, University of Texas Southwestern Medical Center; J. Shelton for assistance with histology; H. I. May for conducting mouse surgeries; J. Heinrich for helping with voluntary exercise; J. Xu, X. Liu and Y. Jung Kim from the Sequencing Core Facility in Children’s Research Institute in University of Texas Southwestern Medical Center for performing the Illumina sequencing. The UTSW mouse MRI facility is supported by an NIH shared instrumentation grant 1S10OD023552-01. H.A.S. is supported by grants from the NIH (1R01HL115275 and 5R01H2131778), National Aeronautics and Space Administration (NNX-15AE06G), American Heart Association (16EIA27740034), Cancer Prevention and Research Institute of Texas (RP160520), Hamon Center for Regenerative Science and Medicine, and Fondation Leducq (Redox Regulation of Cardiomyocyte Renewal). M.S.C. and J.R. are funded by NIH R01GM119336. B.A.R. was supported by grants from the NIH (R01 HD101006, R01 HL072016) and Wellstone (U54HD087351). J.A.H. is supported by grants from NIH (R01 HL120732, R01 HL128215, R01 HL126012). N.U.N.N. is supported by AHA Postdoctoral Fellowship 19POST34450039. Z.W. was supported by a predoctoral fellowship from the American Heart Association and the Harry S. Moss Heart Trust (19PRE34380436). N.T.L. is supported by a Haberecht Wildhare-Idea Research Grant.

Author information

Affiliations

Authors

Contributions

N.U.N.N., D.C.C. and S.A.M. conducted calcineurin and Rcan1-related experiments and interpreted results. N.U.N.N., F.X., Y.N., S.L., S.A.M., J.M., D.C.C., Victor Le, K.A.Z., H.W.E.-F. and W.M.E. conducted immunofluorescent staining. F.X., D.C.C., N.U.N.N. and X.M. designed and performed phosphorylated antibody experiments and interpreted results. N.U.N.N., F.X. and M.E.H. conducted immunoprecipitation and PLA experiments. N.U.N.N., F.X. and S.L. conducted TUNEL assay. Z.W. and N.U.N.N. conducted ChIP–seq experiments. M.K., C.X., C.G.A.-N, W.L.W.T, R.S.F. and Z.W. interpreted ChIP–seq results. E.V., F.X., S.R.A. and N.U.N.N. conducted cardiac fibroblast experiments and interpreted results. N.U.N.N., I.M.-M., Victoria Le and M.S.M. conducted TAC and exercise-related experiments. N.U.N.N., M.S.A., M.S.M. and I.M.-M. conducted BrdU-labelling experiments. N.L., F.X. and N.U.N.N. conducted MADM-related experiments and interpreted results. J.R. and M.S.C. designed and performed calcineurin-binding assays. S.L., and N.U.N.N. performed mouse surgeries. S.L., D.C.C., J.J.S. and N.U.N.N. conducted echocardiography experiments and interpreted results. S.T. and N.U.N.N. managed mouse colonies. N.U.N.N. designed and conducted experiments, interpreted results and contributed to manuscript preparation. J.A.H. interpreted results. B.A.R. interpreted results and contributed to manuscript preparation. H.A.S. designed the experiments, conceived the project and contributed to manuscript preparation.

Corresponding author

Correspondence to Hesham A. Sadek.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Norbert Frey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Additional characterization of Meis1-iKO, Hoxb13-KO, and Hoxb13-iKO mice.

a, Echocardiographic assessment of LVEF in Meis1-iKO hearts at the indicated time points post-MI. b, Immunostaining of Meis1-interacting Hox proteins (red), cardiac troponin T (green), and nucleus (blue) at P1, P7 and 7 days after P1 MI. c, Western blot analysis of Meis1 and Hox proteins during regenerative stages. GAPDH serves as loading control. d, Coimmunoprecipitation of Meis1 from total heart extracts at P1 and P7. e, Coimmunoprecipitation from Fig. 1c, with negative controls (IgG and DKO heart). f, Coimmunoprecipitation from Fig. 1d, with negative controls (IgG and DKO heart). g, PLA of Meis1 and Hoxb13 in P7 DKO heart; serves as negative control for PLA assay in Fig. 1e. h, Schematic of targeting strategy for generation of cardiomyocyte-specific Hoxb13 mutants. Partial map of the Hoxb13 floxed allele (top), and of the knockout allele (bottom) following cardiomyocyte-specific Cre-mediated excision. Exons and loxP sites are indicated as rectangles and triangles, respectively. i, PCR analysis of heart DNA obtained by crossing Hoxb13loxP/loxP (female) mutants with MHC-cre (male) transgenic mice. Left lane shows Hoxb13loxP/loxP. Middle lane indicates cardiomyocyte-specific Cre. Right lane indicates that the deleted allele is present in cardiomyocyte DNA of this offspring. j, Top, western blot of protein extracts from hearts of constitutive (left) or inducible (right) Hoxb13 knockout and Hoxb13fl/fl littermate mice. GAPDH serves as loading control. Bottom, densitometry quantification of the above western blot. k, Control and Hoxb13-KO P14 hearts stained for Meis1 (red), cardiac troponin T (green), and nucleus (blue). Arrowheads indicated Meis1-expressing cardiomyocytes, and arrows indicate Meis1-expressing non-cardiomyocytes. l, Representative echocardiography, ejection fraction and fraction shortening in Hoxb13-KO mice P14 hearts. m, Representative images of Masson’s trichrome staining of control and Hoxb13-iKO hearts at 14 days post-deletion. n, Representative images of immunostaining for TUNEL (green), cardiac troponin T (red) and nucleus (blue) (left) and quantification (right) 14 days post-deletion. Arrow indicates TUNEL-positive cardiomyocytes. Data are mean ± s.e.m.; unpaired two-sided t-test. Data in bf, g, i, k, m were independently repeated three times with similar results. *P < 0.05, **P < 0.01. For gel source data, see Supplementary Fig. 1. For n values, see Methods. Scale bars, 10 μm (b, k, n), 50 μm (g), 1 mm (m).

Source Data

Extended Data Fig. 2 Assessment of Hoxb13-KO and Hoxb13-iKO hearts post-MI.

a, Schematic of MI model in Hoxb13-KO mice. bd, Representative echocardiography (b), heart weight/body weight (c), and wet-to-dry lung weight ratio (d) in Hoxb13-KO mice 16 weeks after MI. e, Representative serial Masson’s trichrome staining in transversal sections (left) and quantification of fibrotic scars (right) in Hoxb13-KO MI hearts at 16 weeks after injury. f, Schematic of MI model in Hoxb13-iKO mice. g, h, Heart weight/body weight (g) and wet-to-dry lung weight ratio (h) in Hoxb13-iKO mice 16 weeks after MI. i, Serial echocardiography of Hoxb13-iKO MI and sham mice. Data are mean ± s.e.m.; unpaired two-sided t-test. **P < 0.01, ***P < 0.001; n.s., not significant. For n values, see Methods. Scale bar 1 mm (e).

Source Data

Extended Data Fig. 3 Concurrent knockout of Meis1 and Hoxb13 preserves cardiomyocyte proliferative capacity.

ag, Assessment of control and DKO hearts at P28. a, Representative images of haematoxylin and eosin-stained heart sections and heart weight/body weight ratio. b, Representative images of WGA staining and cardiomyocyte cross-sectional area quantification. c, Representative images of immunostaining for PH3 (green), cardiac troponin T (red) and nucleus (blue) (left) and the percentage (right) of mitotic cardiomyocytes (arrow). Arrowhead indicates sarcomere disassembly. d, Representative images of immunostaining for aurora B kinase (green), cardiac troponin T (red) and nucleus (blue) (left) showing the percentage (right) of cardiomyocytes undergoing cytokinesis (arrow). e, f, Total number of cardiomyocytes (e) and quantification of nucleation (f). Cnx43, connexin-43. g, Representative echocardiography and LV systolic function. hk, Assessment of control and DKO hearts at 6 months of age. h, Representative images of immunostaining for PH3 (green), cardiac troponin T (red), and nucleus (blue) (left) and the percentage (right) of mitotic cardiomyocytes (arrow). Arrowhead indicates cardiomyocytes with sarcomere disassembly. i, Representative images of immunostaining for aurora B kinase (green), cardiac troponin T (red) and nucleus (blue) (left) and the percentage (right) of cardiomyocytes undergoing cytokinesis (arrow). j, Representative images of WGA (green), cardiac troponin T (red) and nucleus (blue) staining (left) and cardiomyocyte CSA quantification (right). k, LV systolic function quantified by ejection fraction in 6-month-old DKO mice. Data are mean ± s.e.m.; unpaired two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001. For n values, see Methods. Scale bars, 10 μm (c, d, f, h, i), 50 μm (b, j), 1 mm (a).

Source Data

Extended Data Fig. 4 Loss of Meis1 and Hoxb13 attenuates both pathological and physiological hypertrophic responses.

a, Schematic representing the TAC model. b, Representative images of M-mode echocardiography (top) and quantification of ejection fraction (bottom), demonstrating improved LVEF in the DKO mice compared with wild-type mice three weeks after TAC. c, Heart weights, showing significantly higher heart weight in the wild-type group. d, Tibia length was similar in both groups. e, f, The ratio of heart weight/body weight (e) and heart weight/tibial length (f) indicate that the DKO mice have a blunted response to pressure overload. g, Representative images of mouse heart sections stained with haematoxylin and eosin (H&E; top) or Masson’s trichrome (bottom) three weeks after surgery. h, Representative confocal images of cardiomyocyte WGA staining (left) and CSA quantification (right), confirming the blunted hypertrophic response in the DKO hearts in response to pressure overload. i, Schematic representation of the exercise protocol. j, Representative images of echocardiography (top) and ejection fraction quantification (bottom), demonstrating no difference in ejection fraction at baseline and after exercise. k, Running distance of control and DKO mice over four weeks, demonstrating no difference between the two groups. l, Heart weight. m, The ratio of heart weight/body weight suggests that both control and DKO hearts respond to exercise-induced cardiac hypertrophy. n, The ratio of heart weight/tibia length also indicates cardiac hypertrophy in both groups in response to exercise. o, Representative images of mouse heart sections stained with either haematoxylin and eosin (top) or Masson’s trichrome (bottom) four weeks after exercise. p, Confocal images of cardiomyocyte WGA staining and CSA quantification. Data are mean ± s.e.m.; unpaired two-sided t-test. Images in g, o are representative of six independently performed experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001. For n values, see Methods. Scale bars, 50 μm (h, p), 1 mm (g, o).

Source Data

Extended Data Fig. 5 Inducible knockout of Meis1 and Hoxb13 promotes sarcomere disassembly and cardiomyocyte division.

a, Representative images of Masson’s trichrome staining of DiKO hearts four weeks after deletion. b, c, Disassembled sarcomere cardiomyocytes in DiKO hearts one week after deletion, demonstrated by cardiac troponin T (red) (b) or sarcomeric α-actinin (red) (c). d, Representative images of immunostaining for TUNEL (green) and cardiac troponin T (red) in DiKO hearts one week and two months after deletion, as shown in Fig. 2e. e, f, Representative images of immunostaining for PH3 (green), cardiac troponin T (red) and nucleus (blue), showing additional examples of proliferating cardiomyocytes in DiKO hearts at (e) one week and (f) two months after gene deletion. g, Representative images of immunostaining for aurora B kinase (green), cardiac troponin T (red) and nucleus (blue) (left) and the percentage (right) of proliferating cardiomyocytes (arrow) one week after gene deletion. h, Additional examples of cytokinetic cardiomyocytes in DiKO hearts one week after gene deletion. Images in ac are representative of three independently performed experiments with similar results. Scale bars, 10 μm (b, dh), 20 μm (c), 1 mm (a).

Source Data

Extended Data Fig. 6 LV function of DKO hearts after MI.

a, Schematic of MI model in DKO hearts 2 weeks post-MI. b, c, Western blot analysis (b) and quantification (c), showing decrease in expression of p21, p15/p16 in DKO hearts. df, Heart weight/body weight (d), heart weight (e) and body weight (f) in control and DiKO mice 16 weeks after injury. g, Representative images of the whole hearts at 16 weeks post-MI. The white arrow indicates a region with cell death and the yellow arrow indicates the dilated left atrial appendage. h, Additional representative serial Masson’s trichrome-stained transversal sections, as in Fig. 2l. i, j, LV cardiac index (i) and heart rate (j) in DiKO mice 16 weeks after MI measured by MRI. k, LVEF for sham groups. lq, Cardiac function analysis for left-ventricular end-diastolic anterior wall thickness (l), left-ventricular end-diastolic internal diameter (m), left-ventricular end-diastolic volume (n), left-ventricular end-diastolic posterior wall thickness (o), left-ventricular end-systolic internal diameter (p), and left ventricular end-systolic volume (q) in DiKO and control mice after MI. Data are mean ± s.e.m.; unpaired two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001. For gel source data, see Supplementary Fig. 1. For n values, see Methods. Scale bars, 1 mm (g, h).

Source Data

Extended Data Fig. 7 Genome-wide identification of Meis1 and Hoxb13 binding sites in heart using ChIP–seq.

a, Venn diagram showing Meis1 and Hoxb13 binding-site identification. Three replicate sequencing runs were performed for each factor, and the enriched regions were selected only if they were detected in three biological replicates. b, An example of a Meis1 binding site in the cell cycle arrest gene Cdkn1b. c, Gene ontology terms enriched in Meis1-, Hoxb13- and Meis1–Hoxb13-bound peak regions identified from a.

Extended Data Fig. 8 Interaction and dephosphorylation status of Meis1 and Hoxb13 with calcineurin.

ac, Coimmunoprecipitation of Fig. 3b–d with negative controls (IgG and DKO heart). d, PLA of CnA and Hoxb13 at P7 of DKO heart; serves as negative control for PLA assay in Fig. 3e. e, PLA assay using only one antibody; serves as negative control for the assay. f, PLA of CnA and Meis1 at P7 heart showed Meis1–CnA association (red dot). g, h, Schematic representation of Meis1 (g, top) and Hoxb13 (h, top) proteins showing the phosphorylatable serine and threonine residues, amino acid sequence of Meis1 (g, bottom) and Hoxb13 (h, bottom) with potentially phosphorylatable serine and threonine residues in red. Box indicates the predicted nuclear localization signal. Numbers indicate amino acid positions. i, j, Densitometry quantification of western blot analysis in Fig. 3j for Hoxb13 (i) and phosphorylated Hoxb13 S204 (j). k, Western blot analysis of phosphorylated Meis1 protein at P1 and P7. GAPDH serves as loading control. l, Western blot analysis from stable DKO cardiac fibroblasts confirms the loss of Hoxb13 in Cre-treated cells. Data are mean ± s.e.m.; unpaired two-sided t-test. Experiments in af, km were repeated independently three times with similar results. *P < 0.05, **P < 0.01. For gel source data, see Supplementary Fig. 1. For n values, see Methods. Scale bars, 10 μm (f), 50 μm (d, e).

Source Data

Extended Data Fig. 9 Increased calcineurin activity suppresses postnatal cardiomyocyte proliferation.

a, Heart weight/body weight in wild-type, CnA-Tg, Rcan1-Tg, and double-Tg mice at P2 (a) and P21 (b). c, d, Representative images of WGA staining for CSA quantification in Fig. 4a (c) and Fig. 4b (d). e, f, Representative images of immunostaining for PH3 (green) and cardiac troponin T (red) for mitotic cardiomyocytes quantification in Fig. 4c (e) and Fig. 4d (f). g, Schematic of apical resection model in P1 mice. Scale bars, 10 µm (f), 50 µm (c, d). Data are mean ± s.e.m.; unpaired two-sided t-test. ***P < 0.001. For n values, see Methods.

Source Data

Extended Data Table 1 Sequences of peptides used to generate phosphorylation-specific Meis1 and Hoxb13 antibodies

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Nguyen, N.U.N., Canseco, D.C., Xiao, F. et al. A calcineurin–Hoxb13 axis regulates growth mode of mammalian cardiomyocytes. Nature 582, 271–276 (2020). https://doi.org/10.1038/s41586-020-2228-6

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