Meis1 regulates postnatal cardiomyocyte cell cycle arrest

Abstract

The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.

Figure 1. Expression profile of Meis1 in the heart

a, qRT–PCR showing increased expression of Meis1 at postnatal day 7 (P7), a time point that coincides with cell cycle arrest of cardiomyocytes. b, qRT–PCR showing expression levels of Meis1 following Sham or MI at P1 or P7. Samples were collected 7 days after the injury was performed (D7). c, Expression profile of Meis1 in cardiomyocytes. Top row, Meis1 expression is absent at P1. Middle and bottom rows, nuclear localization of Meis1 in cardiomyocytes at P7 and P21, respectively. d, qRT–PCR showing knockdown of Meis1 in rat neonatal cardiomyocytes using Meis1 siRNA. e, Cardiomyocyte mitosis following Meis1 knockdown. Left panel, immunostaining showing co-localization of pH3, TnnT2 and Hoechst (Ho) in rat neonatal cardiomyocytes. Right panel, quantification of the pH3⁺ TnnT2⁺ nuclei in control and Meis1 siRNA treated cardiomyocytes. Quantitative analysis represents counting of multiple fields from three independent samples per group; *P < 0.05, **P < 0.01; error bars represent mean ± s.e.m.

Figure 2. Cardiomyocyte proliferation at P14 following Meis1 deletion

a, Schematic of Meis1 floxed allele. Control mice were αMHC-Cre, Meis1 KO mice were Meis1^f/f αMHC-Cre. b, qRT–PCR demonstrates deletion of Meis1 in isolated cardiomyocytes at P14 (n = 3). c, Trichrome staining of wild-type and Meis1 KO hearts at P14. d, Heart weight (HW) to body weight (BW) ratio in wild-type and Meis1 KO hearts (n = 4–7 per group). e, Left ventricular systolic function quantified by ejection fraction and fractional shortening (n = 4–7 per group). f, Wheat germ agglutinin (WGA) staining and cell size quantification. Quantitative analyses represent counting of multiple fields from three independent samples per group (~50 cells per field assessed, total ~250 cells per group). g, Confocal image with z-stacking showing co-localization of pH3, TnnT2 and Hoechst in a Meis1 KO heart at P14 (top left). Confocal image of a pH3⁺ cardiomyocyte in a Meis1 KO heart (top right). Immunostaining showing sarcomere disassembly in Meis1 KO hearts (arrowhead, bottom left). Normal sarcomeric structure of Meis1 KO myocardium (bottom right). Graph shows quantification of the number of pH3⁺ TnnT2⁺ nuclei. h, Immunostaining showing expression of Aurora B in Meis1 KO cardiomyocytes at P14 and quantification of the number of Aurora B⁺ TnnT2⁺ cardiomyocytes. Quantitative analysis of pH3 and Aurora B⁺ cardiomyocytes represents counting of multiple sections from three independent samples per group (~3 sections per heart) (g, h). i, Immunostaining showing co-localization of BrdU, α—actinin and Hoechst in Meis1 KO heart at P14 and quantification of the number of BrdU⁺ cardiomyocytes. Quantification represents counting of several sections from (3–6) independent samples per group. Total number of cardiomyocytes counted for proliferation indices was 2 × 10³ – 2.5 × 10³ myocytes per section. j, Representative images of control and Meis1 KO isolated cardiomyocytes, and quantification of the number of myocytes from control and Meis1 KO hearts. Approximately 1 × 10³ – 1.5 × 10³ cardiomyocytes were counted using a haemocytometer per group, using 3 independent samples (left). Immunostaining of isolated cardiomyocytes with Connexin 43, and quantification of the number of nuclei in control and Meis1 KO hearts. For nucleation, approximately 1 × 10³ cardiomyocytes were counted per sample, using 3 independent samples per group (right). k, Apoptosis analysis. Image showing co-localization of TUNEL, Desmin, and Hoechst in a control and Meis1 KO heart. Quantification of TUNEL-positive cells. (n = 3). Values presented as mean ± s.e.m., *P, 0.05, **P < 0.01.

Figure 3. Inducible deletion of Meis1 in cardiomyocytes

a, Schematic of inducible Meis1 deletion (Meis1 iKO). Control mice were αMHC-Cre MerCreMer, Meis1 iKO mice were αMHC-Cre MerCreMer Meis1^f/f. b, qRT–PCR demonstrating deletion of Meis1 in isolated cardiomyocytes (n = 3). c, Trichrome stained sections at day 28 following tamoxifen injections. d, Heart weight to body weight ratio in wild-type and Meis1 iKO mice (n = 4 per group). e, Left ventricular systolic function quantified by ejection fraction and fractional shortening (n = 4 per group). f, Wheat germ agglutinin staining (WGA) and cell size quantification. Quantitative analyses represent counting of multiple fields from three independent samples per group (~50 cells per field assessed, total ~250 cells per group). g, Immunostaining image for pH3, TnnT2 and Hoechst, 7 days post tamoxifen induction. Arrowheads mark pH3⁺ myocyte nuclei. Graph shows the number of pH3⁺ TnnT2⁺ nuclei 7 days post tamoxifen treatment in control and Meis1 iKO hearts (n = 6). h, Immunostaining for Aurora B in Meis1 iKO heart. Graph shows the number of Aurora B⁺ cardiomyocytes. Quantitative analysis of pH3⁺ and Aurora B⁺ myocytes represents counting of multiple sections from three independent samples per group (<3 sections per heart). i, Representative images of control and Meis1 iKO isolated cardiomyocytes and quantification of the number of cardiomyocytes. Approximately 1 × 10³ – 1.5 × 10³ cardiomyocytes were counted using a haemocytometer per group, using 3 independent samples. Graph shows quantification of the nucleation of cardiomyocyte nuclei. For nucleation, approximately 1 × 10³ cardiomyocytes were counted per sample, using × independent samples per group. j, Apoptosis following inducible Meis1 deletion in cardiomyocytes. Immunostaining for TUNEL, Desmin, and Hoechst in control and Meis1 iKO hearts and quantification of TUNEL positive cells. Values presented as mean ± s.e.m., *P < 0.05.

Figure 4. Meis1 overexpression in the heart limits neonatal heart regeneration following myocardial infarction

a, Schematic of Meis1 overexpression (OE) in the heart. Control mice were αMHC-tTA, Meis1 (OE) mice were pTRE-Meis1 αMHC-tTA. b, qRT-PCR demonstrates overexpression of Meis1 (n = 3). c, Heart weight to body weight ratio in control and Meis1 (OE) mice (n = 11). d, Haematoxylin and eosin stained sections of wild-type and Meis1 (OE) hearts. e, Left ventricular systolic function quantified by ejection fraction (n = 4–5 per group). f, Wheat germ agglutinin staining and cell size quantification. Quantitative analyses represent counting of multiple fields from three independent samples per group (~50 cells per field assessed, total ~250 cells per group). g, Gene expression of hypertrophy markers in P3 control and Meis1 (OE) hearts. ANP #1 and #2 represent two different primer sets for the same gene. h, Immunostaining image showing co-localization of pH3, TnnT2 and Hoechst in Meis1 (OE) heart at P3. Graph shows quantification of the number of pH3⁺ TnnT2⁺ nuclei. Quantitative analysis represents counting of multiple sections from three independent samples per group (~3 sections per heart). i, Schematic of neonatal MI during the regenerative window at P1. j, Left ventricular systolic function of wild-type and Meis1 (OE) hearts at 21 days post-MI. k, Masson’s trichrome staining at day 21 post-MI. l, qRT–PCR of cyclin dependent kinase inhibitors (CDKIs) in hearts of Meis1 (OE) compared to control. Values presented as mean ± s.e.m; *P < 0.05, **P < 0.01.