Cardiomyocyte Foxp1-Specific Deletion Promotes Post-injury Heart Regeneration via Targeting Usp20-HIF1ɑ-Hand1 Signaling Pathway

Abstract

The adult mammalian heart has limited regenerative capacity to replace lost tissue after a major injury. Forkhead box P1 (Foxp1) regulates embryonic cardiomyocyte proliferation and heart development. However, whether Foxp1 participates in postnatal-injury cardiomyocyte proliferation and heart regeneration remains unclear. This study demonstrates that Foxp1 is downregulated at border zone cardiomyocytes of both neonatal apical resection and adult myocardial infarction. Analysis of the Single-cell transcriptome database reveals reduced Foxp1 expression in the cardiomyocyte population with high regenerative capacity. Cardiomyocyte-Foxp1 loss-of-function significantly promotes, whereas cardiomyocytes-Foxp1 gain-of-function suppresses cardiomyocyte proliferation. Mechanistically, Foxp1 directly regulates ubiquitin specific peptidase 20 (USP20), a de-ubiquitinase that prevents hypoxia inducible factor 1ɑ (HIF1α) degradation. Thus, Foxp1 regulates HIF1α and downstream heart and neural crest derivatives expressed 1 (Hand1) to control the cardiomyocyte proliferation via metabolic transition from fatty acid oxidation to glycolysis. Finally, cardiac type troponin T2 (cTnT)-promoter-driven adeno-associated virus 9 (AAV9) for Hand1 induction in cardiomyocytes significantly promoted cardiac regeneration and functional recovery. These findings may provide novel molecular strategies to promote heart regeneration and therapeutic interventions for heart failure.

Keywords: forkhead box P1; heart regenerations; metabolic transitions; myocardial infarctions.

Figure 1

Loss of Foxp1 in cardiomyocytes increases cell proliferation and promotes heart regeneration in the neonatal apical resection model. A) Uniform manifold approximation and projection (UMAP) visualization of cardiomyocyte clusters colored by identity in the neonatal heart of single nucleus RNA sequencing datasets GSE130699. B) Dot plot of the expression of gene signatures of cardiomyocyte CM1‐CM5 populations according to Cui et al.,^{[ 14 ]} C) Violin plots of Foxp1 expression in high regenerative capacity immature cardiomyocytes (regenerative) and the other non‐regenerative mature CMs (non‐regenerative) of neonatal‐MI hearts. D–F) Foxp1 expression in CMs of hearts 3 days after apical resection (AR) or sham‐operated at postnatal day 3 (P3ARd3) by D) western blot, E) RT‐qPCR, and co‐immunostaining of Foxp1 and ɑ‐sacromeric actinin (ɑ‐SA) of F) heart sections (n = 5). G) Schematic diagram of generation of CM‐specific Foxp1 deletion mice (Foxp1^CMKO) for neonatal cardiac regeneration post‐AR, and administration of tamoxifen (40 µg per day, from postnatal day 0 to 3) to induce Foxp1 deletion in CMs for AR operation, with EdU incorporation quantification for CM proliferation, histology analysis for final cardiac morphology, and echo analysis for cardiac function. H,I) Masson trichrome staining of fibrotic scar area (H) and wheat germ agglutinin (WGA) staining of myocyte cross–sectional size (I) in P3ARd7 and P3ARd21 hearts of Foxp^CMKO mice and wild‐type littermates. The representative images are on the left and the quantification data right (n = 8). J–M) CM proliferation quantified by J) EdU incorporation, K) immunostaining of Ki67 (cell cycles), L) PH3 (karyokinesis), and M) Aurora B (mitosis) of ɑ‐SA⁺ CMs in P3ARd7 hearts of Foxp^CMKO mice and wild‐type littermates. The representative images are on the left and the quantification data right (n = 8). Data are means ± SEM. *p < 0.05; ** p < 0.01, n.s. indicates not significant. Scale bar: (F) and (I) through (M), 50 µm; (H), 2 mm.

Figure 2

Loss of Foxp1 in cardiomyocytes increases cell proliferation to promote heart regeneration and improve cardiac function in the adult myocardial infarction model. A) Schematic diagram of adult cardiac regeneration post‐myocardial infarction (MI), and administration of tamoxifen (100 mg k⁻¹g, ip, every other day for a total 4 injections) for the induction of Foxp1 deletion in CMs for MI operation, with EdU incorporation quantification for CM proliferation, histology analysis for final cardiac morphology and echo evaluation for cardiac function. B,C) Echocardiography parameters left ventricle ejection fraction (B) and fractional shortening C) at post‐MI 28 days (MId28) of Foxp1^CMKO and wild‐type littermates (n = 8). D,E) Masson trichrome staining of fibrotic scar area D) and WGA staining of cross‐sectional size of border zone CMs (E) at MId28 of Foxp1^CMKO and wild‐type littermates. The representative images are on the left and the quantification data right (n = 8). F–I) Border zone CM proliferation in MId14 hearts from Foxp^CMKO mice and wild‐type littermates quantified by F) EdU incorporation, G) co‐immunostaining of Ki67, H) PH3 and I) Aurora B with ɑ‐SA. The representative images are on the left and the quantification data right (n = 8). Data are means ± SEM. *p < 0.05; ** p < 0.01, n.s. indicates not significant. Scale bar: (D), 2 mm; (E) through (I), 50 µm.

Figure 3

Foxp1 gain‐of‐function in cardiomyocytes suppresses proliferation and impairs heart regeneration and function. A–D) CM proliferation quantified by A) EdU incorporation, B) immunostaining of Ki67, C) PH3, and D) Aurora B in P3ARd7 hearts of Foxp^CMTg mice and wild‐type littermates, with representative images on the left and quantification data right (n = 8). E–J) WGA staining of myocyte cross‐sectional size E) and Masson trichrome staining of fibrotic scar area F) in P3ARd7 and P3ARd21 hearts, and border zone CM proliferation quantified by EdU incorporation G), immunostaining of Ki67 H), PH3 I) and Aurora B J) in MId14 hearts from Foxp^CMTg mice and wild‐type littermates. The representative images are on the left and the quantification data right (n = 8). K‐M, Masson trichrome staining of cardiac fibrotic scar area K), and echocardiography parameters left ventricle ejection fraction L) and fractional shortening M) evaluation of cardiac function at MId28 of Foxp1^CMKO mice and wild‐type littermates. The representative images are on the left and the quantification data right (n = 8). Data are means ± SEM. *P<0.05; ** P<0.01, n.s. indicates not significant. Scale bar: (A) through (E) and (G) through (J), 50 µm; (F) and (K), 2 mm.

Figure 4

Hypoxia‐inducible factor 1ɑ (HIF1ɑ) deletion in cardiomyocytes reverses the Foxp1‐dependent elevation of cell proliferation in heart regeneration. A) HIF1ɑ and USP20 protein expression by western blot in CMs from P3ARd3 and sham‐operated hearts of Foxp1^CMKO mice and wild‐type littermates, with representative blots on the top and quantification data bottom (n = 5). B) The protein expression of FOXP1, HIF1ɑ, USP20, and HAND1 in CMs from border zone (BZ) and remote zone (RZ) of MId14 and sham‐operated hearts, with representative blots on the top and quantification data bottom (n = 5). C) The protein expression of FOXP1, HIF1ɑ, USP20, and HAND1 in border zone CMs from MId14 hearts of Foxp1^CMKO mice and wild‐type littermates, with representative blots on the top and quantification data bottom (n = 5). D) CUT&Tag assay showed Foxp1 enrichment in the promoter region of Usp20. E) Schematic diagram of Foxp1 binding sites in the proximal 6 kb promoter of Usp20, with amplified sequence indicated in the dashed box cloned into a vector for luciferase reporter assay. F) Chromatin immunoprecipitation (ChIP)‐qPCR of Foxp1 and the promoter of Usp20 in the bottom with agarose gel on the top (n = 5). G) Luciferase reporter assay of Foxp1 for Usp20 promoter in NIH‐3T3 cells (n = 5). H) HEK293T cells were transfected with Foxp1‐ or Usp20‐siRNA following co‐transfection of HIF1ɑ‐HA and Ubiquitin (Ub)‐His plasmid. The cell lysates were immunoprecipitated by anti‐HA antibody or and the precipitates were immunoblotted by an anti‐His antibody for observation of the effect on HIF1ɑ deubiquitylation. I,J) Masson trichrome staining of cardiac fibrotic scar area I), WGA staining of CM cross‐sectional area J) in P3ARd7 and P3ARd21 hearts of HIF1ɑ; Foxp1^CMKO, Foxp1^CMKO, HIF1ɑ^CMKO and wild‐type mice (n = 8). K–N) CM proliferation quantified by EdU incorporation K), immunostaining of Ki67 L), PH3 M), and Aurora B N) of ɑ‐SA⁺ CMs in P3ARd7 hearts of HIF1ɑ;Foxp1^CMKO, Foxp1^CMKO, HIF1ɑ^CMKO, and wild‐type mice. The representative images on the left and the quantification data are on the right (n = 8). Data are means ± SEM. *p < 0.05; ** p < 0.01, n.s. indicates not significant. Scale bar: (I), 2 mm; (J) through (N), 50 µm.

Figure 5

Cardiomyocytes‐derived Foxp1‐HIF1ɑ signaling regulates cardiac metabolic transition from fatty acid oxidation to glycolysis. A) Non‐targeted metabolomics of P3ARd7 or age‐matched sham‐operated hearts from Foxp1^CMKO, HIF1ɑ^CMKO, Foxp1; HIF1ɑ^CMKO and wild‐type littermates are performed, with the principal component analysis (PCA) indicating good separation of metabolite clusters between each group. B) Non‐targeted metabolomics results of elevated glycolysis and reduced fatty acid (FA) intermediates with differences of AR or sham‐operated wild‐type mice, or HIF1ɑ; Foxp1^CMKO, Foxp1^CMKO, HIF1ɑ^CMKO compared with wild‐type littermates (n = 6). C) The expression of key components of glycolysis genes (Glut‐1, Hk‐2, Pdk‐1, Ldha) and FA oxidation genes (Mlycd, Acsl1, Hsl, Ech1, Fabp3 and Hmgcs2) by RT‐qPCR in the hearts of AR or sham‐operated wild‐type mice, or HIF1ɑ;Foxp1^CMKO, Foxp1^CMKO, HIF1ɑ^CMKO compared with wild‐type littermates (n = 5). Data are means± SEM. *p < 0.05; ** p < 0.01.

Figure 6

Cardiomyocytes‐derived Foxp1‐HIF1ɑ further regulate Hand1, a target gene that controls metabolic transition in cell proliferation and heart regeneration. A) Schematic diagram of HIF1ɑ binding sites within the proximal 3 kb promoter region of Hand1. The dashed box indicates the amplified sequence that was cloned into a luciferase reporter vector. B) Chromatin immunoprecipitation (ChIP)‐qPCR of HIF1ɑ binding to the Hand1 promoter in neonatal cardiomyocytes under 2% oxygen hypoxic conditions. The agarose gel image is shown above and the qPCR results are shown below (n = 4). C) Luciferase reporter assay for Hand1 promoter activity in NIH‐3T3 cells under the indicated treatments (n = 3). D–G) Hand1 expression by western blot and RT‐qPCR in CMs from P3ARd3 or sham‐operated hearts of Foxp1^CMKO (D‐E), HIF1ɑ^CMKO mice F–G) compared with their wild‐type littermates (n = 5). H–K) Hand1 expression by western blot and RT‐qPCR in neonatal mouse CMs (NMCMs) treated with Foxp1‐siRNA (H‐I), HIF1ɑ‐siRNA (J‐K) compared with scramble‐siRNA (n = 5). L,M) Hand1‐siRNA knockdown efficacy in NMCMs by western blot L) and RT‐qPCR M) (n = 5). N) The expression of lipid metabolizing and glycolysis genes in Hand1 knockdown or scramble‐siRNA CMs (n = 5). O‐Q, Cell proliferation quantified by EdU incorporation O), immunostaining of PH3 P) and Aurora B Q) in NMCMs treated by knockdown with Hand1‐siRNA; Foxp1‐siRNA, Foxp1‐siRNA, Hand1‐siRNA compared with scramble siRNA control (n = 5). Data are means± SEM. *p < 0.05; ** p < 0.01, n.s. indicates not significant. Scalebar: (O) through (Q), 50 µm.

Figure 7

Cardiomyocyte‐targeted delivery of Hand1 promoted glycolytic metabolic transition for raising cell proliferation and improving heart regeneration and cardiac dysfunction recovery in post‐MI animals with Foxp1 gain‐of‐function in cardiomyocytes. A) Schematic diagram of intracardial injection of Hand1‐AAV9 driven by cTnT‐promoter in adult MI mice. B) Masson trichrome staining of fibrotic scar size in MId28 hearts of Foxp1^CMTg mice and wild‐type littermates treated by Hand1‐AAV9 or scramble‐AAV9, with representative images on the left and quantification on the right (n = 8). C,D) Echocardiography parameter left ventricle ejection fraction C) and fractional shortening D) evaluation of cardiac function at MId28 hearts of Foxp1^CMTg mice and wild‐type littermates treated by Hand1‐ or scramble‐AAV9 (n = 8). E–I) CM proliferation quantified by EdU incorporation E), immunostaining of Ki67 F), PH3 G), and Aurora B H) of ɑ‐SA⁺ CMs and WGA staining of CM cross‐sectional size I) at MI border zone hearts from Foxp1^CMTg mice and wild‐type littermates treated by Hand1‐ or scramble‐AAV9 (n = 8). Data are means± SEM. *p < 0.05; ** p < 0.01, n.s. indicates not significant. Scale bar: (B), 2 mm; (E) through (I), 50 µm.