Cellular Biology

FoxO1 and FoxM1 Transcription Factors Have Antagonistic Functions in Neonatal Cardiomyocyte Cell-Cycle Withdrawal and IGF1 Gene RegulationNovelty and Significance

Arunima Sengupta, Vladimir V. Kalinichenko, Katherine E. Yutzey
https://doi.org/10.1161/CIRCRESAHA.112.277442
Circulation Research. 2013;112:267-277
Originally published January 17, 2013

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Abstract

Rationale: In the mammalian heart, cardiomyocytes withdraw from the cell cycle and initiate hypertrophic growth soon after birth, but the transcriptional regulatory mechanisms that control these neonatal transitions are not well-defined.

Objective: Forkhead family transcription factors have been implicated as positive (forkhead box [Fox] transcription factor M1) and negative (FoxO1 and FoxO3) regulators of cardiomyocyte proliferation prenatally, but their regulatory interactions and functions in neonatal cell-cycle withdrawal have not been reported previously. Potential regulators of Fox activity, including the metabolic indicator AMP-activated protein kinase (AMPK), and Fox transcriptional targets (p21, p27, insulin-like growth factor 1 [IGF1]) also were examined.

Methods and Results: In cultured neonatal rat cardiomyocytes, AMPK activates FoxOs, and AMPK inhibition is sufficient to induce cell proliferation. In vivo, combined loss of FoxO1 and FoxO3 specifically in cardiomyocytes leads to delayed cell-cycle withdrawal and increased expression of IGF1 and FoxM1. Conversely, cardiomyocyte-specific loss of FoxM1 results in decreased neonatal cardiomyocyte cell proliferation, decreased expression of IGF1, and increased expression of cell-cycle inhibitors p21 and p27. IGF1 is a direct downstream target of cardiac Fox transcription factors, which is negatively regulated by FoxOs and positively regulated by FoxM1, dependent on AMPK activation status.

Conclusions: These data support a regulatory mechanism whereby the balance of FoxO and FoxM1 transcription factors integrates metabolic status, mediated by AMPK, and cell-cycle regulation, through competitive regulation of target genes, including IGF1, in neonatal cardiomyocytes.

Introduction

After birth, cardiomyocytes withdraw from the cell cycle and the heart grows primarily by hypertrophy.1 In the neonatal heart, growth factor signaling is reduced, metabolism shifts from glycolytic to primarily oxidative, and the myocardium has the ability to regenerate.2 Approximately 1 week after birth, binucleated cardiomyocytes withdraw from the cell cycle, the myocardium loses its ability to regenerate, and the heart initiates hypertrophic growth.1,2 Several transcription factors, including GATA4, Nkx2.5, T-box (Tbx)20, Tbx5, HAND1, forkhead box (Fox) p1, and FoxM1, have been implicated in regulating cardiomyocyte cell proliferation in the developing heart.3–5 However, the transcriptional regulatory mechanisms that integrate growth factor signaling, metabolic status, and cell-cycle regulation in the heart after birth are largely unknown.

Fox family transcription factors have been implicated in cardiomyocyte cell-cycle control as well as adult cardiovascular disease. FoxM1 promotes cell-cycle progression in a variety of cellular contexts and is required for cardiomyocyte proliferation in the developing heart.5,6 FoxO factors have multifaceted functions in cell-cycle regulation, metabolism, cell survival, autophagy, and aging.7 FoxOs are activated under conditions of growth factor deprivation, and FoxO1 and FoxO3 are expressed in the developing and diseased heart.8,9 In the prenatal heart, forced expression of FoxOs inhibits cell proliferation and induces expression of cell-cycle inhibitors p21 (p21Cip1) and p27 (p27Kip1).8 In the adult heart, FoxOs promote cardiomyocyte survival under conditions of oxidative stress and also induce autophagy after starvation.10,11 In addition, FoxOs inhibit cardiac hypertrophy and are induced with diabetic cardiomyopathy in adults.9,12 In cancer cells, FoxM1 and FoxOs have antagonistic transcriptional roles in cell-cycle regulation.13,14 However, the functions and potential interactions of these factors in neonatal cardiomyocyte cell-cycle withdrawal have not been reported previously.

Immediately after birth and before feeding, neonatal mammals are subjected to a period of starvation.15 Growth factor deprivation via phosphoinositide 3-kinase/AKT inactivation leads to increased FoxO nuclear localization and transcriptional activity in a variety of cell types, including fetal cardiomyocytes.7,8 Activation of AMP-activated protein kinase (AMPK) occurs as a result of metabolic insufficiency and promotes FoxO3 activity in longevity and tumor suppression,16 but this interaction has not been demonstrated in the heart. Insulin-like growth factor 1 (IGF1) is among the growth factors that inactivate FoxOs and has been implicated in promoting prenatal cardiomyocyte proliferation.8 IGF1 expression also can promote cardiac hypertrophy, cell-cycle reentry, and repair in adults,17,18 but its function and regulation in neonatal cell-cycle withdrawal have not been fully characterized.

In the current study, we examine AMPK function and the requirements for FoxOs and FoxM1 in neonatal cell-cycle withdrawal. In addition, we identify IGF1 as a common target of FoxOs and FoxM1, which is activated by FoxM1 in proliferating cardiomyocytes and repressed by FoxOs during neonatal cell-cycle withdrawal in response to AMPK activation status. These results provide evidence that FoxM1 inactivation and FoxO activation, subject to metabolic regulation, together regulate neonatal cardiomyocyte cell-cycle withdrawal.

Methods

Primary neonatal (1–2 days) rat cardiomyocytes were isolated, infected with FoxO adenoviruses (Ad), and analyzed as described previously.11 Cardiomyocyte-specific conditional loss of FoxOs and FoxM1 was achieved with β-myosin heavy chain (βMHC)-Cre using published mouse lines.5,11 Proliferative indices were calculated as described previously.8,11 Quantitative reverse-transcription polymerase chain reaction, chromatin immunoprecipitation, and IGF-1 reporter assays were performed as previously described.8,11,19 All experimental procedures with animals were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children’s Hospital Medical Center. An expanded Methods section is available in the Online Data Supplement.

Results

AMPK Activity and FoxO Activity Are Increased, Whereas Activity of AKT and Expression of IGF1 and FoxM1 Are Decreased, in Mouse Hearts After Birth

Expression levels of the proliferative factor IGF1 and the activation status of the downstream kinase AKT were determined by Western blot analysis of wild-type mouse heart lysates at embryonic day (E) 14.5, E17.5, postnatal day (pd) 1, pd7, and 1 month. In addition, the activation status of AMPK, an indicator of metabolic insufficiency, was determined relative to the activation status of FoxOs and expression of FoxM1. After birth, IGF1 protein expression is decreased by 50% in pd7 hearts and 1-month-old hearts compared with E14.5. Similarly, the activity of AKT also is decreased by 40% in pd7 hearts and 1-month-old hearts compared with E14.5, as indicated by decreased p-AKT/total AKT (Figure 1A–C). Conversely, AMPK activation is increased postnatally (by 1.9-fold at pd7 and 2.25-fold at 1 month, compared with E14.5), as indicated by increased p-AMPK/total AMPK protein levels (Figure 1A and 1D). The activity of FoxO1 and activity of FoxO3 also are increased postnatally in mouse hearts (Figure 1A), as indicated by decreased levels of inactive phosphorylated FoxO1 (p-FoxO1; Ser-256)/total FoxO1 (30% reduction at pd1 and 60% at 1 month, compared with E14.5; Figure 1E) and inactive p-FoxO3(Ser-318/321)/total FoxO3 (40% reduction at pd1 to 80% at 1 month, compared with E14.5; Figure 1F). In contrast, FoxM1 protein expression is decreased by 80% in postnatal mouse hearts compared with E14.5 (Figure 1A and 1G). Thus, the activity of AMPK and activity of FoxOs increase, whereas the activity of AKT and expression of IGF1 and FoxM1 protein decline, during the first week after birth in mouse hearts in vivo.

Figure 1.
Figure 1.

AMP-activated protein kinase (AMPK) and forkhead box transcription factor (Fox) O activity is increased, whereas the expression of FoxM1 is decreased, in mouse hearts after birth. A, The expression of insulin-like growth factor 1 (IGF1) and the activity of AKT are decreased postnatally in wild-type (WT) mouse hearts in vivo as determined by Western blot. The activity of AMPK is increased postnatally in WT mouse hearts in vivo as indicated by increased p-AMPK/total AMPK protein levels determined by Western blot analyses (indicated by asterisks). The activity of FoxO1 and activity of FoxO3 also are increased postnatally in mouse hearts in vivo as indicated by decreased levels of inactive p-FoxO1 and p-FoxO3 protein levels by Western blot analyses (indicated by asterisks). In contrast, FoxM1 protein expression is decreased in postnatal mouse hearts (indicated by asterisks). B to G, Quantification of the Western blots (n=3) is shown as bar graphs. Statistical significance (*) was determined by Student t test (P<0.05).

Inhibition of AMPK Activity Results in Cell-Cycle Activation and Altered Expression of Cell-Cycle Regulatory Genes in Cultured Rat Neonatal Cardiomyocytes

Neonatal cardiomyocytes normally exit the cell cycle, and postnatal proliferative rates are extremely low.1,20 To determine the effects of altered activity of AMPK on cardiomyocyte cell-cycle withdrawal, rat neonatal cardiomyocytes were treated with either AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR; AMPK activator) or compound C (AMPK inhibitor). AMPK inhibition with compound C increases the cell-cycle activity by 2.6-fold compared with vehicle-treated (dimethyl sulfoxide) cells, as determined by immunofluorescence and cell counts (Figure 2C and C'; Ki67+/α-actinin+ cardiomyocytes).21 Activation of AMPK by AICAR treatment does not inhibit the already low rates of proliferation of neonatal cardiomyocytes compared with vehicle-treated cells. The expression of cell-cycle regulatory genes also was examined in rat neonatal cardiomyocytes treated with either AICAR or compound C. Gene expression of cell-cycle activators, IGF1 and FoxM1, and also of cell-cycle inhibitors, p21 and p27, which are known targets of FoxOs, were determined by quantitative reverse-transcription polymerase chain reaction. The activation of AMPK by AICAR results in decreased expression of FoxM1 and IGF1 with increased expression of p21 (Figure 2E). In contrast, inhibition of AMPK by compound C results in significantly increased expression of FoxM1 and IGF1, with decreased expression of p21 and p27, in accordance with increased cell-cycle activation. Thus, inhibition of AMPK promotes cell-cycle activity, with corresponding effects on cell-cycle regulatory gene expression, in cultured rat neonatal cardiomyocytes.

Figure 2.
Figure 2.

AMP-activated protein kinase (AMPK) regulates cell-cycle activity in rat neonatal cardiomyocytes. A to C, Rat neonatal cardiomyocytes were treated with either AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (activator of AMPK) or compound C (inhibitor of AMPK). Inhibition of AMPK activity with compound C (comp C) results in increased proliferation (Ki67+(green)/α-actinin+(red) proliferating cardiomyocytes, white arrows), quantified in D, in cultured rat neonatal cardiomyocytes compared with vehicle (dimethyl sulfoxide [DMSO])-treated cells. E, Activation of AMPK by AICAR in rat neonatal cardiomyocyte cultures results in decreased expression of FoxM1 and IGF1, with increased expression of p21, as determined by quantitative real time reverse transcription polymerase chain reaction. Inhibition of AMPK with compound C treatment results in increased expression of FoxM1 and IGF1 with decreased expression of p21 and p27. Statistical significance (*) was determined by Student t test (P<0.05).

Activation of AMPK Promotes Nuclear Localization of FoxO1 and FoxO3 in Rat Neonatal Cardiomyocytes

The ability of AMPK to alter subcellular localization of endogenous FoxOs was determined in rat neonatal cardiomyocytes treated with either AICAR (Figure 3B, 3B', 3E, and 3E') or compound C (Figure 3C, 3C', 3F, and 3F'). Nuclear vs cytoplasmic localization of FoxO1 and FoxO3 in treated and control cardiomyocytes was determined by immunofluorescence (Figure 3A, 3A', 3D, and 3D'). Activation of AMPK promotes nuclear localization of endogenous FoxO1 (Figure 3B and 3B') and FoxO3 (Figure 3E and 3E') in rat neonatal cardiomyocytes (α-actinin). Conversely, inhibition of AMPK leads to exclusion of FoxO1 (Figure 3C and 3C') and FoxO3 (Figure 3F and 3F') from the nucleus in rat neonatal cardiomyocytes. Quantitation of these results shows a significant increase in nuclear FoxO1 (75% vs 63%) and FoxO3 (45% vs 25%) in AICAR-treated cardiomyocytes. In contrast, nuclear localization of FoxO1 (43% vs 63%) and FoxO3 (10% vs 25%) in cardiomyocytes is significantly decreased with AMPK inhibition, compared with controls (Figure 3G). Increased nuclear localization of endogenous FoxO1 and FoxO3 by activation of AMPK is consistent with increased transcriptional function and suggests a critical role for AMPK and FoxOs in regulating neonatal cell-cycle withdrawal.

Figure 3.
Figure 3.

Activation of AMP-activated protein kinase (AMPK) promotes AKT inactivation with decreased phosphorylation and increased nuclear localization of endogenous forkhead box transcription factor (Fox) O1 and FoxO3 in neonatal cardiomyocytes. A to F, Rat neonatal cardiomyocytes were treated with either AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (B, B' and E, E') or compound C (C, C' and F, F'), and FoxO1 and FoxO3 proteins (green) were localized compared with controls (A, A' and D, D'). Activation of AMPK promotes nuclear localization of FoxO1 (B, B', white arrow) and FoxO3 (E, E', white arrow) in rat cardiomyocytes (α-actinin, red) as determined by immunofluorescence. Conversely, inhibition of AMPK excludes FoxO1 (C, C') and FoxO3 (F, F') from the nucleus in rat neonatal cardiomyocytes. G, Quantitation of (A) to (E) shows a significant increase in FoxO1+ and FoxO3+ cardiomyocytes in AICAR-treated cultures and a significant decrease with AMPK inhibition, compared with controls. H to L, Western blot analysis shows decreased AKT activity (I) with increased FoxO1 (J) and FoxO3 (K) activity in cardiomyocytes treated with AICAR. With compound C (Comp C) treatment, AKT activity is increased (I), FoxO1 phosphorylation is increased (J), FoxO3 phosphorylation is increased (J), and FoxM1 expression is increased (L). Statistical significance (*) was determined by Student t test (P<0.05).

To determine a direct link between AMPK and FoxO activation in rat neonatal cardiomyocytes, we examined the activation status of AKT, FoxOs, and the expression levels of FoxM1 in rat neonatal cardiomyocyte cultures treated with either AMPK activator AICAR or AMPK inhibitor compound C. Activation of AMPK by AICAR decreases AKT activity, as indicated by decreased p-AKT/total AKT, with concomitant increased FoxO1 and FoxO3 activity, as indicated by decreased AKT-specific phosphorylation of FoxO1 (Ser-256) and FoxO3 (Ser-318/321) (Figure 3H–3K). Conversely, inactivation of AMPK by compound C increases AKT activity, as indicated by increased p-AKT/total AKT, with concomitant decreased FoxO1 and FoxO3 activity, as indicated by increased AKT-specific phosphorylation of FoxO1 and FoxO3 (Figure 3H–3K). In addition, FoxM1 protein expression increases with compound C treatment consistent with the decreased activation of FoxO1 and FoxO3 (Figure 3H and 3L and Figure 2C and 2D). Thus AMPK activation inhibits AKT activity and subsequent phosphorylation, nuclear localization, and downstream target gene expression of FoxOs in rat neonatal cardiomyocytes.

FoxO Inhibition Increases Proliferation, Whereas Induction of Proliferation With AMPK Inhibition Is Attenuated With Increased FoxO Activity, in Rat Neonatal Cardiomyocyte Cell Cultures

The function of FoxO transcription factors in regulation of neonatal cardiomyocyte cell-cycle withdrawal was examined in cardiomyocytes infected with recombinant Ad expressing FoxO1 or FoxO3.11,22,23 FoxO gain of function was achieved using Ad expressing either constitutively active FoxO1 (ADA) or constitutively active FoxO3 (TmO3). FoxO loss of function was achieved by infection with Ad expressing δ256-FoxO (dominant-negative), which inhibits transcriptional activity of FoxOs. Cardiomyocytes infected in parallel with β-galactosidase Ad serve as a control. Inhibition of FoxO activity (Figure 4A; δ256) results in increased cardiomyocyte cell-cycle activation (Ki67+/α-actinin+; 35% vs 11%; Figure 4Ad and 4Ad'). However, no changes were observed in cells infected with Ad expressing either ADA or TmO3 compared with Ad β-galactosidase-infected-controls (Figure 4A and 4B). Thus, inhibition of FoxO transcriptional activity promotes cardiomyocyte cell-cycle activation.

Figure 4.
Figure 4.

Forkhead box transcription factor (Fox) Os inhibit cell-cycle activity in rat neonatal cardiomyocytes. A, Inhibition of FoxO activity (dominant-negative FoxO-Δ256 adenovirus) results in increased cardiomyocyte proliferation (d, d'; Ki67+ (green)/α-actinin+ (red); white arrows). No proliferative changes were observed in cells infected with constitutively active ADA-FoxO1 (b, b’) or TmO3-FoxO3 (c, c') compared with adenovirus (Ad) β-gal–infected (a, a') controls. B, Quantitative representation is in (A). C and D, Increased cardiomyocyte proliferation by compound C (a, a') is attenuated in the presence of ADA-FoxO1 (b, b') or TmO3-FoxO3 (c, c'). D, Quantitative representation is in (C). E, Cardiomyocyte proliferation is increased by dnFoxOΔ256 (c, c') in the presence or absence AMP-activated protein kinase (AMPK) activation by AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (d, d') consistent with the requirement for FoxOs in cell-cycle inhibition mediated by AMPK activation in neonatal cardiomyocytes. F, Quantitative representation is in (E). Statistical significance (*) was determined by Student t test (P<0.05).

The ability of FoxO activation to block induction of neonatal cardiomyocyte cell-cycle activation by AMPK inhibition was examined. Rat neonatal cardiomyocytes were infected with either ADA or TmO3 Ad, compared with Ad β-galactosidase–infected controls, for 24 hours and then treated with compound C. The induction of cardiomyocyte cell-cycle activation attributable to inhibition of AMPK activity (Figure 4Ca and 4Ca'), as detected by Ki67 antibody reactivity, is attenuated in presence of constitutively active FoxO1 (Figure 4Cb and 4Cb'; 20% vs 37%) or FoxO3 (Figure 4Cc and 4Cc'; 12% vs 37%). Quantitative representation of Figure 4C is shown in Figure 4D. Thus, FoxO activation is sufficient to inhibit cardiomyocyte cell-cycle activity under conditions of AMPK inhibition.

The necessity of FoxO function in AMPK-mediated cell-cycle withdrawal in neonatal cardiomyocytes was examined. Rat neonatal cardiomyocytes were infected with dominant-negative Δ256-FoxO Ad or control Ad β-galactosidase for 24 hours and then were treated with the AMPK activator, AICAR. Inhibition of FoxO activity in Δ256-FoxO Ad-infected cardiomyocytes results in increased cardiomyocyte cell-cycle activation (Ki67+/α-actinin+; Figure 4Ad, 4Ad', 4Ec, and 4Ec') compared with control cells (Figure 4Aa, 4Aa', 4Ea and 4Ea'). Normally, AMPK activation leads to decreased proliferation, but cardiomyocyte cell-cycle activation is increased with dominant-negative Δ256-FoxO in the presence of AICAR (Figure 4Eb and 4Eb' [14%] 4Ed and 4Ed' [32%] and Figure 4F). Thus, AMPK-mediated cell-cycle withdrawal is dependent on FoxO activity in neonatal cardiomyocytes.

Cardiomyocyte-Specific Loss of FoxO1 and FoxO3 Results in Increased Mitotic Activity, Whereas Cardiomyocyte-Specific Loss of FoxM1 Results in Decreased Mitotic Activity, in Neonatal Mouse Hearts In Vivo

The requirements for FoxO function in neonatal cardiomyocyte cell-cycle withdrawal in vivo were determined in mice with combined deficiency of FoxO1 and FoxO3 specifically in cardiomyocytes. Because both FoxO1 and FoxO3 are expressed in the heart and are functionally redundant in many cell types, both were deleted together to determine the effect of loss of FoxOs on cardiomyocyte cell-cycle withdrawal. βMHC-Cre was used to specifically delete FoxO1 and FoxO3 from cardiomyocytes beginning at late fetal stages, and the mice were viable.11 Here, we report the effects of cardiomyocyte-specific loss of FoxOs on neonatal cardiomyocyte cell-cycle withdrawal. To determine cardiomyocyte mitotic activity in vivo, immunostaining was performed to colocalize phospho-histone H3+ (proliferation marker) and MF20+ (cardiomyocyte-specific marker) cells in pd1, pd3, pd7, and 1-month-old βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl mouse hearts compared with those of littermate FoxO1fl/fl/FoxO3fl/fl controls.

Cardiomyocyte proliferation is increased in the βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl mouse hearts compared with FoxO1fl/fl/FoxO3fl/fl control hearts at pd1 (4.3% vs 2.8%) and pd3 (1.8% vs 0.7%) as determined by phospho-histone H3 immunofluorescence (Figure 5A–5C). However, by pd7 and in adults, there were no significant differences in cardiomyocyte mitotic activity between the 2 genotypes. Thus, cardiomyocyte-specific loss of FoxO1 and FoxO3 results in increased cell proliferation in the first week after birth, but the cardiomyocytes exhibit delayed cell-cycle withdrawal apparent at pd7.

Figure 5.
Figure 5.

Cardiomyocyte-specific loss of forkhead box transcription factor (Fox) O1 and FoxO3 results in increased mitotic activity, whereas cardiomyocyte-specific loss of FoxM1 results in decreased mitotic activity, in neonatal mouse hearts in vivo. A and B, At postnatal day (pd) 3, βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl mouse hearts show increased mitotic activity in cardiomyocytes (phospho-histone H3 [PHH3+] (red)/MF20+ (green) cells) of mouse hearts, compared with FoxO1fl/fl/FoxO3fl/fl controls, as determined by immunofluorescence. C, Quantitation of pd1 and pd3 hearts shows increased mitotic activity in cardiomyocytes of βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl mouse hearts compared with FoxO1fl/fl/FoxO3fl/fl control hearts. D and E, Cardiomyocyte mitotic activity is decreased in the βMHC-Cre;FoxM1fl/fl mouse hearts at pd3, compared with FoxM1fl/fl control hearts, as determined by immunofluorescence. F, Quantitation of pd1 and pd3 hearts shows decreased mitotic activity in cardiomyocytes of βMHC-Cre;FoxM1fl/fl mouse hearts compared with FoxM1fl/fl control hearts. At pd7 and 1 month, no differences in cardiomyocyte mitotic activity were detected among the genotypes. Statistical significance (*) was determined by Student t test (P<0.05).

During development, FoxM1 is required for cardiomyocyte proliferation.5,24 FoxM1 expression is downregulated at birth in the heart, with little or no expression in the adult.5,25 To determine FoxM1 function in the regulation of the timing of neonatal cardiomyocyte cell-cycle withdrawal in vivo, FoxM1fl/fl mice were bred with βMHC-Cre mice to delete FoxM1 specifically from cardiomyocytes beginning at late fetal stages in resulting βMHC-Cre;FoxM1fl/fl animals. In contrast to βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl hearts, βMHC-Cre;FoxM1fl/fl neonatal hearts have decreased cardiomyocyte proliferation compared with FOXM1fl/fl control hearts at pd1 (2.4% vs 0.7%) and pd3 (1.5% vs 0.8%), as determined by immunofluorescence (Figure 5D–5F). Similar to βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl hearts, no significant differences in cardiomyocyte proliferation were observed between βMHC-Cre;FoxM1fl/fl and FoxM1fl/fl animals at pd7 and 1 month (Figure 5F). During the neonatal period, cardiomyocyte cell size and indicators of cardiac hypertrophy (βMHC, atrial natriuretic factor, brain natriuretic protein) are not altered in cardiomyocyte-specific FoxO-deficient and FoxM1-deficient mice compared with littermate controls (Online Figure I). In addition, cell proliferation of noncardiomyocyte lineages was apparently unaffected with cardiomyocyte-specific loss of FoxOs or FoxM1 (data not shown); thus, further studies are necessary to determine whether FoxOs and FoxM1 regulate cell proliferation in cardiac fibroblasts and smooth muscle cells. These results demonstrate that FoxOs and FoxM1 have opposing effects in neonatal cardiomyocytes, supporting a mechanism whereby FoxOs promote and FoxM1 inhibits neonatal cell-cycle withdrawal.

FoxOs and FoxM1 Are Differentially Required for Cell-Cycle Regulatory Gene Expression in Neonatal Cardiomyocytes In Vivo

During the neonatal period in mice, genes that promote cell proliferation are downregulated and cell-cycle inhibitory genes are induced. IGF1 and FoxM1 promote prenatal cardiomyocyte proliferation, and p21 and p27 are direct downstream targets of FoxOs that inhibit cardiomyocyte proliferation.8,26 Therefore, we examined the expression levels of these cell-cycle regulatory genes in βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl neonatal hearts with delayed cell-cycle withdrawal, compared with βMHC-Cre;FoxM1fl/fl neonatal hearts with accelerated cell-cycle withdrawal (Figure 6). Expression of IGF1 (≈2-fold; Figure 6A) and FoxM1 (≈ 4-fold; Figure 6B) is increased in βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl neonatal hearts. In contrast, cardiomyocyte-specific loss of FoxM1 results in decreased IGF1 gene expression (≈80% reduced; Figure 6A) with increased gene expression of both FoxO1 and FoxO3 (≈3–4 fold, Figure 6E; ≈10-fold, Figure 6F). As expected, expression of both FoxO1 (Figure 6E) and FoxO3 (Figure 6F) is decreased in mice with conditional targeting of FoxOs in cardiomyocytes with βMHC-Cre, and there is negligible expression of FoxM1 in mice with conditional targeting of FoxM1 in cardiomyocytes with βMHC-Cre (Figure 6B). p27 is a direct transcriptional target of FoxOs, and p27 expression is decreased by 60% to 70% with cardiomyocyte-specific loss of FoxO1 and FoxO3 in neonatal (pd1 and pd3) hearts, as determined by quantitative reverse-transcription polymerase chain reaction (Figure 6D). However, gene expression of p21 remains unchanged, probably because of the very low level of expression in the control hearts. In contrast, expression of p21 (Figure 6C) and p27 (Figure 6D) is increased 2- to 4.5-fold in βMHC-Cre;FoxM1fl/fl hearts at pd1-3, consistent with observed premature cell-cycle withdrawal in these animals. These results are in accordance with increased mitotic activity observed with cardiomyocyte-specific loss of FoxO1 and FoxO3, in contrast to decreased neonatal cardiomyocyte mitotic activity with loss of FoxM1. In addition, these data provide initial evidence for competitive regulation of shared target genes in the determination of the timing of cardiomyocyte cell-cycle withdrawal.

Figure 6.
Figure 6.

Expression of IGF1 is increased with deficiency of forkhead box (Fox) transcription factor Os and is decreased with loss of FoxM1, whereas expression of cell-cycle inhibitors is increased with loss of FoxM1 in neonatal mouse hearts. A and B, Expression of IGF1 (A) and FoxM1 (B) is increased in βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl hearts and is decreased in βMHC-Cre;FoxM1fl/fl hearts at postnatal day (pd) 1 and pd3, compared with Cre-negative controls, as determined by quantitative reverse-transcription polymerase chain reaction. C and D, Expression of cell-cycle inhibitors p21 and p27 are increased in FoxM1-deficient cardiomyocytes, and p27 expression is decreased in FoxO-deficient cardiomyocytes at pd1 and pd3. E and F, Expression of FoxO1 and FoxO3 is decreased in βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl hearts and is increased in βMHC-Cre;FoxM1fl/fl hearts. These results are in accordance with increased proliferation in βMHC-Cre;FoxO1fl/fl/FoxO3fl/fl hearts and decreased proliferation in the βMHC-Cre;FoxM1fl/fl hearts during pd1 to pd3. Statistical significance (*) was determined by Student t test (P<0.05).

FoxM1 and FoxO1 Bind Directly to and Have Opposite Effects on IGF1 Promoter Activity

Cardiomyocyte-specific loss of FoxOs and FoxM1 has opposing effects on IGF1 gene expression in neonatal cardiomyocytes, consistent with altered timing of cell-cycle withdrawal (Figure 6A). Therefore, IGF1 genomic sequences were examined for conserved Forkhead (FOX) binding consensus sequences and for transcriptional regulation by FoxO1 and FoxM1. The mouse IGF1 gene sequence (NC_000076.6) contains a conserved FOX DNA binding sequence TAAACA located at -550 relative to the transcriptional start site (Figure 7A and 7B).19,27,28 Cotransfection experiments performed in C2C12 cells demonstrate that murine IGF1 (−1600) sequences linked to a pGL3 basic reporter are transactivated by FoxM1 in a concentration-dependent manner (1.5–4.5-fold; Figure 7C). Mutagenesis of the conserved FOX binding site prevents transactivation of IGF1 regulatory sequences by FoxM1 (Figure 7C). Conversely, cotransfection assays performed in HEK293 cells demonstrate that FoxO1 suppresses IGF1-luciferase reporter activity (Figure 7D). Together, these results show that FoxM1 and FoxO1 have opposite effects on transactivation of the IGF1 promoter in transfected cells.

Figure 7.
Figure 7.

Forkhead box (Fox) transcription factor M1 and FoxO1 have opposite effects on transcriptional activity of the IGF1 promoter and binding is differentially regulated by AMP-activated protein kinase (AMPK) in neonatal cardiomyocytes. A, The mouse IGF1 gene contains a conserved Forkhead (FOX) DNA binding domain located at -550 relative to the transcriptional start site. Arrows indicate polymerase chain reaction primer sequences for amplification of FOX binding sites in IGF1 genomic sequence for chromatin immunoprecipitation (ChIP) assay. B, The murine FOX consensus sequence (AAACA) is conserved at -137 in rat (NC_005106.3) and at -190 in human (NC_000012.11) IGF1 genes. C, Cotransfection experiments performed in C2C12 cells demonstrate that murine IGF1 (−1600) sequences linked to a pGL3-basic reporter are transactivated by FoxM1 in a concentration-dependent manner. Mutagenesis of the conserved FOX binding sites (indicated by asterisks in B) leads to little transactivation of IGF1 regulatory sequences by FoxM1. D, Cotransfection experiments performed in HEK293 cells show that FoxO1-ADA can suppress IGF1-luciferase reporter activity. E, ChIP assays were performed in rat neonatal cardiomyocytes treated with either AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or compound C and immunoprecipitated with FoxO1 or FoxM1 antibodies to determine the fold enrichment of binding to the IGF1 promoter region. ChIP assays demonstrate ≈13-fold enrichment of FoxM1 binding to the IGF1 promoter region with AMPK inhibition by compound C, whereas ≈6-fold FoxO1 enrichment was observed in the presence of the AMPK activator (AICAR). Statistical significance (*) was determined by Student t test (P<0.05).

To determine whether FoxO1 and FoxM1 bind to the identified IGF1 promoter sequences, chromatin immunoprecipitation assays were performed in rat neonatal cardiomyocytes under different proliferative conditions (Figure 7E). Binding of FoxO1 and FoxM1 to IGF1 promoter sequences was examined in neonatal rat cardiomyocytes with altered AMPK activity resulting from treatment with either AICAR or compound C. Immunoprecipitation with FoxO1 or FoxM1 antibodies was used to determine the fold enrichment of binding to IGF1 promoter sequences relative to the IgG control. In cardiomyocytes proliferating because of AMPK inhibition, FoxM1 binding to the IGF1 promoter region is increased by ≈9-fold. In contrast, AMPK activation results in a ≈6-fold enrichment of FoxO1 bound to the IGF1 promoter. Thus, FoxM1 and FoxO1 bind directly to the IGF1 promoter, and the specific Fox transcription factor bound to these sequences is subject to cardiomyocyte metabolic status. Together, these data are consistent with a mechanism whereby differential binding of the IGF1 promoter by FoxM1 during development and differential binding by FoxO1 during the neonatal period regulate IGF1 gene expression and contribute to neonatal cell-cycle withdrawal.

Discussion

Here, we demonstrate that FoxOs and FoxM1 regulate cardiomyocyte proliferation and cell-cycle withdrawal in the neonatal period. In the days after birth, FoxM1 is downregulated and FoxOs are activated concomitant with increased AMPK activation, decreased AKT activity, decreased IGF1 expression, and induction of cell-cycle inhibitors. Studies in cardiomyocyte-specific FoxM1-deficient and FoxO-deficient mice, together with experiments in cultured neonatal cardiomyocytes, support a model in which the balance of growth factor signaling and activity of FoxM1 vs FoxOs regulates cardiomyocyte proliferation in the fetal and neonatal periods (Figure 8). In fetal cardiomyocytes, growth factor signaling is high, FoxM1 is expressed, IGF1 is expressed, AKT is active, and cardiomyocytes are highly proliferative. During the neonatal period, growth factor signaling is reduced, AMPK is activated, AKT is inactivated, FoxOs are activated, FoxM1 is downregulated, IGF1 is downregulated, cell-cycle inhibitors are upregulated, and cardiomyocytes withdraw from the cell cycle. Increased FoxO activity inhibits FoxM1 and IGF1 gene expression, while activating cell-cycle inhibitor genes p21 and p27. Chromatin immunoprecipitation and transfection studies demonstrate that FoxM1 binds and is a transcriptional activator of IGF1 regulatory sequences, whereas FoxO1 binds and represses the same IGF1 regulatory sequences, subject to regulation by AMPK. Together, these data support a regulatory mechanism whereby FoxO and FoxM1 transcription factors integrate metabolic status and cell-cycle withdrawal in neonatal cardiomyocytes.

Figure 8.
Figure 8.

Model for forkhead box (Fox) transcription factor O/FoxM1 function in controlling cell proliferation of neonatal cardiomyocytes. In fetal cardiomyocytes (CMs), high growth factor signaling results in AMP-activated protein kinase (AMPK) inactivation, AKT activation, increased FoxM1 expression, and inhibition of FoxO activation, which, in turn, augment IGF1 gene expression to promote high levels of proliferation. In contrast, in neonatal CMs, AMPK activation leads to decreased AKT activity with increased FoxO nuclear localization that directly inhibits FoxM1 and IGF1 gene expression to promote cell-cycle withdrawal. Therefore, the balance between FoxOs and FoxM1 controls IGF1 levels to promote cell-cycle withdrawal in neonatal cardiomyocytes.

Multiple regulatory pathways contribute to neonatal cardiomyocyte cell-cycle withdrawal and control cardiomyocyte cell-cycle activity after birth.29,30 Loss of the cell-cycle inhibitors p27 or p130, or alternatively forced expression of the cell-cycle activator cyclinA2, leads to delayed cardiomyocyte cell-cycle withdrawal after birth, similar to that observed with cardiomyocyte-specific loss of FoxOs in the current study.20,30 However, the mechanism by which cell-cycle withdrawal is delayed, but not prevented, in these models is not known. Increased expression of cyclinD2 or Myc can promote neonatal cardiomyocyte cell-cycle activity, but also leads to multinucleation of cardiomyocytes later in life.29 Additional pathways, including the Hippo/Yap pathway, have been implicated in cardiomyocyte cell-cycle inhibition, and overexpression of activated Yap leads to prolonged cell-cycle activity and cardiomyocyte hyperplasia after birth.31 FoxOs inhibit cell-cycle progression through a variety of mechanisms, including activation of p21 and p27, as well as inhibition of cyclinD1/2, gene expression.32 Although the regulatory hierarchies that control neonatal cell-cycle withdrawal are not fully defined, FoxOs appear to be critical mediators of this process through inhibition of cell-cycle activators in addition to activation of cell-cycle inhibitors.

There is increasing evidence that the balance of FoxOs and FoxM1 regulates cell-cycle activity in a variety of developmental and disease processes. In cancer, FoxM1 promotes cell proliferation, whereas FoxOs can act as tumor suppressors.6,32 In developing cardiomyocytes, loss of FoxM1 in mice results in myocardial hypoplasia and fetal death, whereas FoxO inhibition leads to increased cardiomyocyte proliferation at E17.5.5,8 FoxOs and FoxM1 have opposing effects on cell-cycle inhibitors in that FoxM1 promotes the protein degradation of p21 and p27, whereas FoxOs are transcriptional activators of p21 and p27 gene expression.6,32 In breast cancer cells, FoxO3 represses FoxM1 expression, whereas VEGF and estrogen receptor α genes are transcriptionally inhibited by FoxO3 and are activated by FoxM1.14,33 Here, we provide evidence for a similar antagonistic relationship in neonatal cardiomyocytes in which FoxOs repress FoxM1 gene expression in addition to competing for binding of IGF1 gene regulatory sequences.

IGF1 gene expression is dynamically regulated in heart development and disease. Before birth, IGF1 activates phosphoinositide 3-kinase/AKT signaling, leading to phosphorylation, nuclear exclusion, and inactivation of FoxOs in cardiomyocytes.8 FoxM1 activation of IGF1 gene expression in prenatal cardiomyocytes has not been examined directly, but it is a possible mechanism by which FoxM1 promotes cell proliferation in the developing heart.5 During the neonatal period, IGF1 gene expression is decreased, potentially because of decreased expression of FoxM1, supporting a feedback mechanism of IGF1 downregulation and FoxO activation. Forced expression of IGF1 can prolong neonatal cell cycling,30 but it is not known whether this is mediated through FoxO inactivation. Additional factors also contribute to IGF1 gene regulation. A nuclear factor of activated T-cell-responsive regulatory element was previously identified in IGF1 promoter sequences,19 and Yap, a transcriptional regulator of the Hippo pathway, also has been implicated as a positive regulator of IGF1 signaling in cardiomyocytes.31 In the adult heart, there are conflicting reports regarding whether IGF1 signaling is damaging or cardioprotective during injury or oxidative stress, and it is likely that secreted and locally activated isoforms have distinct functions.18 IGF1 also has been implicated in cardiac myocyte regeneration and repair by acting on cardiac stem cells. However, the potential interactions of FoxOs and IGF1 signaling in cardiac disease and repair have not yet been explored.

AMPK activation is an indicator of energy depletion that induces oxidative metabolism in addition to regulating mitochondrial biogenesis, autophagy, cell growth, and proliferation.34 In the heart, AMPK is activated during the neonatal period coincident with energy depletion and induction of oxidative metabolism. In addition, AMPK inhibition in neonatal cardiomyocytes is sufficient to promote cell-cycle activation, consistent with a role for AMPK activation in neonatal cardiomyocyte cell-cycle withdrawal. Here, we show that AMPK inhibition leads to increased activation of AKT with increased phosphorylation of FoxO1 at Ser-256 and of FoxO3 at Ser-318/321 AKT target sites, concomitant with nuclear exclusion and inactivation. In contrast, AMPK directly phosphorylates FoxO3 at alternative sites, leading to increased transcriptional activity in response to nutrient deprivation,16 and AMPK activation causes cell-cycle arrest in a variety of cell types.35,36 AMPK activation of FoxOs also promotes oxidative metabolism, but this has not yet been demonstrated directly in neonatal cardiomyocytes. In the adult heart, AMPK37 and FoxOs11 have been implicated in cardioprotection under conditions of oxidative stress or cardiac injury, but intersecting regulatory mechanisms have not yet been defined. Interestingly, increased activity of AMPK and FoxOs, along with decreased IGF1, are associated with prolonged lifespan attributable to caloric restriction and also with cardiovascular aging.18 Thus, there is accumulating evidence that the AMPK/FoxO/IGF1 regulatory network has multiple roles in cardiac development and disease from birth to old age.

The neonatal heart undergoes multiple critical transitions, including cell-cycle withdrawal, metabolic substrate shifts, and loss of regenerative potential, apparent 1 week after birth. Here, we show that the AMPK activation of FoxOs promotes cell-cycle withdrawal and inhibits IGF1 gene expression in neonatal cardiomyocytes. It would be interesting to determine whether the activation status of this pathway affects the ability of the neonatal heart to regenerate or the loss of regenerative potential approximately 1 week after birth. The balance of AMPK and FoxO activation vs IGF1 signaling has multiple functions during cardiomyocyte development and disease processes that must be taken into account when considering therapeutic approaches to heart failure and enhancement of repair. In addition, AMPK activation by metaformin has been used to treat diabetes mellitus and metabolic syndrome, but it also has likely implications for cardiac disease.34 Likewise, IGF1 treatment has been proposed as a mechanism that promotes cardiac repair, but it also could lead to increased oxidative injury attributable to inhibition of AMPK and FoxOs.18 Thus, therapeutic approaches that affect AMPK/FoxO/IGF1 signaling pathways could have varied and complex effects on cardiac disease and repair processes.

Acknowledgments

The authors thank Michelle Sargent for cardiomyocyte isolation, Christina Alfieri and Jonathan Cheek for technical support, and Craig Bolte for assistance with the FoxM1 mutant mice.

Sources of Funding

This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute grant P01 HL069779 to K.E. Yutzey, aR01 HL84151 to V.V. Kalinichenko, and an American Heart Association–Great Rivers Affiliate Post doctoral Fellowship 11POST7210026 to A. Sengupta.

Disclosures

None.

Footnotes

  • In October 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.5 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.277442/-/DC1.

  • Non-standard Abbreviations and Acronyms

    Ad
    adenovirus
    AICAR
    AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside
    AMPK
    AMP-activated protein kinase
    b gal
    b galactosidae
    βMHC
    β-myosin heavy chain
    FoxM1
    forkhead box transcription factor M1
    FoxO1
    forkhead box transcription factor O1
    FoxO3
    forkhead box transcription factor O3
    IGF1
    insulin-like growth factor 1
    p21
    p21cip1
    p27
    p27 kip1
    pd
    postnatal day
    PHH3
    phospho-histone H3

  • Received July 11, 2011.
  • Revision received November 12, 2012.
  • Accepted November 14, 2012.

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Novelty and Significance

What Is Known?

  • After birth, cardiomyocytes withdraw from the cell cycle and the heart grows primarily by hypertrophy.

  • In the neonatal heart, growth factor signaling is reduced, metabolism shifts from glycolytic to primarily oxidative, and the myocardium has the ability to regenerate.

  • Forkhead box (Fox) transcription factor Os are activated under conditions of growth factor deprivation, and FoxO1 and FoxO3 are expressed in the developing and in the diseased heart.

What New Information Does This Article Contribute?

  • In vivo, combined loss of FoxO1 and FoxO3, specifically in cardiomyocytes, leads to delayed cell-cycle withdrawal and increased expression of insulin-like growth factor-1 (IGF1) and FoxM1, whereas loss of FoxM1 results in decreased neonatal cardiomyocyte cell proliferation, decreased expression of IGF1, and increased expression of cell-cycle inhibitors p21 and p27.

  • IGF1 gene expression is activated by FoxM1 in proliferating cardiomyocytes and is repressed by FoxOs during neonatal cell-cycle withdrawal in response to AMP-activated protein kinase activation status.

  • FoxM1 inactivation and FoxO activation, subject to metabolic regulation, together regulate neonatal cardiomyocyte cell-cycle withdrawal.

Here, we report that FoxO and FoxM1 transcription factors regulate cardiomyocyte proliferation and cell-cycle withdrawal in the neonatal period. Immediately after birth and before feeding, the mammalian heart is subjected to a period of starvation in which growth factor signaling is reduced and cardiomyocytes are under metabolic stress. At the same time, expression of FoxM1, which promotes cardiomyocyte proliferation, is downregulated and FoxOs are activated, concomitant with induction of cell-cycle inhibitors. Mice lacking FoxM1 in cardiomyocytes exhibit decreased cell proliferation after birth, whereas cardiomyocyte-specific loss of FoxO1 and FoxO3 delays neonatal cell-cycle withdrawal. In cultured neonatal cardiomyocytes, FoxO activity is subject to AMP-kinase activation, an indicator of metabolic stress. IGF1 can promote cardiac hypertrophy, cell-cycle reentry, and repair in adult hearts. Our results provide evidence that IGF1 is a direct downstream target of cardiac Fox transcription factors, which is negatively regulated by FoxOs and positively regulated by FoxM1, dependent on AMP-activated protein kinase activation status. Together, these data support a regulatory mechanism in which FoxO and FoxM1 transcription factors integrate metabolic status and cell-cycle withdrawal in neonatal cardiomyocytes.

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  • FoxO1 and FoxM1 Transcription Factors Have Antagonistic Functions in Neonatal Cardiomyocyte Cell-Cycle Withdrawal and IGF1 Gene RegulationNovelty and Significance
    Arunima Sengupta, Vladimir V. Kalinichenko and Katherine E. Yutzey
    Circulation Research. 2013;112:267-277, originally published January 17, 2013
    https://doi.org/10.1161/CIRCRESAHA.112.277442
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