Pregnancy-induced Physiological Hypertrophic Preconditioning Attenuates Pathological Myocardial Hypertrophy by Mnemonic Activation of FoxO3a/GSK3β Pathway

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Pregnancy-induced Physiological Hypertrophic Preconditioning Attenuates Pathological Myocardial Hypertrophy by Mnemonic Activation of FoxO3a/GSK3β Pathway

https://doi.org/10.21203/rs.3.rs-2009966/v1

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Previous studies show a woman’s age at final pregnancy is correlated with post-reproductive longevity and nulliparity is associated with higher risk of incident heart failure, and we previously reported a cardioprotective phenomenon termed myocardial hypertrophic preconditioning, but it is unknown whether pregnancy-induced physiological hypertrophic preconditioning can also protect the heart against subsequent pathological hypertrophic stress. Here, we found that compared with nulliparous mice, pathological cardiac hypertrophy induced by Ang Ⅱ infusion or TAC was significantly attenuated and heart failure induced by TAC was markedly improved in mice with pregnant preconditioning. Activation of FoxO3a was significantly enhanced in the hearts of postpartum mice. FoxO3a inhibited myocardial hypertrophy by suppressing signaling pathway of phosphorylated glycogen synthase kinase-3β (p-GSK3β)/β-catenin/Cyclin D1. Silencing or overexpression of FoxO3a attenuated or enhanced the anti-hypertrophic effect of pregnant preconditioning in mice with pathological stimulation. Our findings demonstrate that pregnancy-induced myocardial hypertrophic preconditioning confers resistance to subsequent hypertrophic stress and slows progression to heart failure through activation of FoxO3a/GSK3β pathway.

Myocardial hypertrophy

Heart failure

Pregnancy Hypertrophic preconditioning

FoxO3a

Pathological cardiac hypertrophy has detrimental effects on cardiac function and makes a major contribution to the eventual onset of heart failure¹. We previously reported a phenomenon termed hypertrophic myocardial preconditioning, in which short-term imposition of pathological hypertrophic stress on the heart has a protective effect against subsequent hypertrophic stress and slows progression to heart failure², and these results were verified by other researchers^3,4,5,6. Recently, we found that myocardial hypertrophy preconditioning induced by exercise exerts a cardioprotective effect on pathological stress via antihypertrophic memory^7,8, indicating a cardioprotection can be induced by preconditioning of physiological myocardial hypertrophy. However, there has been little investigation into the effects of pregnancy related physiological myocardial hypertrophy on subsequent resistance to pathological hypertrophic stress.

Significant physiological changes of the cardiovascular system occur during pregnancy, including cardiac hypertrophy, sustained activation of the renin–angiotensin–aldosterone system, and a marked increase in plasma volume, indicating that the heart is under stress⁹. These physiological responses to increased demand are beneficial for both the mother and fetus. There is evidence that short-term, high-intensity stress on the heart can induce cardioprotective preconditioning², but it is unknown whether less intense physiological stress acting over a longer period, such as pregnancy, can also protect the heart against subsequent pathological hypertrophic stress.

A few studies have provided evidence for a cardioprotective effect of pregnancy. Kara et al. reported that fetal cells enter the maternal circulation and migrate to sites of injury in the maternal myocardium to assist in repair¹⁰. It was also reported that peripartum cardiomyopathy has the highest recovery rate amongst all types of heart failure¹¹, and pregnancy-induced hypertensive disorders are associated with a better outcome in patients with peripartum cardiomyopathy¹². Interestingly, it was reported that the age of women at final pregnancy is correlated with their post-reproductive longevity¹³, and nulliparity is associated with higher risk of incident heart failure with preserved ejection fraction¹⁴, which suggest that senescence is slowed after pregnancy. These reports led us to postulate that pregnancy induces cardioprotective memory in the mother, possibly by activation of anti-hypertrophic factors, and increases resistance to subsequent cardiac stress, although it is unclear how long such an effect persists.

Forkhead box class O3a (FoxO3a) is an important member of the FOXO subfamily of forkhead transcription factors. Extensive researches have shown that the increased activation of FoxO3a involves with anti-hypertrophic effect in heart^15,16, and is strongly associated with human longevity^17,18. In addition, it has been demonstrated activation of FoxO3a in the hearts of postpartum mice¹⁹, but it is unclear whether enhanced cardiac activation of FoxO3a during the postpartum period confers resistance to pathological hypertrophic stress.

Based on the above points, this study was performed to test the hypothesis that pregnancy-induced myocardial hypertrophy regression engenders anti-hypertrophic memory and cardiac resistance to subsequent pathological hypertrophic stress, as well as slows progression to heart failure. We also explored the involvement of FoxO3a in the underlying mechanisms.

All procedures were performed in accordance with our Institutional Guidelines for Animal Research and this investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (National Institutes of Health Publication, 8th Edition, 2011). This study was approved by ethics review board of Southern Medical University. (Experimental protocols were shown in Fig. S1A-D, sequences of primers for real-time PCR were shown in Table S1).

Model of physiological myocardial hypertrophy due to pregnancy

C57BL/6 female mice (8–10 weeks old, weighing 20–25 g) were provided by the Animal Center of Southern Medical University. Mice were randomly assigned to two groups: pregnant group and control group (virgin mice). And the successful pregnancy was identified by observing vaginal plug at the first day after mating. The related data were obtained at various time points. Briefly, blood pressure, plasma angiotensin Ⅱ (Ang Ⅱ), heart rate, body weight and heart weight were obtained in pregnant group at the following time points: baseline (1 day before mating), early pregnancy (5-7 days after pregnancy), middle-late pregnancy (14-18 days after pregnancy) and post 3w (3 weeks after delivery).

Noninvasive blood pressure measurements

Blood pressure and heart rate were measured in conscious mice using a noninvasive tail-cuff system (Softron BP-2010; Kyoto, Japan). Before the measurement, animals were placed in a temperature-controlled holder (kept at 37 °C) for 10 minutes to obtain enough blood flow, and recordings were performed in steady-state conditions. Blood pressure and heart rate values were averaged from three continuous measurements.

Induction of myocardial hypertrophy by angiotensin Ⅱ Infusion

After anesthesia was induced in C57BL/6 female mice (14-16 weeks, 20-25 g) with a 1% inhaled isoflurane, an osmotic minipump (model 2004; Alzet) was implanted subcutaneously to deliver angiotensin Ⅱ (Ang Ⅱ) at 2 mg/kg/day or the vehicle (200 µl of normal saline). Tail blood pressure was measured in a time course. Four weeks after implantation, the mice were sacrificed by an overdose of pentobarbital sodium (150 mg/kg, ip). Experimental protocol is shown in Figure S 1A. Mice were divided into 4 treatment groups. (1) The sham control (C-sham, nulliparous mice) group received no intervention for 6 weeks and then saline was infused for 4 weeks, (2) The Ang Ⅱ control (C-Ang Ⅱ, nulliparous mice) group received no intervention for 6 weeks and then AngⅡ was infused for 4 weeks, (3) The sham pregnant preconditioning (P-sham) group underwent pregnancy for 3 weeks and then received saline infusion for 4 weeks starting from 3 weeks after delivery, (4) The Ang Ⅱ pregnant preconditioning (P-Ang Ⅱ) group underwent pregnancy for 3 weeks and then received Ang Ⅱ infusion for 4 weeks starting from 3 weeks after delivery.

Transverse aortic constriction

Transverse aortic constriction (TAC) was performed as described previously². Briefly, mice were intraperitoneal anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg). After left-sided thoracotomy in the 2nd intercostal space, the transverse aorta was ligated with a 7-0 silk by using a blunt 27G needle, and the needle was then withdrawn. Sham-operated animals underwent the same procedure, except that the ligature was tied loosely. The experiment protocol is shown in Figure S 1B. The mice were divided into the following 4 group. (1) The sham control (C-sham, nulliparous mice) group received no intervention for 6 weeks and then sham for 4 weeks. (2) The TAC control (C-TAC, nulliparous mice) group received no intervention for 6 weeks and then TAC for 4 weeks. (3) The pregnant preconditioning sham (P-sham) group underwent pregnancy for 3 weeks and sham for 4 weeks starting from 3 weeks after delivery. (4) The pregnant preconditioning TAC (P-TAC) group underwent pregnancy for 3 weeks and TAC for 4 weeks starting from 3 weeks after delivery. Before sacrifice, left ventricular hemodynamics were assessed by using a Millar catheter and Blood Pressure Module software, as described elsewhere².

Measurement of cell surface area

To evaluate myocyte surface area, heart sections were incubated with wheat germ agglutinin (WGA; W21405, Invitrogen/Molecular Probes, Carlsbad, CA, USA) according to standard procedures, and then sections were viewed under a confocal laser scanning microscopy and the surface area were measured by using NIH Image J software. H&E staining was carried out according to the manufacturer’s instructions.

Echocardiography

Mice were anesthetized with 1% inhaled isoflurane. Echocardiography was performed using a Vevo 2100 system with a 30 MHz transducer (Fujifilm VisualSonics, Ontario, Canada). Chest hairs were epilated with a topical depilatory agent. Limb leads were employed for electrocardiogram gating. Two-dimensional images were obtained in parasternal short axis projection of the left ventricle (LV) with guided M-mode recordings at the level of papillary muscles. LV cavity size (LV end-diastolic diameter LVEDD, LV end-systolic diameter LVESD) and wall thickness [LV diastolic posterior wall thickness (LVPWd), LV posterior wall systolic thickness (LVPWs)] were measured in M-mode tracings. LV fractional shortening was calculated from the M-mode measurements: LVFS = (LVEDd – LVEDs)/LVEDd*100.

ELISA for Determination of Ang Ⅱ

Plasma Ang Ⅱ levels were measured with the Enzyme Immunoassay kit according to the assay procedure provided by the manufacturer (Phoenix Pharmaceuticals, Catalog # EK-002-12; Massachusetts, USA).

Construction of recombinant adeno-associated virus carrying FoxO3a or sh-FoxO3a and administration to mice

pAAV2/9-CMV-ZsGreen adeno-associated virus (AAV) vectors carrying FoxO3a (NM_019740, 2019 bp) or short hairpin FoxO3a (sh-FoxO3a) and a negative control vector (scramble) were purchased from Vigene Company (Shandong, China). For in vivo infection, either AAV-FoxO3a or AAV-sh-FoxO3a or AAV-scramble (3.3× 10¹¹ pfu/ml) was administered to 6-8-week-old female mice by direct injection into the LV myocardium at 3 sites (10 μl/site) using a syringe with a 32-gauge needle. Two weeks later, the animals were mated. Three weeks after delivery, the mice were subjected to infusion of Ang Ⅱ or TAC or saline (sham) for 4 weeks. The experimental protocols are shown in Fig. S1C and D.

Western blot

Total protein was obtained from heart homogenates. The protein was separated by 8-15% SDS–polyacrylamide gels, and then was transferred to a polyvinyl difluoride membrane. The membrane was blocked with 5% albumin from bovine serum at room temperature for 2 hours, and then incubated overnight at 4°C with the following primary antibodies: anti-GAPDH (#2118L, CST, USA), anti-GAPDH antibody (ARG10112; Arigo, Taiwan, China), anti-β-catenin (#610154; BD Transduction Laboratories, San Jose, CA, USA), anti-FoxO3a (#2497S, CST, USA), anti-p-FoxO3a (Ser253, #9466S, CST, USA), anti-p-GSK3β (#9331, CST, USA), anti-p-GSK3β (Ser9, #9323S, CST, USA), anti-GSK3β (#9315S, CST, USA), anti-GSK3β (#sc-7291, Santa Cruz, USA), anti-Cyclin D1 (ab134175, Abcam, Cambridge, UK), and anti-OTR (ab181077, Abcam, Cambridge, UK). The blots were detected using a Super Signal ECL kit (Invitrogen, Carlsbad, CA, USA) in a Western blotting detection system (Kodak Digital Science, Rochester, NY, USA) and quantified by densitometry using the Image J Analysis software (National Institutes of Health, Bethesda, MD, USA).

Polymerase chain reaction (PCR)

Total RNA was isolated from murine hearts using a total RNA isolation system (Omega, USA). Quantitative real time PCR (qPCR) reactions were performed using the LightCycler® 480 system (Basel City, Swiss). Primer sequences for Nppa, Myh7, SERCA, FoxO3a, OTR and GAPDH are available in Table S1. Gene expression was normalized to GAPDH and levels of the genes were quantified using the delta-delta-CT method.

Statistical analysis

Results are reported as the mean ± SEM unless otherwise noted. Unpaired Student’s t-test and Mann–Whitney U-test were used to compare two groups. Multiple comparisons were carried out by one-way analysis of variance combined with Dunnett's multiple comparisons test (SPSS 16.0). Kaplan-Meier analysis was performed for comparison of overall survival. In all analyses, P<0.05 was considered statistically significant.

Pregnant preconditioning attenuates pathological myocardial hypertrophy induced by angiotensin-Ⅱ (Ang Ⅱ).

We firstly investigated the characteristics of cardiac hypertrophy in pregnant mice. Consistent with previous reports, we found that tail blood pressure decreased slightly in the middle to late stages of pregnancy and returned to baseline by 3 weeks after delivery (Fig. S2A). While plasma Ang II concentrations were increased at middle-late pregnancy stage and recovered to baseline (control) at 3 weeks after delivery (Fig. S2B). Heart rate, body weight (BW), heart weight (HW), HW/tibia length (HW/TL) ratio, and cardiomyocyte surface area were all increased significantly in pregnant mice, while there were no significant changes in the expression of fetal genes Nppa (natriuretic peptide type A) and Myh7 (myosin heavy chain 7) or the lung weight/BW ratio (LW/BW) (Fig. S2C-J).

We also investigated the time window for pregnant hypertrophic preconditioning. When transverse aortic construction (TAC) was performed in postpartum 1 week and mice were observed for 8 weeks, there were no significant changes in HW/TL ratio and cardiomyocyte surface area of mice at 1 week after TAC, and the cumulative survival rate of mice for 8 weeks of TAC was significantly lower in the postpartum group than in the control group (Fig. S3A-D). These findings suggest that peripartum pathological hypertrophic stress is harmful. Considering pregnancy-induced cardiac hypertrophy regressed completely to normal by 3 weeks after delivery, and pregnancy associated cardiovascular complications such as peripartum cardiomyopathy or preeclampsia or myocardial ischemia/reperfusion injury are prone to emerge during the peripartum period²⁰^,²¹^,⁹, we selected three weeks postpartum as the time for application of pathological hypertrophic stress (Ang Ⅱ infusion and pressure overload).

In mice with myocardial hypertrophy induced by continuous infusion of Ang Ⅱ for 4 weeks, tail blood pressure was significantly increased (Fig. S4A-B). The HW/BW ratio, cardiomyocyte cross-sectional area and the expression of fetal genes (Nppa and Myh7) were significantly smaller in the preconditioning Ang Ⅱ group than in the control Ang Ⅱ group (Fig. 1A-D). Echocardiography showed a smaller increase of diastolic left ventricular posterior wall thickness (LVPWd) in the preconditioning Ang Ⅱ group than in control Ang Ⅱ group (Fig. S5A-B), but there were no significant differences on left ventricular end-diastolic diameter (LVEDd), end-systolic diameter (LVESd), LV fractional shortening (LVFS) and the LW/BW ratio between the two groups (Fig. S5C-E).

Pregnant preconditioning attenuates cardiac remodeling and slows progression of cardiac dysfunction induced by pressure overload.

Because Ang Ⅱ infusion was insufficient to induce heart failure in mice, we employed TAC to induce both cardiac hypertrophy and heart failure for further assessment of the cardioprotective effects of pregnant preconditioning. Four weeks after TAC, the HW/BW ratio and the cardiomyocyte cross-sectional area were significantly smaller and the expression of Nppa and Myh7 genes were lower in the preconditioning TAC group than in the control TAC group (Fig. 1E-H), and the preconditioning TAC group also showed significantly smaller LVPWd, LVEDd and LVESd, and larger LVFS (Fig. 2A-D). In addition, LV end-diastolic pressure (LVEDP) was lower in the preconditioning TAC group, while LV dp/dt max (maximum rate of rising in left ventricular pressure) and LV dp/dt min (maximum rate of descending in left ventricular pressure) were higher (Fig. 2E-H). TAC induced congestive heart failure with an increase of the LW/BW. Four weeks after TAC, the LW/BW ratio was markedly smaller in the preconditioning TAC group than in the control TAC group (Fig. 2I). These results demonstrated that myocardial hypertrophic preconditioning due to pregnancy could relieve cardiac remodeling and dysfunction induced by pathological hypertrophy.

FoxO3a and its anti-growth signaling were activated in the heart of mice with pregnant preconditioning.

According to previous reports, FoxO3a and oxytocin receptor (OTR) are involved in myocardial hypertrophy, therefore we further confirm their expressions in pregnancy-induced physiological hypertrophic preconditioning. We noted that myocardial gene expressions of the FoxO3a and OTR were significantly higher at 3 week postpartum mice than in control group, and their protein expressions were increased in heart of 1 week postpartum mice (Fig. S6A-B, Fig. 3A). Furthermore, we found that phosphorylation of FoxO3a was decreased in a time-dependent manner from baseline to pregnancy at 18d and 3 weeks after delivery (Fig. 3B), suggesting pregnant preconditioning increases the activity of FoxO3a. It is known that glycogen synthase kinase-3β (GSK3β) related signaling molecules regulate both pathological and physiological cardiac growth. Here we investigated whether pregnant hypertrophic preconditioning is associated with those signaling molecules. Western blotting demonstrated that pressure overload in the nulliparous mice for 4 weeks significantly resulted in increased myocardial expressions of p-FoxO3a, p-GSK3, β-catenin, and Cyclin D1, while these increases were prevented by pregnant hypertrophic preconditioning (Fig. 3C-E).

Silencing or overexpression of FoxO3a affects growth signaling.

At 2 weeks after direct intramyocardial injection of (adenovirus-associated virus) AAV-shFoxO3a (short hairpin of FoxO3a) or AAV-FoxO3a (overexpression of FoxO3a) in female mice, we found that silencing of FoxO3a led to an increased myocardial expression of p-GSK3, β-catenin, and Cyclin D1 compared with the scramble group (Fig. 4A-C). In contrast, overexpression of FoxO3a was associated with down-regulation of p-GSK3, β-catenin, and Cyclin D1 (Fig. 4D-F).

Silencing or overexpression of Foxo3a influences cardioprotection by pregnant preconditioning.

In postpartum mice subjected to TAC for 4 weeks, FoxO3a knockdown in heart increased the HW/BW ratio, cardiomyocyte cross-sectional area and fetal genes (Fig. 5A-D). Correspondingly, echocardiographic measurements revealed increases in LVPWd, LVESd and LVEDd, and a reduction in LVFS in FoxO3a knockdown mice (Fig. 5E-H). LVEDP were increased in FoxO3a knockdown mice, while LV dp/dt max and LV dp/dt min were reduced (Fig. 5J-K). In addition, there is an increase in LW/BW ratio in FoxO3a knockdown mice (Fig. 5L). In contrast, overexpression of FoxO3a attenuated TAC-induced increases of the HW/BW ratio, cardiomyocyte cross-sectional area, fetal genes of Nppa and Myh7, LVPWd, LVESd, LVEDd, LVEDP and LW/BW ratio, and TAC-induced reduction of LVFS, LV dp/dt max and LV dp/dt min (Fig. 5A-L).

Effects of silencing or overexpression of FoxO3a on growth signaling in the hearts of female mice subjected to TAC.

Western blotting demonstrated that silencing of FoxO3a in heart of mice subjected to TAC led to increased expression of p-FoxO3a, p-GSK3, β-catenin and Cyclin D1, and reduced expression of FoxO3a compared with the scramble + TAC group (Fig. 6A-C). In contrast, overexpression of FoxO3a was associated with a reduction of p-FoxO3a, p-GSK3, β-catenin and Cyclin D1, and an increase of FoxO3a in the hearts of female mice subjected to TAC (Fig. 6A-C).

This study provided first experimental evidence that myocardial hypertrophic preconditioning occurs during pregnancy. We demonstrated that hypertrophic preconditioning induced by pregnancy led to an increase of cardiac resistance to subsequent pathological hypertrophic stress and delayed progression from hypertrophy to heart failure, indicating the existence of pregnant hypertrophic preconditioning as a physiological phenomenon. We also found that activation of FoxO3a was significantly enhanced in the hearts of postpartum mice and silencing or overexpression of FoxO3a attenuated or enhanced the anti-hypertrophic effect of pregnant preconditioning in mice treated with Ang Ⅱ infusion or TAC, which may partly explain the cardioprotective effect of pregnant hypertrophic preconditioning. Furthermore, we demonstrated that activation of FoxO3a contributed to the anti-remodeling effect of hypertrophic preconditioning by suppressing signaling pathways of GSK3β/β-catenin/Cyclin D1 involved in cell growth.

A cardioprotective effect of pregnancy has already been reported in the setting of cardiovascular disease, but not in relation to myocardial hypertrophy. During pregnancy, fetal cells readily entered the maternal circulation and colonize various maternal organs for decades as microchimeras, with beneficial effects for the mother²². Kara and colleagues reported that fetal cells migrated to the maternal myocardium and are recruited to sites of injury to assist in repair, which may help to explain why patients with peripartum cardiomyopathy have a high rate of recovery from heart failure¹⁰. Persistence of a lower mean arterial pressure and reduced arterial stiffness following pregnancy may also have a favorable effect on maternal cardiovascular remodeling²³. In addition, it is well known that the incidence of coronary heart disease is lower in premenopausal women than in men of the same age, a difference which cannot be completely explained by the influence of female hormones. Interestingly, longevity is associated with fertility in women ^13,24, and the maternal age at final pregnancy shows a positive correlation with post-reproductive survival²⁵, with late first and last pregnancies being protective against all-cause mortality²⁶. Besides, shorter total reproductive duration and nulliparity was associated with higher risk for incident heart failure¹⁴. These reports have suggested the possibility of pregnant preconditioning, but it was unknown whether this phenomenon had an anti-hypertrophic effect until we provided experimental evidence in the present study.

Unloading of mechanical stress leads to regression of hypertrophy and functional recovery. Thus, regression of myocardial hypertrophy occurs in patients with aortic stenosis following aortic valve replacement, but regression is faster in women than men, partly because they are more likely to show adaptive cardiac remodeling with less fibrosis before surgery²⁷. Regression of cardiac remodeling is associated with a distinct, phase-dependent gene expression profile that inhibits hypertrophic and triggers atrophic signaling pathways²⁸. Maternal blood volume decreases rapidly after delivery, which represents physiological postpartum unloading.

The increased expression of FoxO3a is associated with anti-hypertrophy effect in the heart¹⁶, and a dominant-negative FoxO3a mutant was reported to inhibit endogenous FoxO3a activity and suppress the anti-hypertrophic effect of Sirtuin 3²⁹. Recent evidence suggests that activation of FoxO3a triggered by methyltransferases and unloading of mechanical stress is involved in muscle atrophy^28,30, and genetic polymorphisms of FoxO3a is associated with longevity in humans^17,18.

GSK3β was implicated in the growth of the heart and can block hypertrophic response by antagonizing the actions of calcineurin. In histone deacetylase-2 transgenic mice subjected to hypertrophic stress, severe cardiac hypertrophy was associated with inactivation of GSK3β, while activation of GSK3β attenuates hypertrophy³¹. It was also reported that deletion of GSK3β in cardiac fibroblasts leads to cardiac fibrosis and contractile dysfunction after myocardial infarction³². Moreover, intramyocardial injection of GSK3β-overexpressing stem cells improved mortality and attenuated myocardial dysfunction after myocardial infarction in mice³³. In contrast to the anti-hypertrophic role of GSK3β, β-catenin is believed to be a pro-hypertrophic factor. Stimulation of the Wnt/Frizzled pathway activates disheveled protein, which subsequently inhibits GSK3β, while ablation of the disheveled-1 gene attenuates pressure overload-induced cardiac hypertrophy and reduces β-catenin expression³⁴. It was reported that β-catenin gene transfer increased hypertrophic stress-induced growth³⁵, and we have also previously demonstrated that β-catenin promotes cardiac fibrosis³⁶. Additionally, Wnt signaling modulates activation of GSK3β, while our previous findings and evidence from other investigators indicate that the GSK3β/Cyclin D1 signaling pathway is closely associated with cardiovascular disease^37,35. Thus, it has generally been recognized that both FoxO3a and the Wnt/GSK3β/β-catenin/Cyclin D1 pathway are involved in myocardial hypertrophy, but there have been no reports of any association between them. In this study, we used overexpression and silencing of myocardial FoxO3a to reveal that FoxO3a promoted the activation of GSK3β while reduced the expression of β-catenin and Cyclin D1, and consequently was involved in the cardioprotective effect of pregnant preconditioning.

In addition to FoxO3a, other mechanisms may contribute to the cardioprotective effect of pregnant myocardial hypertrophy. For example, heart rejuvenating in circulation was reported to reverse age-related cardiac hypertrophy³⁸. Several experimental studies have demonstrated that pregnancy has a rejuvenating effect on various organs in female mice^39,10. Substantial researches are needed in the future to clarify the mechanisms of pregnant myocardial hypertrophy.

In summary, myocardial hypertrophic preconditioning induced by pregnancy confers resistance to subsequent hypertrophic stress and slows progression to heart failure through activation of FoxO3a and inhibition of p-GSK3β/β-catenin/Cyclin D1 pathway.

Acknowledgement

This work was supported by grants from the National Natural Science Foundation of China (81770271 to Y.L.), the Joint Funds of the National Natural Science Foundation of China (U1908205 to Y.L.), the National Natural Science Foundation of China (82170278 to Y.L.), and the Municipal Project of Research and Utilization of Healthcare Key Technology in Guangzhou (202206010199 to Y.L.)., the Scientific Research Project of Gannan Medical University (ZD201825 to J.X.).

conflict of Interest

The authors declare that they have no conflict of interest.

author contributions

J.X. and C.Z. performed experimental design and executed most of the experiments, data analyses, and wrote the manuscript; M.S., W.L., and H.L. contributed to the technical, and material support.; L.Y. contributed to the concept design, data interpretation, writing and revising the manuscript. All authors reviewed the results and approved the final version of the manuscript.

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Pregnancy-induced Physiological Hypertrophic Preconditioning Attenuates Pathological Myocardial Hypertrophy by Mnemonic Activation of FoxO3a/GSK3β Pathway

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