Involvement Of Akt/FoxO1 Pathway In Mitosis Of Cardiomyocytes

Cardiac myocytes proliferate most rapidly during the hyperplastic phase of heartdevelopment, however, the level of cell cycle activity is drastically down regulatedafter birth. Further growth of the heart is achieved by hypertrophic growth of cardiacmyocytes. The mechanism that controls the switch from hyperplastic proliferation tohypertrophic growth in cardiac myocytes is unknown. Understanding this fundamentalmechanism of cardiac myocyte biology would be most beneficial for studies directedtowards myocardial regeneration.Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes.Therefore, the concept of stimulation myocyte mitotic divisions has dramaticimplications for cardiomyocyte regeneration and hence, cardiovascular disease. Cellproliferation is a complex process involving a large network of regulatory proteins.Although a number of molecules which play an important role in the progression of thecell cycle have been isolated and a growing body of information on mechanisms of thecell cycle regulation has been accumulated, little is known about the mechanism ofcardiomyocytes withdrawal from the cell cycle. Data obtained from transgenic modeland other experimental results suggest that myocytes are able to proliferate postnatallyin the presence of elevated insulin-like growth factor 1 (IGF-1) levels.Multiple extracellular signals are responsible for triggering cardiac growth,including mechanical stretch and autocrine, paracrine, and endocrine factors. Akt andphosphoinositide-dependent protein kinase (PDK)1 may play important roles inregulation of cardiomyocyte growth, and their expression may be tightly linked tocardiac development. These proteins are critical components of the phosphoinositide3-kinase (PI3K) signaling pathway. Akt, also known as protein kinase B, has emergedas a focal point for signal transduction pathways responsible for cell survival. Akt is initially activated at the cell membrane in response to stimulation by growth factorssuch as IGF-1. After activation, Akt phosphorylates multiple cytoplasmic substratesand translocates to the nucleus, where Akt is thought to regulate gene transcription. Incardiomyocytes, Akt prevents apoptosis from ischemia reperfusion, volume and/orpressure overload, hypoxia, hypoglycemia, or cardiotoxic drugs. PI-3K/Akt pathwayplays a role in the regulation of cell proliferation through the induction of cyclin D1 inhuman keratinocytes upon arsenite treatment. The PI 3-kinase/Akt pathway is known todirect cellular processes like differentiation and stress resistance through a tightregulation of the forkhead family of transcription factors (FoxO1/3a/4). The ontogenyof this important signaling pathway in the heart has not been fully characterized.In this study, we investigated the impact of insulin and wild type Aktoverexpression on the ability of H9c2(2-1) cells to proliferation, and we also tested theexpression of FoxO1 and the location of phosph-Akt in H9c2(2-1)cells after activationof Akt.Materials and methods1. AnimalsFemales of developmental Wistar rats were supplied by the Department ofLaboratory of Animals, China Medical University.2. Ascending aortic constriction.AAC was performed in 4-6-week-old female rats as described. Ligation of theaorta was performed by using a 7-0 polypropylene suture through a Thefelon tubearound the root of aortic artery to produce a discrete stenosis. After ligation, thepneumothorax was reduced before chest closure and extubation. Sham rats underwent acomparable operation in which the aortic was isolated and a band was twined aroundthe aorta but not ligated and subsequently was removed.3. EchocardiographyRat transthoracic echocardiography was performed in lightly anesthetized rats asdescribed by using echocardiography systems (Phillips) and a 14-MHz lineartransducer (15L8). Briefly, anesthesia was administered by 0.4% pentobarbital. Theheart was imaged in the 2D parasternal short-axis view, and an M-modeechocardiography of the midventricle was recorded at the level of papillary muscles. LV end-diastolic and-systolic diameter (LVEDD and LVESD, respectively,end-diastolic LV anterior wall thickness (LVAW), and LV posterior wall thickness(LVPW) were measured from images obtained by M-mode echocardiography.4. Double immunofluorescent stainingHearts were harvested and quick-frozen in liquid nitrogen, and were then stored at-70℃. Cryostat sections were cut at 4μm and mounted onto gelatine-coated coverslips.Cover The sections were fixed with 2-4% paraformaldehyde or acetone for 15 min atroom temperature. After rinsing with three changes of PBS, the specimens wereblocked in 5% BSA in PBS/Triton for 60 min. Then the specimens were incubated withmouse/rabbit/goat primary antibody at their appropriate dilutions overnight at 4℃.After rinsing, the specimens were incubated in FITC/Cy3-conjugated secondaryantibody for 1-2 hr at room temperature in dark. Finally, the sections were mountedwith glycerine.5. RNA isolation and RT-PCR.Total RNA was isolated using the TRIzol reagent (Invitrogen) as described by themanufacturer. For RT-PCR, TaKaRa RNA LA PCR^TM Kit (AMV)Ver.1.1 was used asdescribed by the manufacturer. Briefly, 1μg total RNA was reverse-transcribed intocDNA with 1 unit/ml AMV reverse transcriptase at 42℃for 50min. Primers sequencesfor rat FoxO1, Akt, Plk1, Wee1 and GAPDH are available upon request. The PCRswere carried out at 55-60℃with LA Taq Polymerase.6. Western blotting analysisExpression of the p-Akt and plk was analysed after lysing heart tissue or culturedcells in lysis buffer. The extracts were first pre-cleared by centrifugation at 10000g in acentrifuge for 4 min at 4℃. The extracts were analysed by SDS\PAGE (12% gel) andtransferred to nitrocellulose membranes (Immobilon, Millipore, Bedford, MA, U.S.A.)which were blocked for 1 hr at RT in TBS with 5% BSA\0.2% Tween 20. The plk wasdetected with the plk-N19 polyclonal antibody (Santa Cruz Biotechnology Inc, U.S.A.).Phosphorylation of Akt was detected with Akt1/2/3 polyclonal antibody (Santa CruzBiotechnology Inc, U.S.A.). Binding of the primary antibodies was assessed by theappropriate secondary antibody and ECL( Santa Cruz Biotechnology Inc, U.S.A.)substrate. 7. Cell Culture and transfectionH9c2(2-1) cardiomyocytes that originally developed from rat DBIX heart (ATCC)were purchased from CAS Cell Bank. These cells were cultured and used for the genetransfection experiments. The cells were grown to 70% confluence in DMEMsupplemented with 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, and 2mmol/L L-glutamine (all from Gibco), 5% CO₂ at 37℃. The pCIS2-Akt-WT plasmid(gift from Prof. Michael J. Quon at National Institutes of Health) were transfected intoH9c2 cells using KeyGene Transâ…¢transfection reagent according to manufacturers.8. Immunoprecipitation200μl H9c2 cell lysate were incubated with H3P monoclonal antibody with gentlerocking overnight at 4℃. Then either protein A or G agarose beads were added, andincubated with gentle rocking for 1-3 hrs at 4℃.After heated to 95-100℃for 2-5 min,the sample was loaded on 12%SDS-PAGE, and then analyzed by Western blotting.9. Statistical analysisResults for continuous Variable are expressed as means±SE. Comparisonsbetween groups were analyzed by two-tailed Student's t or x² test with SPSS13.0software, as appropriate. P＜0.01 was deemed significant.Results1. Cardiomyocytes proliferation in neonatal hearts of ratThe double immunofluorescence staining revealed that the Mitotic Index ofcardiomyocytes in the 0-day-old rat heart was approximately 0.905±0.087%, and wasdeclined 2.4-fold (0.372±0.094%) in the 14-day-old rat heart.2. The mRNA expression of Akt and plk were down-regulated with postnataldevelopment of rat heart, however the mRNA expression of FoxO1 was up-regulated.The expression of plk protein and the phosphorylation level of Akt protein were alldown-regulated with postnatal development of rat heart.3. The mRNA expression of FoxO1 was down-regulated in the pressure-overloadhypertrophy heart of rat compared with sham, and the mRNA expression of Wee1 andAkt was up-regulated or unchanged respectively, however the phosphorylation level ofAkt protein was obviously up-regulated.4. Either transfection of pCIS2-Akt-WT plasmid or stimulation of insulin on H9c2 cells increased the level of phosphorylation of Akt.5. Actived Akt promoted mitosis of H9c2 cells, and was located in the nucleus ofH9c2 cells.The mRNA expression of FoxO1 was down-regulated by nucleus-locatedactived Akt in H9c2 cells.DiscussionMammalian cardiomyocytes lose their ability to proliferate and exit the cell cyclein vivo during the first 2 weeks of age and thus the mammalian heart is incapable ofregeneration after ischemic or other forms of injury. In our study, the presence ofdividing cardiomyocytes in 0-day-old, 14-day-old and adult hearts was establishedusing H3P and a-SMA.H3P was used to reveal the presence of mitotic cells anda-SMA the presence of cardiomyocytes. The results imply that the majority ofcardiomyocytes in the rat heart have stopped dividing by day 14.The molecular mechanisms responsible for the arrest of cardiomyocyteproliferation during the postnatal period are largely unknown. Earlier studies haveshown that the activity of cardiomyocyte DNA polymerase decreases during the firstpostnatal weeks. Recently, cyclin-dependent kinase (cdk) activities in cardiomyocyteswere also reported to decline during the early neonatal period. In particular, thephosphotransferase activity of cdk4 governing the G1 phase of cell cycle is detectableuntil only day 1, whereas the cdk2 activity, regulating the G1-S-phase transition,decreases markedly after day 2. We found that the mRNA expression of Akt and plkwere down-regulated during postnatal development of rat heart, however the mRNAexpression of FoxO1 was up-regulated. So the diferential expression of Akt and FoxO1was related to the cell cycle quit of cardiomyocytes during the early neonatal period ofrat heart. We also established that the expression of plk protein and the phosphorylationlevel of Akt protein were all down-regulated with postnatal development of rat heart.The G2/M boundary and the ability to undergo cytokinesis have long been recognisedas critical checkpoints to the proliferation of mammalian cardiomyocytes. Thuscardiomyocytes in several mammalian species become polyploid and/or multinucleateafter birth. So we postulate that the down-regulation of plk was involved in the arrest ofcardiomyocyte division and the formation of binucleate cell.The PI 3-kinase/Akt pathway is known to direct cellular processes likedifferentiation and stress resistance through the modulation of FoxO transcription factors activity. In fact, FoxOs activate or repress transcriptional programs involved inmetabolic control, cell cycle, oxidative stress sensing, and apoptosis, depending on theintegration of external signals received by growth factors like insulin, IGF-I, or VEGF.However, the intimal mechanisms regulating FoxO activity are still unclear. Weestablished that ether transfection of pCIS2-Akt-WT plasmid or stimulation of insulinon H9c2 cells increased the level of phosphorylation of Akt, and that actived andnucleus-located Akt promoted mitosis of H9c2 cells through down-regulating theexpress of FoxO1.Conclusion1. The diferential expression of Akt and FoxO1 was related to the cell cycle quitof cardiomyocytes during postnatal development of rat heart. The down-regulation ofplk was involved in the arrest of cardiomyocyte division and the formation ofbinucleate cell.2. The up-regulation of phosphorylation level of Akt protein followed bydown-regulation of FoxO1 possibly participated in the proliferation regulation ofcardiomyocytes during pressure-overload hypertrophy.3. Actived and nucleus-located Akt promoted mitosis of H9c2 cells throughdown-regulating the express of FoxO1.