C/EBPβ Controls Exercise-Induced Cardiac Growth and Protects against Pathological Cardiac Remodeling-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文

DOI: 10.1016/j.cell.2010.11.036

Pontus BoströmNina MannJun WuPablo QuinteroEva PlovieDaniela PanákováRana K. GuptaChunyang XiaoCalum A. MacRaeAnthony RosenzweigBruce M. Spiegelman

Pontus BoströmNina MannJun Wu ...+7 Bruce M. Spiegelman

Cell

Dec 2010

344被引用

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摘要原文

The heart has the ability to grow in size in response to exercise, but little is known about the transcriptional mechanisms underlying physiological hypertrophy. Adult cardiomyocytes have also recently been proven to hold the potential for proliferation, a process that could be of great importance for regenerative medicine. Using a unique RT-PCR-based screen against all transcriptional components, we showed that C/EBPβ was downregulated with exercise, whereas the expression of CITED4 was increased. Reduction of C/EBPβ in vitro and in vivo resulted in a phenocopy of endurance exercise with cardiomyocyte hypertrophy and proliferation. This proliferation was mediated, at least in part, by the increased CITED4. Importantly, mice with reduced cardiac C/EBPβ levels displayed substantial resistance to cardiac failure upon pressure overload. These data indicate that C/EBPβ represses cardiomyocyte growth and proliferation in the adult mammalian heart and that reduction in C/EBPβ is a central signal in physiologic hypertrophy and proliferation.PaperClip/cms/asset/500d1125-0749-4d6a-9ee9-54a659244e27/mmc5.mp3Loading ...(mp3, 3.06 MB) Download audio The heart has the ability to grow in size in response to exercise, but little is known about the transcriptional mechanisms underlying physiological hypertrophy. Adult cardiomyocytes have also recently been proven to hold the potential for proliferation, a process that could be of great importance for regenerative medicine. Using a unique RT-PCR-based screen against all transcriptional components, we showed that C/EBPβ was downregulated with exercise, whereas the expression of CITED4 was increased. Reduction of C/EBPβ in vitro and in vivo resulted in a phenocopy of endurance exercise with cardiomyocyte hypertrophy and proliferation. This proliferation was mediated, at least in part, by the increased CITED4. Importantly, mice with reduced cardiac C/EBPβ levels displayed substantial resistance to cardiac failure upon pressure overload. These data indicate that C/EBPβ represses cardiomyocyte growth and proliferation in the adult mammalian heart and that reduction in C/EBPβ is a central signal in physiologic hypertrophy and proliferation. Endurance exercise of mice induces cardiomyocyte proliferation Endurance exercise causes a reduction of C/EBPβ expression and an increase of CITED4 Reduction of C/EBPβ in vitro induces cellular hypertrophy and proliferation via CITED4 Genetic reduction of C/EBPβ in vivo results in a phenocopy of the exercised heart Cardiac muscle adapts to increased pressure and/or volume with hypertrophy. This holds true for physiological stimuli, such as exercise, as well as for pathological provocations, including hypertension. Though the earliest stages appear quite similar at a morphological level, pathological cardiac hypertrophy leads to cardiovascular diseases such as heart failure and arrhythmia, whereas physiological hypertrophy does not. Thus, understanding the mechanistic distinction between these two types of cardiac growth has very important clinical implications. Numerous prior reports document transcription factors involved in pathological hypertrophy (Akazawa and Komuro, 2003Akazawa H. Komuro I. Roles of cardiac transcription factors in cardiac hypertrophy.Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (274) Google Scholar, Frey and Olson, 2003Frey N. Olson E.N. Cardiac hypertrophy: the good, the bad, and the ugly.Annu. Rev. Physiol. 2003; 65: 45-79Crossref PubMed Scopus (1108) Google Scholar), but relatively little is known about the molecular mechanisms controlling physiological hypertrophy. Cardiomyocytes in adult mammals retain a limited ability to proliferate in response to specific stimuli (Bergmann et al., 2009Bergmann O. Bhardwaj R.D. Bernard S. Zdunek S. Barnabé-Heider F. Walsh S. Zupicich J. Alkass K. Buchholz B.A. Druid H. et al.Evidence for cardiomyocyte renewal in humans.Science. 2009; 324: 98-102Crossref PubMed Scopus (2002) Google Scholar, Bersell et al., 2009Bersell K. Arab S. Haring B. Kühn B. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury.Cell. 2009; 138: 257-270Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, Kajstura et al., 2010Kajstura J. Urbanek K. Perl S. Hosoda T. Zheng H. Ogorek B. Ferreira-Martins J. Goichberg P. Rondon C. D'Amario D. et al.Cardiomyogenesis in the Adult Human Heart.Circ. Res. 2010; 107: 305-315Crossref PubMed Scopus (244) Google Scholar). This has potential importance because cardiomyocyte proliferation could serve as a basis for regeneration of an injured heart if it could be enhanced and harnessed. However, whether this process can be modulated through physiological interventions remains unclear. Moreover, whereas recent studies have implicated some signal transduction pathways involved in adult cardiomyocyte proliferation (Bersell et al., 2009Bersell K. Arab S. Haring B. Kühn B. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury.Cell. 2009; 138: 257-270Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar), little is known about the detailed molecular mechanisms and particularly the transcriptional components that are decisive in this process. Furthermore, there is little data available on factors that control cardiomyocyte proliferation in the adult heart. One of few known regulators of cardiomyocyte proliferation is Gata4, which was recently reported to mark proliferative cells during cardiac regeneration in the zebrafish (Kikuchi et al., 2010Kikuchi K. Holdway J.E. Werdich A.A. Anderson R.M. Fang Y. Egnaczyk G.F. Evans T. Macrae C.A. Stainier D.Y. Poss K.D. Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes.Nature. 2010; 464: 601-605Crossref PubMed Scopus (697) Google Scholar). This factor is also a well-known regulator of cardiomyocyte differentiation in adult hypertrophy, together with SRF, Nkx2.5, Tbx5, and Gata6 (Akazawa and Komuro, 2003Akazawa H. Komuro I. Roles of cardiac transcription factors in cardiac hypertrophy.Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (274) Google Scholar). Here, we studied a murine model of physiological hypertrophy induced by endurance exercise, using a method termed Quanttrx, which we recently developed for genome-wide, quantitative measurement of the expression of all transcriptional components (Gupta et al., 2010Gupta R.K. Arany Z. Seale P. Mepani R.J. Ye L. Conroe H.M. Roby Y.A. Kulaga H. Reed R.R. Spiegelman B.M. Transcriptional control of preadipocyte determination by Zfp423.Nature. 2010; 464: 619-623Crossref PubMed Scopus (330) Google Scholar). The database underlying this method includes all known transcription factors, transcriptional coregulatory proteins, and all proteins that contain a motif that has been associated with transcriptional components, whether their function is known or not. We report here that endurance exercise effected a hypertrophic and proliferative program in cardiomyocytes, which was dependent on a reduction in the expression of the transcription factor C/EBPβ, and a linked increase in the expression of CITED4. Furthermore, mice with genetically reduced C/EBPβ levels recapitulated many of the cardiac phenotypes seen in exercised mice. Importantly, mice with reduced C/EBPβ were resistant to the development of cardiac dysfunction in response to pressure overload on the heart. To study transcriptional regulators of physiological cardiac hypertrophy, a ramp swimming exercise model was used (Taniike et al., 2008Taniike M. Yamaguchi O. Tsujimoto I. Hikoso S. Takeda T. Nakai A. Omiya S. Mizote I. Nakano Y. Higuchi Y. et al.Apoptosis signal-regulating kinase 1/p38 signaling pathway negatively regulates physiological hypertrophy.Circulation. 2008; 117: 545-552Crossref PubMed Scopus (50) Google Scholar). As shown in Figure S1 (available online), mild cardiac hypertrophy was induced without alterations in the expression of pathological gene markers such as ANP and BNP. There was also a 45% increase in cardiomyocyte cell size (Figure S1) without any detectable difference in fibrosis or angiogenesis (Figure S1). Hence, we may conclude that healthy physiological cardiac hypertrophy occurred in these animals. Further characterization revealed that expression of the cell proliferation marker PCNA was increased in the hearts from exercised mice (Figure 1A ). To establish the cellular origin of the increased proliferation markers, confocal microscopy was employed in cardiac tissue stained for both the cardiomyocyte-selective protein α-actinin and the proliferation marker ki67. Robust ki67 staining was seen in 1%–5% of cardiomyocytes (Figure 1B). Importantly, there was a significant increase in cardiomyocyte ki67 staining in the exercised mice compared to the nonexercised controls, as judged from blinded analyses of histological sections using digital software. The more specific mitosis marker, phospho-Histone3 (pH3), was also elevated (Figure 1C), though the absolute number of positive cells was relatively low. Figure S1 shows pH3-positive cardiomyocytes in the different stages of mitosis. Thus, markers of both cell size and proliferation are elevated in the cardiomyocytes of mice in this model of physiological hypertrophy. Previous studies have utilized the density of cardiomyocyte nuclei as an indicator of cardiomyocyte proliferation (Bersell et al., 2009Bersell K. Arab S. Haring B. Kühn B. Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury.Cell. 2009; 138: 257-270Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). We could detect a small increase in nuclear density (100% and 132% in controls versus exercised mice, respectively; p = 0.07), but this did not reach statistical significance. However, it is worth noting that the observed increase in cardiomyocyte size would be expected to confound/dilute the calculated changes in nuclear density. DNA biosynthesis was therefore assayed using bromodeoxyuridine (BrdU) injections in mice undergoing this exercise protocol. We could detect double-α-actinin and BrdU-positive cardiomyocytes at a frequency of 1%–6% in exercised mice. Figure 1D shows a substantial increase in BrdU-positive cardiomyocytes in the hearts of exercised mice compared to the controls. Importantly, Z stack visualization of positive cells clearly demonstrates a distinct cardiomyocyte localization of the BrdU (Figure S1). Finally, staining for the cytokinesis marker Aurora B kinase was used to demonstrate that the exercise protocol did indeed promote completed cardiomyocyte cell division (Figure 1E). Conversely, we could not detect any of these changes in proliferation markers in a murine model of pathological hypertrophy (data not shown). Taken together, these data strongly suggest that adult cardiomyocytes increase in both size and proliferation rate in response to this form of endurance exercise. To investigate the transcriptional basis of physiological cardiac hypertrophy, we utilized Quanttrx, a qPCR-based screen that we recently developed against all known and predicted transcriptional components (Gupta et al., 2010Gupta R.K. Arany Z. Seale P. Mepani R.J. Ye L. Conroe H.M. Roby Y.A. Kulaga H. Reed R.R. Spiegelman B.M. Transcriptional control of preadipocyte determination by Zfp423.Nature. 2010; 464: 619-623Crossref PubMed Scopus (330) Google Scholar). We used cardiac samples from the mice characterized (above) together with cardiac samples from mice subjected to transaortic constriction (TAC) for 2 weeks. As shown in Figure S1, the TAC procedure induced a similar degree of hypertrophy as seen in the exercised animals but with elevation of ANP/BNP, thus indicating pathological hypertrophy. There were no signs of heart failure or other pathological changes in the mice subjected to TAC at this time point. The Quanttrx screen identified 175 genes significantly regulated in the exercise model and 96 in the pathological (TAC) model (Table S1 and Table S2). Importantly, there was little overlap in the expression profile between the two models. In fact, there was a significant, negative correlation between the changes induced in the respective model (Figure S2; r = −0.32; p < 0.001). Thus, these models of pathological and physiological hypertrophy express distinct programs of transcriptional components. Of the 175 transcription factors regulated by physiological hypertrophy, 47 (27%) had no known function (no PubMed annotation). However, 10 transcription factors with known functions in cardiac differentiation and/or adult hypertrophy were differentially regulated in the swim model. These included well-known genes such as Nkx2.5, Gata4, Tbx5, Mef2c, and Gata6. Furthermore, 13% of the differentially expressed factors had known or suggested roles in cellular proliferation. Less than 1% of the genes regulated in the pathological model had any known role in cellular proliferation. Based on the magnitude of expression changes and absolute cardiac expression, we chose 30 genes out of this preliminary list of 272 and performed qPCR analyses in completely new samples of either physiological (endurance exercise) or pathological (TAC) hypertrophy. Eight out of these 30 showed robust validation (Figure S2 and Figure 2A ), and these were further analyzed for cardiomyocyte-specific expression. Figure S2 shows expression of these genes in cardiomyocytes and noncardiomyocytes after density gradient separation. Five out of eight were expressed primarily in the cardiomyocyte fraction, and these were subsequently analyzed using adenoviral-mediated forced expression and/or siRNA-mediated knockdown, using changes in cell size of rat neonatal cardiomyocytes as our endpoint. Gbx2, Cited4, Mlx, and Meox1 failed to demonstrate any effect on cardiomyocyte sizes (Figure S2), but reduction of C/EBPβ caused a clear increase in cell size (Figure 3A ).Figure 3Reduction of C/EBPβ in Primary Cardiomyocytes Results in Hypertrophic Cell Growth and ProliferationShow full captionPrimary rat neonatal cardiomyocytes treated with either a C/EBPβ siRNA or a C/EBPβ-expressing adenovirus with respective controls. All experiments were performed 48 hr after transfection/transduction.(A) Immunohistochemistry against α-actinin followed by cell area quantifications as described in methods. At least 100 cells were quantified in all groups.(B) Protein biosynthesis measured as 35S-Met incorporation into the protein pool after a 1 hr pulse. Data are presented as percent of control.(C) Quantification of cell numbers in primary after transfection with indicated siRNA constructs.(D) Western blot analysis of PCNA followed by quantification (n = 4). Data are presented as PCNA relative to β-actin after background subtraction.(E) Primary rat neonatal cardiomyocytes were treated with BrdU 24 hr after transfection with indicated siRNA. Cells were then stained against BrdU and α-actinin, and BrdU-positive cardiomyocytes were counted and normalized to total number of cardiomyocytes.Error bars represent standard error of mean. ∗p < 0.05 versus respective control using Student's t test. See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Primary rat neonatal cardiomyocytes treated with either a C/EBPβ siRNA or a C/EBPβ-expressing adenovirus with respective controls. All experiments were performed 48 hr after transfection/transduction. (A) Immunohistochemistry against α-actinin followed by cell area quantifications as described in methods. At least 100 cells were quantified in all groups. (B) Protein biosynthesis measured as 35S-Met incorporation into the protein pool after a 1 hr pulse. Data are presented as percent of control. (C) Quantification of cell numbers in primary after transfection with indicated siRNA constructs. (D) Western blot analysis of PCNA followed by quantification (n = 4). Data are presented as PCNA relative to β-actin after background subtraction. (E) Primary rat neonatal cardiomyocytes were treated with BrdU 24 hr after transfection with indicated siRNA. Cells were then stained against BrdU and α-actinin, and BrdU-positive cardiomyocytes were counted and normalized to total number of cardiomyocytes. Error bars represent standard error of mean. ∗p < 0.05 versus respective control using Student's t test. See also Figure S3. C/EBPβ is a member of the bHLH gene family of DNA-binding transcription factors and has known roles in cell proliferation and differentiation in other tissues (Sebastian and Johnson, 2006Sebastian T. Johnson P.F. Stop and go: anti-proliferative and mitogenic functions of the transcription factor C/EBPbeta.Cell Cycle. 2006; 5: 953-957Crossref PubMed Scopus (86) Google Scholar), including adipose cells and liver. However, it has not been studied in the context of the cardiomyocyte. C/EBPβ mRNA expression was reduced by 61% in hearts in the exercise model when assayed in new samples, but this effect was not observed in a model of pathological hypertrophy using transverse banding (TAC) performed in parallel (Figure 2A). Downregulation of C/EBPβ was also seen in response to the exercise training at the protein level (Figure 2B). Expression of the short LIP C/EBPβ isoform that is inhibitory toward C/EBPβ function was undetectable in these cardiac samples (Figure 2B). Confocal microscopy illustrated that it was indeed cardiomyocyte-specific expression of C/EBPβ that was reduced with exercise (Figure 2C). Consistent with this, C/EBPβ expression was enriched in the cardiomyocyte fraction, versus noncardiomyocyte cells, following gradient purification of rat neonatal cardiomyocytes (Figure 2D and Figure S2). Two additional mouse models of exercise were tested for effects on C/EBPβ expression: acute and low-intensity treadmill running. Neither treatment resulted in cardiac hypertrophy (data not shown), but reduction of C/EBPβ expression occurred in the acute setting (40 min of treadmill running), as seen in Figure 2E. Thus, C/EBPβ expression is reduced relatively early in endurance exercise. The functional consequences of experimental reduction of C/EBPβ were assessed in detail in primary rat cardiomyocytes. An siRNA was used to reduce C/EBPβ levels to approximately the levels of mRNA observed after endurance exercise (43%) (Figure S3). As demonstrated in Figures 3A and 3B, the forced decrease in C/EBPβ mRNA was sufficient to drive an increase in cell size and in the rate of protein biosynthesis. No statistically significant change in cell size or protein biosynthesis was seen with forced C/EBPβ expression (p = 0.42 and p = 0.054, respectively). The reduction of C/EBPβ expression was also sufficient to ablate the increase in ANP levels seen with pathological provocation (Figure S3). Interestingly, the C/EBPβ reduction via siRNA also led to an increase in cell number (Figure 3C). This was accompanied by increased PCNA expression and increased BrdU incorporation into DNA, highly suggestive of cardiomyocyte proliferation (Figures 3D and 3E). These data indicate that a reduction in C/EBPβ expression is sufficient to induce hypertrophy and proliferation in cultured cardiomyocytes. The initial Quanttrx screen revealed a specific set of regulated genes with known functions in cardiomyocyte differentiation and hypertrophy (Figure 4A and Table S1). Examples of such genes were Gata4, Tbx5, and Nkx2.5, which all induce hypertrophy (Akazawa and Komuro, 2003Akazawa H. Komuro I. Roles of cardiac transcription factors in cardiac hypertrophy.Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (274) Google Scholar). Beyond these 11 genes, we also investigated expression levels of the genes that are well known to be altered in cardiac hypertrophy or differentiation, including α-MHC, TnI, and TnT, which were all upregulated in the hearts of exercised mice (Figure 4A). The transcriptional coregulator PGC-1α has previously been demonstrated to have an important role in preventing cardiac dysfunction after the development of hypertrophy (Arany et al., 2006Arany Z. Novikov M. Chin S. Ma Y. Rosenzweig A. Spiegelman B.M. Transverse aortic constriction leads to accelerated heart failure in mice lacking PPAR-gamma coactivator 1alpha.Proc. Natl. Acad. Sci. USA. 2006; 103: 10086-10091Crossref PubMed Scopus (276) Google Scholar), and exercised mice indeed displayed increased cardiac expression levels of PGC-1α together with its downstream targets VEGF, Ndufs1, Ndufv2, and ATP5o (Figure 4A). Thus, by combining the Quanttrx screen hits with markers of hypertrophy and the PGC-1α, as well as its downstream targets, we defined an exercise-induced gene set of 19 genes (Figure 4A). The changes in mRNA expression for Nkx2.5, Tbx5, Mef2c, Gata4, and PGC-1α following TAC surgery are shown in Figure S4.Figure S4AKT Overexpression In Vitro and Its Effect on C/EBPβ and Target Genes, Related to Figure 4Show full captionExpression levels of exercise-induced genes after TAC surgery. PGC1α overexpression and cell sizes.(A) Normalized mRNA levels of indicated genes following C/EBPβ adenoviral overexpression in primary cardiomyocytes.(B and C) Rat neonatal cardiomyocytes transduced with indicated adenoviral constructs followed by RT-PCR analysis of indicated genes normalized to 18S.(D) mRNA expression levels of indicated genes from cardiac samples of sham- or TAC operated mice 2 weeks after intervention (n = 4).(E) Rat neonatal cardiomyocytes transduced with PGC1α or GFP expressing adenovirus followed by size measurements. ∗ indicates p < 0.05 using students t test (A, D and E) and § marks p < 0.05 versus AKTwt + GFP using one-way ANOVA (B-C).Error bars represent standard error of mean.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Expression levels of exercise-induced genes after TAC surgery. PGC1α overexpression and cell sizes. (A) Normalized mRNA levels of indicated genes following C/EBPβ adenoviral overexpression in primary cardiomyocytes. (B and C) Rat neonatal cardiomyocytes transduced with indicated adenoviral constructs followed by RT-PCR analysis of indicated genes normalized to 18S. (D) mRNA expression levels of indicated genes from cardiac samples of sham- or TAC operated mice 2 weeks after intervention (n = 4). (E) Rat neonatal cardiomyocytes transduced with PGC1α or GFP expressing adenovirus followed by size measurements. ∗ indicates p < 0.05 using students t test (A, D and E) and § marks p < 0.05 versus AKTwt + GFP using one-way ANOVA (B-C). Error bars represent standard error of mean. As shown in Figure 4B, reduction in C/EBPβ levels in primary cardiomyocytes resulted in a strikingly similar expression pattern. In fact, 11/19 of these genes were regulated in the same manner as in the exercise model (p < 0.001 using chi-square statistics). Furthermore, adenoviral expression of C/EBPβ induced essentially the reverse expression pattern (Figure S4). These data strongly suggest that C/EBPβ acts upstream of other transcriptional components that are also regulated in physiological hypertrophy. Especially interesting was the C/EBPβ-dependent regulation of Gata4, which has recently been demonstrated to play a major role in zebrafish cardiomyocyte proliferation during myocardial regeneration (Kikuchi et al., 2010Kikuchi K. Holdway J.E. Werdich A.A. Anderson R.M. Fang Y. Egnaczyk G.F. Evans T. Macrae C.A. Stainier D.Y. Poss K.D. Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes.Nature. 2010; 464: 601-605Crossref PubMed Scopus (697) Google Scholar). Many of the genes regulated by both endurance exercise and the siRNA-mediated C/EBPβ reduction are known to be transcriptional targets of the transcription factor SRF, with SRE elements in their promoters (Akazawa and Komuro, 2003Akazawa H. Komuro I. Roles of cardiac transcription factors in cardiac hypertrophy.Circ. Res. 2003; 92: 1079-1088Crossref PubMed Scopus (274) Google Scholar). C/EBPβ has been demonstrated to physically interact with SRF (Hanlon and Sealy, 1999Hanlon M. Sealy L. Ras regulates the association of serum response factor and CCAAT/enhancer-binding protein beta.J. Biol. Chem. 1999; 274: 14224-14228Crossref PubMed Scopus (35) Google Scholar), but the functional consequences of this interaction are not clear, especially in cardiomyocytes in which the role of C/EBPβ is unknown. Previous reports on the C/EBPβ-SRF interaction have suggested a positive effect on SRE-dependent elements, but the increased expression levels of Gata4 and α-MHC (Figure 4A) following C/EBPβ siRNA here suggested a negative regulation of SRE-dependent genes in cardiomyocytes. A physical interaction between the SRF and C/EBPβ proteins was indeed observed using coimmunoprecipitation from primary cardiomyocytes (Figure 4C). Next, chromatin immunoprecipitation (ChIP) assays were performed, examining the binding of SRF to the Gata4 and α-MHC promoters following gain and loss of the C/EBPβ protein. As shown in Figure 4D, CEBPβ reduction using siRNA dramatically increased SRF binding to the promoters of both α-MHC and Gata4, in which SRF is known to play a positive regulatory role. Conversely, adenoviral expression of C/EBPβ reduced SRF binding to these promoters. Thus, C/EBPβ interferes with SRF binding to the promoters of critical cardiac genes (Figure 4E). The recent discovery of the ErbB4 pathway in cardiomyocyte proliferation led us to investigate whether C/EBPβ could be affected by such signaling. This was especially interesting as ErbB4 signals via AKT1, a kinase known to function in physiological hypertrophy (DeBosch et al., 2006DeBosch B. Treskov I. Lupu T.S. Weinheimer C. Kovacs A. Courtois M. Muslin A.J. Akt1 is required for physiological cardiac growth.Circulation. 2006; 113: 2097-2104Crossref PubMed Scopus (396) Google Scholar). We thus investigated the connection of this pathway to C/EBPβ using both forced expression of wild-type AKT1 and a dominant-negative AKT1 (Nagoshi et al., 2005Nagoshi T. Matsui T. Aoyama T. Leri A. Anversa P. Li L. Ogawa W. del Monte F. Gwathmey J.K. Grazette L. et al.PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury.J. Clin. Invest. 2005; 115: 2128-2138Crossref PubMed Scopus (187) Google Scholar). As demonstrated in Figure S4, increasing AKT1 activity did indeed reduce C/EBPβ expression levels together with an expression profile resembling what was observed with exercise with increased expression of Tbx5, Gata4, TnI, and α-MHC. Conversely, the dominant-negative AKT1 mutant increased C/EBPβ in parallel with decreased mRNA levels of Tbx5, Gata4, TnI, TnT, and α-MHC. We could thus conclude that AKT1 activity evokes the same gene expression profile as seen in exercise. We next used C/EBPβ adenovirus together with AKT1 overexpression to establish whether C/EBPβ indeed act downstream on AKT1 in the regulation of these target genes. As demonstrated in Figure S4, C/EBPβ overexpression ablated the positive expression effects of AKT1 on Tbx5, Gata4, TnI, TnT, and α-MHC. Taken together, these data suggest that AKT1 regulates C/EBPβ expression, connecting C/EBPβ to a previously characterized physiological growth pathway in the heart. The SRF-dependent gene set described above includes well-known regulators of cardiomyocyte hypertrophy, but apart from Gata4, they are not known to participate in a proliferative phenotype, such as we see with C/EBPβ reduction. We thus analyzed the list of exercise-induced cardiac genes (Figure S2) for those controlled by C/EBPβ that might influence or control cardiomyocyte proliferation. Preliminary studies using gain and loss of function for six factors apparently downstream of CEBPβ led us to focus on CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4). Indeed, CITED4 showed a robust increase in exercised hearts (Figure 5A ), and its expression level was markedly altered with C/EBPβ gain or loss of function in vitro (Figure 5B). Strikingly, forced CITED4 expression (Figure S5) increased cardiomyocyte cell numbers together with an increased percentage of ki67-positive cells (Figures 5C and 5D). Conversely, siRNA directed against CITED4 (Figure S5C) reduced both cell numbers and ki67 staining (Figures 5C and 5D). Gene expression analysis also revealed a gene set of proproliferative genes increased in endurance exercise (Figure S5), in which the important cell cycle/proliferation factors CyclinD1 and n-myc also displayed increased expression with forced CITED4 expression together with SRF (Figure S5). To investigate the functional relationship between C/EBPβ and CITED4, we used siRNA directed against CITED4 together with C/EBPβ siRNA. As shown in Figure 5E, CITED4 knockdown could completely abolish the effect of cellular proliferation seen with the C/EBPβ reduction. Thus, C/EBPβ is likely to mediate its antiproliferative effects, at least in part, via CITED4 expression. We next tested the role of C/EBPβ reduction in zebrafish, a simple and experimentally accessible in vivo system. Anti-C/EBPβ morpholino oligonucleotides were injected into zebrafish embryos transgenic for cmlc2-GFP at the one-cell stage (Figure 6A ). Figure 6B shows zebrafish hearts dissected at 36 hr and stained for nuclei (DAPI, blue) and plasma membrane (integrin, red). Quantification of these images revealed increased cardiomyocyte cell number but did not change cardiomyocyte size up to 72 hr postfertilization (Figure

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C/EBPβ Controls Exercise-Induced Cardiac Growth and Protects against Pathological Cardiac Remodeling-论文阅读讨论-ReadPaper - 轻松读论文 | 专业翻译 | 一键引文 | 图表同屏 (2024)

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