1. Volume 143, Issue 7, Pages 1072-1083 (December 2010)
C/EBPβ Controls Exercise-Induced Cardiac Growth and Protects against Pathological Cardiac Remodeling 
Pontus Boström, Nina Mann, Jun Wu, Pablo A. Quintero, Eva R. Plovie, Daniela Panáková, Rana K. Gupta, Chunyang Xiao, Calum A. MacRae, Anthony Rosenzweig, Bruce M. Spiegelman 
Cell 
Volume 143, Issue 7, Pages (December 2010)
DOI: /j.cell
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
A Swim Model Induces Physiological Cardiac Hypertrophy and Cardiomyocyte Proliferation
(A) Mice swam using a ramp protocol for 2 weeks with respective controls (n = 4) and were assayed for PCNA protein levels with subsequent quantification. Data are presented as PCNA per α-tubulin after subtraction of background. Data are representative for two independent mouse cohorts.
(B and C) Representative image and quantification of Ki67 (B) and phosphohistone3 (C) staining from exercised mice (green) counterstained with α-actinin (red) and DAPI (blue) in cardiac tissue.
(D) Exercised and control mice were injected with BrdU 3 days prior to the final exercise day. The image shows a representative section from the exercised cohort. BrdU is labeled in green, α-actinin in red, and DAPI in blue.
(E) Representative image and quantification of Aurora B kinase (green) staining from exercised mice counterstained with α-actinin (red) and DAPI (blue) in cardiac tissue. All quantifications were based on at least 80 (blinded) images from each group (from at least four mice). Any uncertainty concerning whether a ki67-, AuroraB-, or BrdU-positive cell was completely within an α-actinin-positive area was resolved using confocal Z stacks.
Error bars represent standard error of mean. ∗p < 0.05 versus respective control using Student's t test. See also Figure S1.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 2
C/EBPβ Is Expressed in Cardiomyocytes and Downregulated with Endurance Exercise
(A) Cardiac C/EBPβ mRNA levels in a model for physiological (swim) and pathological (TAC) hypertrophy with respective controls (n = 4) as described in methods.
(B) Western blot analysis of C/EBPβ and β-actin in the swim cohort (above) with subsequent quantification. Data are presented as percent of control with C/EBPβ relative to β-actin after background subtraction.
(C) Confocal microscopy after immunohistochemistry against C/EBPβ (green), α-actinin (red), and DAPI (blue) in cardiac tissue with subsequent quantification in indicated groups. Data are presented as the ratio of C/EBPβ-positive cardiomyocyte nuclei.
(D) Gradient fractionation of primary rat neonatal cardiac cells followed by expression analysis of cardiomyocyte and fibroblast markers.
(E) Cardiac C/EBPβ mRNA levels from three different exercise regimens: acute = 40 min treadmill running (n = 6); “low intensity” = 2 weeks of 30 min daily running (n = 6); “endurance” = the swimming protocol as described in methods (n = 4).
Error bars represent standard error of mean. ∗p < 0.05 versus respective control using Student's t test. See also Figure S2.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3
Reduction of C/EBPβ in Primary Cardiomyocytes Results in Hypertrophic Cell Growth and Proliferation
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.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4
C/EBPβ Controls an Exercise-Induced Gene Set by Inhibiting SRF DNA Binding
(A and B) QPCR analysis of indicated genes in hearts from endurance-exercised mice (A) and in primary cardiomyocytes treated with C/EBPβ or control siRNA for 48 hr (B). ∗p < 0.05 versus respective control using Student's t test.
(C) Immunoprecipitation against SRF or a nonimmune IgG in primary cardiomyocytes followed by western blot against SRF, C/EBPβ, and the control protein α-tubulin as indicated.
(D and E) Chromatin immunoprecipitation in primary cardiomyocytes treated with either C/EBPβ siRNA (D) or C/EBPβ adenoviral overexpression (E) with respective controls. Precipitated DNA was then analyzed with RT-PCR using primers directed against promoter regions in the Gata4 and myh6 gene. ChIP experiments were repeated at least three times with similar results.
Error bars represent standard error of mean. ∗p < 0.05 versus respective control using one-way ANOVA statistics. See also Figure S4.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5
CITED4 Is Upregulated with Endurance Exercise, Regulated by C/EBPβ, and Its Forced Expression Induced Cardiomyocyte Proliferation In Vitro
(A) mRNA levels of cardiac CITED4 in control and endurance-exercised (swim) mice normalized to 18S expression.
(B) mRNA expression of CITED4 in primary rat neonatal cardiomyocytes after siRNA knockdown (left) or adenoviral overexpression (right) of CITED4. Expression levels are normalized to 18S.
(C and D) Rat neonatal cardiomyocytes treated with either an adenovirus overexpressing CITED4 (left) or a siRNA directed against CITED4 (right). Cells were then stained against α-actinin and ki67, and positive cells were counted in 20× view fields from 15 random images per group.
(E) Rat neonatal cardiomyocytes treated with control siRNA, C/EBPβ siRNA + control siRNA, or C/EBPβ siRNA + Cited4 siRNA, followed by assay of BrdU incorporation as previously described. Data is pooled from three experiments and presented as percent of control.
Error bars represent standard error of mean. ∗p < 0.05 versus respective control, and § versus C/EBPβ siRNA using one-way ANOVA statistics. T test was used for (A–D). See also Figure S5.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 6
C/EBPβ Reduction in the Zebrafish Embryo Results in Cardiomyocyte Proliferation
An anti-C/EBPβ morpholino was injected into one-cell stage cmlc-GFP transgenic zebrafish embryos.
(A) Western blot analysis against indicated proteins in zebrafish embryos 48 hpf.
(B) Dissection of beating hearts at 36 hr followed by integrin staining (blue = DAPI, green = GFP, and red = zn-8).
(C) Twenty minutes BrdU labeling prior to dissection of beating hearts and BrdU (green)/zn-8 (red) staining.
(D) Quantification (B) from totally 30 hearts per group.
(E) Quantification of integrin- and BrdU-positive cardiomyocytes (C) from n = 6 embryos per group. Data are presented as percent of control and representative of two independent experiments.
(F) RT-PCR analysis of indicated transcripts.
Error bars represent standard error of mean. ∗p < 0.05 versus respective control using Student's t test. See also Figure S6.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 7
C/EBPβ+/− Mice Have Cardiac Hypertrophy and Increased Proliferation Markers and Are Resistant to Pressure Overload
(A) Cardiomyocyte size was measured in cardiac tissue from wild-type (n = 5) and C/EBPβ+/− (n = 7) mice using WGA membrane staining.
(B and C) Quantification of nuclear density assayed (B) and BrdU incorporation (C) from indicated mice.
(D) Typical image of Aurora B kinase (green) positive cardiomyocyte from C/EBPβ+/− mouse heart, counterstained with anti-α-actinin (red) and dapi (blue).
(E) Immunohistochemistry against phosphohistone H3 (green) in primary cardiomyocyte from an adult C/EBPβ+/− mouse heart counterstained with α-actinin (red) and dapi (blue).
(F) Wild-type and C/EBPβ+/− (n = 6 and 7) mice subjected to TAC and assayed for fractional shortening at indicated times.
(G) Lung weight 52 days after TAC intervention.
(H) Kaplan-Meier plot of cardiac failure-free fraction at indicated times in wild-type and C/EBPβ+/− mice following TAC procedure.
Error bars represent standard error of mean. ∗p < 0.05 versus respective control using Student's t test. ∗∗p < See also Figure S7.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

9. Figure S1
Characteristics of the Physiological and Pathological Cardiac Hypertrophy Mice Model (Swimming and TAC), Related to Figure 1
Additional immunohistochemistry of proliferation markers, including Z stack orthogonal views
(A–D) Data from exercised and TAC-operated mice with respective controls (n = 4).
(A) Heart weights normalized to body weights (multiplied by 1000).
(B) Left Ventricular Posterior Wall in diastole (LWPWd) + Left Ventricular Septum in diastole (LVSd). Data presented as mm.
(C) Cell size measurement of cardiomyocytes after cardiac WGA staining in indicated groups. More than 100 cells from each group (n = 4) was measured.
(D) mRNA levels of ANP and BNP normalized to 18 s levels in indicated groups.
(E) Quantification of CD31 positive cell area relative to total tissue area from exercised and control mice.
(F) Quantification of cells positive with staining using an anti-fibroblast antibody (abcam) in exercised and control mice.
(G) Confocal images of Phospho-histone3 staining (green) counterstained with dapi (blue) and α-actinin (green) in cardiac tissue from exercised mice. Upper left: Cardiomyocyte in prophase with nuclear condensation. Upper right: Cardiomyocyte at end of prophase. Lower left: Cardiomyocyte undergoing karyokinesis and lower right: Cardiomyocyte in telophase after nuclear separation.
(H) Confocal stack of BrdU staining with orthogonal view displaying a nuclei within cardiomyocyte (upper panel) and a nuclei outside cardiomyocyte (lower panel). ∗ indicates p < 0.05 using students t test. Error bars represent standard error of mean.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

10. Figure S2
Preliminary Follow-Up Data on Eight Transcription Factors Identified Using the Quanttrx Screen, Related to Figure 2
(A) qPCR validation of Quanttrx-identified transcription factors with expression changes in either the physiological or pathological hypertrophy model (n = 4). All changes are p < 0.05 using students t test.
(B) Cardiomyocytes and non-cardiomyocytes separated from rat neonatal hearts using density gradient centrifugation. Samples were analyzed for indicated gene expression normalized to 18S, and cardiomyocytes set to 1.
(C) Adenoviral overexpression of indicated genes in rat neonatal cardiomyocytes followed by size measurements. Data is expressed as percent of GFP control.
(D) Western Blot against C/EBPβ in cardiomyocytes (CM) and non-cardiomyocytes with actin as loading control.
(E) Correlation between the percent change seen in the exercise model (y axis) and the TAC-model (x axis). All Quanttrx transcription factors significantly altered in either model are included. ∗ indicates p < 0.05 using students t test. Error bars represent standard error of mean.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

11. Figure S3
C/EBPβ siRNA and Adenoviral Overexpression Data In Vitro, Related to Figure 3
ANP/BNP levels and target gene expression with C/EBPβ overexpression
(A) SiRNA knockdown of C/EBPβ in primary cardiomyocytes assayed using RT-PCR. Two siRNA constructs and two control siRNA's was used. Data is normalized to respective control and 18S. T test was used.
(B) Western blot against C/EBPβ and tubulin from primary cardiomyocytes transfected with control or C/EBPβ siRNA #2.
(C) mRNA levels of ANP and BNP in rat neonatal cardiomyocytes transfected with control or C/EBPβ siRNA with or without 24 hr treatment with phenylephrine (PE).
(D) Overexpression with C/EBPβ adenovirus in primary cardiomyocytes followed by Western blot analysis against C/EBPβ. ∗ indicates p < 0.05 versus respective control and § versus control siRNA +PE using one-way ANOVA.
Error bars represent standard error of mean.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

12. Figure S4
AKT Overexpression In Vitro and Its Effect on C/EBPβ and Target Genes, Related to Figure 4
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.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

13. Figure S5
CITED4 Gain- and Loss-of-Function Characteristics In Vitro and Proliferation Target Gene Expression, Related to Figure 5
(A) Pro-proliferative genes upregulated in endurance exercise from expression analysis experiment.
(B) Western blot against CITED4 from rat neonatal cardiomyocytes treated with control or CITED4 expressing adenovirus.
(C) mRNA analysis of CITED4 after transient transfection with control or CITED4 siRNA as annotated.
(D) Normalized SRF mRNA levels with GFP or CITED4 adenoviral overexpression.
(E and F) mRNA levels of n-myc and cyclinD1 in heart samples following endurance exercise (E) or in rat neonatal cardiomyocytes overexpressing CITED4 (F). ∗ indicates p < 0.05 using students t test. Error bars represent standard error of mean.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

14. Figure S6
Characteristics of the C/EBPβ Heterozygous Mouse, Including Cardiac C/EBPβ Levels, Target Gene Expression, Heart Weight, Exercise Capacity, and Proliferation Markers, Related to Figure 6
(A and B) C/EBPβ mRNA (A) and protein (B) levels in heart from wild-type (n = 5) and C/EBPβ+/− (n = 7) mice. mRNA levels were normalized to the 18S gene.
(C) Expression of exercise-induced genes in the C/EBPβ+/− mice compared to wild-type littermates (n = 5 and 7 respectively).
(D) Heart weights of C/EBPβ+/− mice compared to wild-type littermates (n = 4 and 5 respectively) at baseline.
(E) Maximal exercise capacity measured as described in the method section, in wt and C/EBPβ+/− mice.
(F) Representative images of BrdU staining from heart tissue in indicated mice.
(G) PCNA Western blot from wt and C/EBPβ+/− mice with subsequent quantification. Data is presented as PCNA-background normalized to β-actin.
(H) Quantification of ki67 positive cardiomyocyte nuclei (n = 5 and 7 mice based on at least 20 images per mouse). ∗ indicates p < 0.05 using students t test. Error bars represent standard error of mean.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

15. Figure S7
Ultrasound Data from C/EBPβ+/− and Control Mice Subjected to TAC, Related to Figure 7
Heart weight and C/EBPβ levels of exercised C/EBPβ+/− mice.
(A and B) Echocardiographic characteristics of wild-type and C/EBPβ+/− mice after TAC intervention (day 0).
(A) Diastolic (left) and systolic (right) Left intraventricular diameter (LVD).
(B) The sum of either diastolic (left) or systolic (right) Intraventricular septum (IVS) and Left Ventricular Posteriar Wall (LVPW) width.
(C) Heart weight in wild-type and C/EBPβ+/− mice after TAC intervention, normalized to body weight.
(D) C/EBPβ expression levels in C/EBPβ+/− and wild-type mice after the endurance exercise protocol.
(E) Heart weights normalized to body weight in C/EBPβ+/− and wild-type mice after the endurance exercise protocol. ∗ indicates p < 0.05 using students t test.
Error bars represent standard error of mean.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

16. Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions