Advertisement

Myocardial transcription factors are modulated during pathologic cardiac hypertrophy in vivo

  • Author Footnotes
    ⁎ A.A. is supported by a joint program grant from the National Heart, Lung, and Blood Institute (1 K08 HL72774-01) and The Thoracic Surgery Foundation for Research and Education. This work was supported in part by grant NIH-R01- HL61284 (J.F.).
    Anthony Azakie
    Correspondence
    Address for reprints: Anthony Azakie, MD, 513 Parnassus Ave, Room S-549, Box 0117, University of California San Francisco, San Francisco, CA 94143.
    Footnotes
    ⁎ A.A. is supported by a joint program grant from the National Heart, Lung, and Blood Institute (1 K08 HL72774-01) and The Thoracic Surgery Foundation for Research and Education. This work was supported in part by grant NIH-R01- HL61284 (J.F.).
    Affiliations
    University of California, San Francisco, Department of Surgery, San Francisco, Calif

    University of California, San Francisco, Department of Pediatrics, San Francisco, Calif.
    Search for articles by this author
  • Jeffrey R. Fineman
    Affiliations
    University of California, San Francisco, Department of Pediatrics, San Francisco, Calif.
    Search for articles by this author
  • Youping He
    Affiliations
    University of California, San Francisco, Department of Surgery, San Francisco, Calif
    Search for articles by this author
  • Author Footnotes
    ⁎ A.A. is supported by a joint program grant from the National Heart, Lung, and Blood Institute (1 K08 HL72774-01) and The Thoracic Surgery Foundation for Research and Education. This work was supported in part by grant NIH-R01- HL61284 (J.F.).
Published:November 06, 2006DOI:https://doi.org/10.1016/j.jtcvs.2006.08.005

      Objectives

      In the current study we describe and characterize a novel ovine model of biventricular hypertrophy and heart failure and evaluate the role of selected cardiac transcription factors in the regulation of cardiac gene expression during pathologic hypertrophy in vivo. The cardiac troponin T promoter is used as a model gene.

      Methods and Results

      Transient transfections of ovine cardiomyocytes in culture show that Sp1, transcriptional enhancer factor-1, and myocyte enhancer factor-2 activate cardiac troponin T promoter constructs. Cotransfection of Sp3 inhibits cardiac troponin T promoter activity and represses Sp1-mediated activation of the cardiac troponin T promoter. By chromatin immunoprecipitation, transcriptional enhancer factor-1, myocyte enhancer factor-2, NKX2.5, GATA-4, and Sp factors bind the cardiac troponin T promoter in vivo. To assess the role of cardiac transcription during pathologic hypertrophy, in vivo, we created surgical aorta-pulmonary shunts in utero in fetal lambs. Two weeks after spontaneous delivery, shunted lambs showed failure to thrive, significant biventricular hypertrophy, and heart failure. Shunted hearts had significant increases in myosin and cardiac troponin T protein expression. There was a shift in expression to the high-molecular-weight fetal isoforms. Transcriptional enhancer factor-1, myocyte enhancer factor-2, GATA-4, NKX2.5, and Sp1 transcription factor levels were increased in all heart chambers of shunted animals. Sp3 expression was decreased in shunted ventricles. Immunoprecipitated Sp3 was associated with significant increases in histone acetyl transferase activity and decreases in histone-deacetylase activity.

      Conclusion

      The shunted neonatal lamb is a valid, novel model of pathologic biventricular hypertrophy. During pathologic hypertrophy myocardial transactivators are upregulated while repressors are downregulated.

      CTSNet classification

      Abbreviations and Acronyms:

      ADP (adenosine diphosphate), ChIP (chromatin immunoprecipitation), cTnT (cardiac troponin T), HAT (histone acetyl transferase), HDAC (histone deacetylase), MEF2 (myocyte enhancer factor 2), PAR (poly(adenosine diphosphate ribose)), PARP (poly(adenosine diphosphate ribose) polymerase), TEF-1 (transcriptional enhancer factor-1)
      Cardiac failure and dysfunction can result from numerous clinicopathologic conditions including hypertension, ischemic heart disease, valvular heart disease, cardiomyopathy, and congenital heart disease. The need for effective broad-based therapy for patients with heart failure far outweighs and has outpaced current therapeutic strategies. Further understanding of the complex cellular mechanisms that lead to heart failure may aid in the development of novel and effective therapies.
      Various pathologic signals, stressors, or biomechanical forces elicit different intracellular signaling pathways that ultimately stimulate fetal cardiac gene expression (in children and adults) and pathologic hypertrophy, an early milestone in heart failure.
      • Hunter J.J.
      • Chien K.R.
      Signaling pathways for cardiac hypertrophy and failure.
      • Chien K.R.
      Stress pathways and heart failure.
      Although numerous biomechanical forces and pathologic conditions can result in myocardial dysfunction, it is hypothesized that there is a final common pathway in which transcriptional alterations in sarcomeric gene expression, common to many forms of pathologic hypertrophy (concentric, eccentric), result in contractile dysfunction of the heart. Thus, contractile dysfunction of the heart can be viewed as a transcriptional disorder.
      • McKinsey T.A.
      • Olson E.N.
      Toward transcriptional therapies for the failing heart: chemical screens to modulate genes.
      • Backs J.
      • Olson E.N.
      Control of cardiac growth by histone acetylation/deacetylation.
      Transcription factors are nuclear proteins that bind promoter regulatory elements to activate or repress gene expression. The combinatorial interactions between transcription factors and promoter elements that are required for the regulation of cardiac gene expression during normal cardiac development probably operate during pathologic cardiac remodeling and hypertrophy. NKX2.5, myocyte enhancer factor 2 (MEF2), HAND, and GATA transcription factors have been implicated in transcriptional control, pattern formation, and segmental control of cardiac development.
      • Charron F.
      • Nemer M.
      GATA transcription factors and cardiac development.
      • Jamali M.
      • Rogerson P.J.
      • Wilton S.
      • Skerjanc I.S.
      Nkx2-5 activity is essential for cardiomyogenesis.
      • Martin J.F.
      • Schwarz J.J.
      • Olson E.N.
      Myocyte enhancer factor (MEF) 2C: a tissue-restricted member of the MEF-2 family of transcription factors.
      • McBride K.
      • Nemer M.
      Regulation of the ANF and BNP promoters by GATA factors: lessons learned for cardiac transcription.
      Using the cardiac troponin T (cTnT) promoter as a model gene, we have shown that transcriptional enhancer factor-1 (TEF-1), MEF2, and Sp family of transcription factors also contribute to the developmental regulation of cardiac gene expression in avian embryonic cardiomyocytes.
      • Azakie A.
      • Lamont L.
      • Fineman J.R.
      • He Y.
      Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.
      • Azakie A.
      • Larkin S.B.
      • Farrance I.K.
      • Grenningloh G.
      • Ordahl C.P.
      DTEF-1, a novel member of the transcription enhancer factor-1 (TEF-1) multigene family.
      These myocardial transcription factors, which are important for normal growth, development, and maintenance of the cardiac phenotype, may also be implicated during induction of fetal genes and the upregulation of constitutive genes that is seen with pathophysiologic hypertrophy.
      • Chien K.R.
      Stress pathways and heart failure.
      • Chien K.R.
      • Knowlton K.U.
      • Zhu H.
      • Chien S.
      Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.
      Recent interest in “transcriptional disorders of the heart” has also implicated cardiac histone acetylation in the development of pathologic cardiac hypertrophy and heart failure. Histone acetyl transferases (HATs) are believed to acetylate histone proteins, relax chromatin, and expose “prohypertrophic” genes for activation by cardiogenic transcription factors.
      • McKinsey T.A.
      • Olson E.N.
      Cardiac histone acetylation—therapeutic opportunities abound.
      • Yanazume T.
      • Hasegawa K.
      • Morimoto T.
      • Kawamura T.
      • Wada H.
      • Matsumori A.
      • et al.
      Cardiac p300 is involved in myocyte growth with decompensated heart failure.
      Histone deacetylases (HDAC classes I and II) have also been implicated in regulation of myocardial gene expression and cardiac hypertrophy.
      • Backs J.
      • Olson E.N.
      Control of cardiac growth by histone acetylation/deacetylation.
      • McKinsey T.A.
      • Olson E.N.
      Cardiac histone acetylation—therapeutic opportunities abound.
      • Zhang C.L.
      • McKinsey T.A.
      • Chang S.
      • Antos C.L.
      • Hill J.A.
      • Olson E.N.
      Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy.
      Class I HDACs may act as prohypertrophic agonists.
      • Kee H.J.
      • Sohn I.S.
      • Nam K.I.
      • Park J.E.
      • Qian Y.R.
      • Yin Z.
      • et al.
      Inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding.
      • Chen F.
      • Kook H.
      • Milewski R.
      • Gitler A.D.
      • Lu M.M.
      • Li J.
      • et al.
      Hop is an unusual homeobox gene that modulates cardiac development.
      • Lau O.D.
      • Kundu T.K.
      • Soccio R.E.
      • Ait-Si-Ali S.
      • Khalil E.M.
      • Vassilev A.
      • et al.
      HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF.
      Thus, isoform-specific inhibitors of class I HDACs and/or cardiac-specific HATs may have an important and specific inhibitory effect on the development of pathologic cardiac hypertrophy, contractile dysfunction, and heart failure.
      • Kee H.J.
      • Sohn I.S.
      • Nam K.I.
      • Park J.E.
      • Qian Y.R.
      • Yin Z.
      • et al.
      Inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding.
      • Chen F.
      • Kook H.
      • Milewski R.
      • Gitler A.D.
      • Lu M.M.
      • Li J.
      • et al.
      Hop is an unusual homeobox gene that modulates cardiac development.
      • Lau O.D.
      • Kundu T.K.
      • Soccio R.E.
      • Ait-Si-Ali S.
      • Khalil E.M.
      • Vassilev A.
      • et al.
      HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF.
      • de Ruijter A.J.
      • van Gennip A.H.
      • Caron H.N.
      • Kemp S.
      • van Kuilenburg A.B.
      Histone deacetylases (HDACs): characterization of the classical HDAC family.
      • Antos C.L.
      • McKinsey T.A.
      • Dreitz M.
      • Hollingsworth L.M.
      • Zhang C.L.
      • Schreiber K.
      • et al.
      Dose-dependent blockade to cardiomyocyte hypertrophy by histone deacetylase inhibitors.
      Recently, Kee and colleagues
      • Kee H.J.
      • Sohn I.S.
      • Nam K.I.
      • Park J.E.
      • Qian Y.R.
      • Yin Z.
      • et al.
      Inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding.
      have shown that inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding. Similarly, Kong and colleagues
      • Kong Y.
      • Tannous P.
      • Lu G.
      • Berenji K.
      • Rothermel B.A.
      • Olson E.N.
      • et al.
      Suppression of class I and II histone deacetylases blunts pressure-overload cardiac hypertrophy.
      reported that suppression of class I and II HDACs blunts pressure-overload cardiac hypertrophy. Suppression of ventricular growth was well tolerated in terms of both clinical outcome and cardiac performance measures. Interstitial fibrosis was diminished in hearts treated with HDAC inhibitors, and collagen synthesis in isolated cardiac fibroblasts was suppressed. There was no evidence of cell death or apoptosis. Systolic function in the setting of blunted hypertrophic growth was preserved, as shown by echocardiography and hemodynamic data. The hypertrophy-associated switch of adult and fetal sarcomeric isoforms was attenuated, which likely contributed to the observed preservation of systolic function in HDAC inhibitor-treated hearts. Both these studies suggest that HDAC inhibition is a viable therapeutic strategy that holds promise in the treatment of pressure load-induced heart disease. The effects of HDAC inhibition on volume-loaded heart dysfunction are not known.
      The aims of the current study are (1) to biochemically and morphologically characterize a clinically relevant model of pathologic hypertrophy and congestive heart failure and (2) to determine the changes in expression levels and posttranslational modifications of selected cardiac transcription factors. In utero placement of aorta–pulmonary arterial shunts has been reported as a model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs
      • Reddy V.M.
      • Meyrick B.
      • Wong J.
      • Khoor A.
      • Liddicoat J.R.
      • Hanley F.L.
      • et al.
      In utero placement of aortopulmonary shunts A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs.
      and is used in this study as a model of pathologic cardiac hypertrophy. Surgically created aorta-pulmonary shunts in fetal lambs produced left ventricular volume overload and right ventricular pressure overload, leading to altered contractile protein gene expression, biventricular hypertrophy, and congestive heart failure. In the shunted lamb model, pathologic cardiac remodeling and deleterious cardiac gene expression occurred in association with changes in the levels and modification states of cardiac-specific transcription factors (GATA-4, DTEF-1, Sp1, Sp3, and NKX2.5). Furthermore, we show that, functionally, TEF-1, MEF2, and Sp1 factors, which bind the cTnT promoter within the context of chromatin, transactivate the cTnT promoter in ovine cardiomyocytes, implicating them as important regulators of the pathologic hypertrophic process.

      Materials and Methods

      Reagents

      Media, serum, enzymes, reagents, and materials for tissue culture were purchased from the Cell Culture facility at the University of California, San Francisco. Oligonucleotides were purchased from Operon Technologies Inc (Alameda, Calif) or Invitrogen (Carlsbad, Calif).

      Plasmids and Constructs: Tissue Culture, Cell Transfections, and Reporter Gene Assays

      The promoter/reporter constructs used in this study consist of 268 nucleotides of the chicken cTnT promoter upstream of the transcription initiation site (Gallus domesticus cardiac isoform of troponin T [TNT] gene, exon 1, and promoter sequence. ACCESSION# M5790). Luciferase reporter constructs were created and subcloned into the plasmid pGL2 (Promega, Madison Wis), as previously described.
      • Azakie A.
      • Lamont L.
      • Fineman J.R.
      • He Y.
      Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.
      Hearts were harvested from neonatal lambs and cardiomyocyte cultures established by standard techniques.
      • Azakie A.
      • Larkin S.B.
      • Farrance I.K.
      • Grenningloh G.
      • Ordahl C.P.
      DTEF-1, a novel member of the transcription enhancer factor-1 (TEF-1) multigene family.
      The differentiation state of cultured cardiomyocytes was determined by light microscopy, presence of beating cardiomyocytes, immunocytochemistry for MF20, and Western blotting for myosins, cTnT, GATA-4, MEF2, and NKX2.5. Plasmid constructs were transfected according to the manufacturer’s protocol (Effectene Transfection kit; Qiagen, Valencia, Calif) with minor modifications.
      • Azakie A.
      • Lamont L.
      • Fineman J.R.
      • He Y.
      Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.
      Reporter gene activity was determined with the Dual Luciferase reporter assay (DLR-Promega, Madison, Wis). Firefly light intensity was read on a Luminometer TD-20/20, DLR Ready (Turner Designs Instrument, Sunnyvale, Calif). Cotransfection with 20 ng phRL-TK reporter was used to standardize for transfection variability. Cotransfection efficiency of Sp, MEF2, and DTEF-1 expression constructs was verified with pSV-β-galactosidase (Promega, Madison Wis) and/or pAAV-GFP staining. Reporter gene activity was expressed as mean ± standard deviation. Statistical comparisons were performed by paired t tests.

      Nuclear Extracts

      Cardiac muscle was harvested from 2-week-old lambs (control and shunted) at 4°C. Nuclear extracts were prepared by standard methods (Active Motif, Inc, Carlsbad, Calif).

      Western Blot Analysis and Densitometry

      Total protein concentration was quantitated with the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, Calif). An equal amount of protein was loaded in each lane for Western blots: sodium dodecylsulfate–polyacrylamide gel electrophoresis was used to separate nuclear proteins (10 μg) on a 10% gel and followed by transfer to a polyvinylidene difluoride membrane (Amersham Biosciences, GE Healthcare Bio-Sciences Corp, Piscataway, NJ). Membranes were then blocked and incubated with antibody as previously described.
      • Azakie A.
      • Lamont L.
      • Fineman J.R.
      • He Y.
      Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.
      Reactive bands were visualized with the SuperSignal West Femto Maximum Sensitivity Substrate Kit (Pierce, Rockford, Ill) and Kodak 440CF image station (Kodak, New Haven, Conn). The image was selected and analyzed with the public domain program NIH Image, and band intensity was quantified. Parallel sodium dodecylsulfate gels and immunoblots of β-actin and glyceraldehyde-3-phosphate dehydrogenase controls were performed to verify sample integrity and loaded quantity.

      Surgical Preparations and Care: Ewes

      Pregnant, mixed-breed Western ewes (135-140 days’ gestation; term = 145 days) were operated on under sterile conditions. Through a left lateral fetal thoracotomy, an 8.0-mm polytetrafluoroethylene vascular graft (2-mm length; W. L. Gore & Associates, Inc, Flagstaff, Ariz) was sewn between the ascending aorta and main pulmonary artery. All lambs were spontaneously delivered and were instrumented as previously described.
      • Reddy V.M.
      • Meyrick B.
      • Wong J.
      • Khoor A.
      • Liddicoat J.R.
      • Hanley F.L.
      • et al.
      In utero placement of aortopulmonary shunts A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs.

      Heart Tissue Preparation

      Whole heart and chamber-specific weights were measured in all shunted and control specimens. Samples of right atrium, right ventricle, left atrium, left ventricle, and ventricular septum were excised, weighed, and used to make fresh nuclear preparations. Whole tissue and excess nuclear preparations were snap-frozen in liquid nitrogen. Samples were stored at −70°C until used for analysis.

      Determination of DNA Content

      DNA content was determined by standard methods (Genomic DNA mini-prep; Bay Gene Inc, San Francisco, Calif). Values were normalized to gram of cardiac muscle.

      Statistical Analysis

      Comparisons between shunt and age-matched controls were made by an unpaired t test.
      All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco. All animals were put to death by appropriate methods as described in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

      Results

      In Utero Placement of Aorta–Pulmonary Shunts Results in Growth Failure, Four-chamber Cardiomegaly, and Biventricular Hypertrophy

      Shunted lambs showed clinical evidence of failure to thrive. Compared with control animals, shunted lambs were tachypneic and demonstrated less activity and lower tolerance to stress after 1 week of age. By 2 weeks of age, shunted lambs had significantly slower weight gain than controls. The hearts of shunted lambs were significantly larger than those of controls (Figure 1, Table 1, and Figure E1). All four chambers showed substantial increases in mass without significant increases in DNA content, suggesting that hypertrophy and not hyperplasia was the predominant stress response. The left ventricle was dilated and showed evidence of eccentric hypertrophy, while the right ventricle was predominantly thick-walled with evidence of concentric hypertrophy.
      Figure thumbnail gr1
      Figure 1Gross pathology of shunted hearts at 2 weeks of age. Left panel, Photograph of shunted heart. Right panel, Photograph of control heart.
      TABLE 1General hemodynamics in 2-week-old lambs
      Control lambsShunted lambs
      Mean arterial blood pressure (mm Hg)65 ± 755 ± 7
      Pulmonary arterial pressure (mm Hg)15 ± 222 ± 4
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Left pulmonary blood flow (mL · kg−1 · min−1)60 ± 21157 ± 28
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Heart rate (beats/min)171 ± 24174 ± 25
      Left atrial pressure (mm Hg)3 ± 1.77 ± 3.4
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Right atrial pressure (mm Hg)2.2 ± 1.44.8 ± 3.1
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Pulmonary/systemic blood flow1.002.91 ± 01
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Right ventricular weight (g)11 ± 221 ± 5
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Left ventricular weight (g)19 ± 431 ± 7
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Total cardiac weight (g)49 ± 680 ± 8
      P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Values are means ± SD; n = 6 shunted lambs and 7 twin or age-matched controls.
      low asterisk P < .05 shunt versus control lambs; weight comparisons were performed for 5 control and 5 shunted lambs.
      Figure thumbnail gre1
      Figure E1Shunted hearts showed 4-chamber cardiomegaly.

      Hemodynamics of 2-week-old Shunted Lambs

      Table 1 summarizes the hemodynamics of shunted versus control lambs at 2 weeks of age. Shunted animals had significantly higher mean pulmonary artery pressures with a mean pulmonary/systemic flow ratio of 2.9:1. Left and right atrial pressures were significantly higher in shunted animals.

      Sarcomeric Protein and Myocardial Transcription Factor Levels Are Altered During Pathophysiologic Shunting in the Neonatal Ovine Heart

      Western blots of shunted (n = 5) and control (n = 5) hearts were analyzed by band densitometry. The hearts of 2-week-old shunted lambs showed significant increases in myosin and cTnT expression (Figure 2 and Figure 3, A and B). Fetal, high-molecular-weight isoforms of cTnT were upregulated, consistent with the activation of a pathologic hypertrophic program (Figure 2). The increase in sarcomeric protein expression and associated fetal isoforms was seen in all four chambers. Associated with the increase in sarcomeric proteins, a variety of cardiac transcription factors known to govern cardiac gene expression during embryogenesis, growth, and development were upregulated (Figure 2, Figure 3). TEF-1 factors, which regulate muscle gene promoters (eg, cTnT, α- and β-myosin heavy chains, tropomyosins, β-acetylcholine receptor, and α-actin genes) through muscle CAT sites, were significantly upregulated in the right side of the heart (Figure 3, C). There was a trend to increased expression in the left atrium and left ventricle. Both GATA-4 and NKX2.5 levels were significantly increased in all four chambers (Figure 3, D and E). The magnitude of the increase in NKX2.5 levels, although statistically significant, was small (Figure 3, E). Densitometric analysis of Sp factor expression showed that Sp1, a transcriptional activator of cardiac promoters, was increased in all four chambers; Sp3, a transcriptional repressor of cTnT promoter constructs, was significantly downregulated during pathologic hypertrophy.
      Figure thumbnail gr2
      Figure 2Characteristic Western blot of sarcomeric proteins and selected transcription factors in 2-week-old control and shunted lambs. Antibody used for Western blot analysis is listed on left. MF20 (antimyosin) and cTnT levels are increased in shunted animals, consistent with the hypertrophy and increased mass seen on gross pathologic examination. Fetal/embryonic isoforms of cTnT are expressed in the hypertrophied, shunted hearts. There are associated increases in DTEF-1, MEF2 (not shown), GATA4, NKX2.5, and Sp1 levels and concomitant decreases in Sp3 levels within the cardiac chambers (see ). β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control proteins are comparable in shunted and control specimens. RA, Right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; Sept, interventricular septal muscle; MW (kDa), molecular weight in kilo-Daltons.
      Figure thumbnail gr3
      Figure 3Bar graphs of densitometric analysis of selected myocardial transcription factors during pathologic biventricular hypertrophy in neonatal lambs. Bands seen on Western blots of myocardial specimens from hearts of control (n = 5) and shunted (n = 5) neonatal lamb were analyzed by quantitative densitometry. Myosins and cardiac troponin protein expression are increased in all four chambers. TEF-1 proteins are primarily increased in the right side of the heart whereas NKX 2.5, GATA-4, and Sp1 are upregulated and Sp3 is downregulated in all four cardiac chambers. A, Myosin expression detected with MF20 antibody. B, Cardiac troponin T (cTnT). C. TEF-1 proteins. D, GATA-4. E, NKX2.5. F, Sp1. G, Sp3. β-actin (H) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (I) were used as controls. RA, Right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; Septum, interventricular septal muscle.
      Immunohistochemistry was performed to determine the cell type and compartment within which transcription factors were localized (Figure E2, Figure E3, Figure E4). Previous studies have shown that MEF2, GATA-4, and NKX2.5 predictably localize to the nucleus of myocytes and act as transcriptional activators.
      • Charron F.
      • Nemer M.
      GATA transcription factors and cardiac development.
      • Jamali M.
      • Rogerson P.J.
      • Wilton S.
      • Skerjanc I.S.
      Nkx2-5 activity is essential for cardiomyogenesis.
      • Martin J.F.
      • Schwarz J.J.
      • Olson E.N.
      Myocyte enhancer factor (MEF) 2C: a tissue-restricted member of the MEF-2 family of transcription factors.
      • Cripps R.M.
      • Zhao B.
      • Olson E.N.
      Transcription of the myogenic regulatory gene Mef2 in cardiac, somatic, and visceral muscle cell lineages is regulated by a Tinman-dependent core enhancer.
      • Han Z.
      • Olson E.N.
      Hand is a direct target of Tinman and GATA factors during Drosophila cardiogenesis and hematopoiesis.
      • Harvey R.P.
      NK-2 homeobox genes and heart development.
      • Olson E.N.
      • Perry M.
      • Schulz R.A.
      Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.
      TEF-1, Sp1, and Sp3 are expressed within the myocyte population of the ovine heart and localize to the nucleus. By chromatin immunoprecipitation, DTEF-1, MEF2, GATA-4, NKX2.5, Sp1, Sp3, and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) bind the ovine cTnT promoter “in vivo,” within the context of chromatin (Figure E5). Furthermore, anti-PAR and pan-acetyl antibody immunoprecipitated chromatin from cardiomyocytes that yielded a positive chromatin immunoprecipitation (ChIP) assay for cTnT binding, implicating that these epigenetic modifications (poly-ADP-ribosylation and acetylation) were present in the transcriptional proteins that bound the cTnT promoter in vivo. Co-immunoprecipitation experiments with MEF2 and neonatal cardiomyocytes confirmed that Sp factors, PARP, NKX2.5, and DTEF-1 directly interacted to potentially form a transcriptional complex (data not shown).
      Figure thumbnail gre2
      Figure E2Immunohistochemistry of sheep myocardium double-stained with MF20 (red) antibody and Sp1 antibody (green immunofluorescence).
      Figure thumbnail gre3
      Figure E3Immunohistochemistry of sheep myocardium double-stained with MF20 (red) antibody and Sp3 antibody (green immunofluorescence).
      Figure thumbnail gre4
      Figure E4Immunohistochemistry of sheep myocardium double-stained with MF20 (red) antibody and DTEF-1 antibody (green immunofluorescence).
      Figure thumbnail gre5
      Figure E5Transcriptional complexes bind the cTnT promoter in vivo. Polymerase chain reaction using primers specific for cTnT promoter on DNA purified after chromatin immunoprecipitation. After cross-linking and sonication, chromatin complexes from neonatal sheep heart nuclear preparations were purified. Chromatin was incubated with antibodies to DTEF-1, MEF2, GATA-4, Nkx2.5, Sp1, Sp3, poly-ADP-ribose polymerase (PARP), poly-ADP-ribose (PAR), β-actin, cTnT, and preimmune serum. Separation of immune complexes by elution from protein A/G/agarose beads was followed by reversal of protein-DNA crosslinks and DNA purification. Lane A depicts chromatin input DNA plus antibody. Lanes A-E include the ChIP “output” DNA for polymerase chain reactions. Immunoprecipitation of chromatin preparations was performed with antibodies listed on the left-sided column. Lane F contains ChIP buffer as the “DNA source.” Lane G contains the control, “input DNA” to verify the quality of nuclear preparations. a, Output DNA; b, input DNA; c, polymerase chain reaction buffer and primers.

      Transcriptional Effects on cTnT Promoter Activity

      Having shown that selected cardiac transcription factors are modulated during pathologic hypertrophy and bind the cTnT promoter (and are associated with epigenetic modifications of acetylation and poly-ADP-ribosylation), in vivo, we sought to determine their transactivating effects. Expression constructs of DTEF-1A and B, MEF2C, Sp1, and Sp3 were cotransfected with cTnT promoter constructs in primary neonatal sheep cardiomyocytes in culture (Figure 4). Relative activity was indexed to a reporter gene driven by thymidine kinase to control for transfection variability. Both isoforms of DTEF-1, A and B, transactivated the 268cTnT promoter approximately 3.2-fold and 3.6-fold above baseline, respectively (268cTnT = 16 ± 3 relative light units vs 268cTnT + DTEF1A= 51 ± 7 relative units; 268cTnT + DTEF-1B = 58 ± 6 relative units, n = 5 transfections per construct, P < .01). Cotransfection of MEF2C with the 268cTnT promoter construct resulted in a 4.3-fold increase in baseline reporter gene activity (268cTnT + MEF2C = 70 ± 6 relative units, n = 5, P < .01.). Cotransfection of both MEF2C and DTEF-1B expression constructs produced a 5.7-fold increase in reporter gene activity (92 ± 8 relative units, n = 5, P < .01). Sp1 acted as a transcriptional activator of −268cTnT promoter constructs that were transiently transfected in ovine cardiomyocytes in culture, producing a 2.2-fold increase in baseline reporter gene activity (36 ± 6 relative units, n = 5, P < .01). Sp3 repressed baseline −268cTnT promoter activity by over 50% and inhibited Sp1-mediated transcriptional activation of the promoter (Figure 4).
      Figure thumbnail gr4
      Figure 4Luciferase reporter gene activity driven by −268cTnT promoter constructs that are transiently transfected in primary cultures of neonatal ovine cardiomyocytes. Baseline –268cTnT relative activity is increased when Sp1, DTEF-1A or B, and MEF2C are cotransfected. Cotransfection of Sp3 results in repression of baseline cTnT promoter activity. Sp3 inhibits Sp1-mediated gene activation. MEF2 and DTEF-1 have a synergistic effect on reporter gene activity. Horizontal axis, Cotransfected expression constructs. Vertical axis, Relative reporter gene activity indexed to TK-driven reporter to control for transfection variability. Cells alone, pGL2 basic vector, and pcDNA4HisMax expression constructs have undetectable activity (not shown). Anti-luciferase staining of cardiomyocyte cultures shows reporter gene expression restricted to myocyte population.

      Discussion

      In utero placement of aorta–pulmonary shunts produces pathologic biventricular hypertrophy and clinical congestive heart failure in neonatal lambs. There are associated increases in constitutively expressed sarcomeric proteins and induction of fetal isoforms of contractile proteins in all four cardiac chambers. In this model, there is upregulation of myocardial transcriptional activators and downregulation of repressors. Furthermore, cardiac transcription factors also undergo changes in interactions with cardiac histone acetylating and deacetylating enzymes, as well as ADP-ribose polymerases, each of which are potential pharmacologic targets of PARP, HDAC, and HAT inhibitors.
      • Backs J.
      • Olson E.N.
      Control of cardiac growth by histone acetylation/deacetylation.
      • Kim M.Y.
      • Mauro S.
      • Gevry N.
      • Lis J.T.
      • Kraus W.L.
      NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1.

      In Vivo Model of Pathologic Cardiac Hypertrophy

      Murine models of cardiac hypertrophy are favorable because the mouse is amenable to transgenic manipulation, allowing for the identification of molecular mechanisms of pathophysiologic processes. Aortic banding or constriction in the mouse, which has a heart rate of 500 beats/min and chamber dimensions in the millimeter range, is a commonly used animal model for the study of pathologic hypertrophy. Echocardiographic or hemodynamic analysis of murine models, although well established, requires special equipment and expertise. Compared with mouse models of pathologic hypertrophy, the ovine model has some advantages. It is amenable to standard echocardiographic and catheterization techniques and is not limited to the study of only pressure loading of the left side of the heart, but can be used to study other biomechanical stressors including volume loading of the heart. Furthermore, pathologic hypertrophy can be generated in either the left or right ventricle, or both, thus allowing for the determination of common or asymmetric transcriptional responses of either side of the heart.
      The ovine model used in this study, that is, in utero placement of a large aorta-pulmonary shunt in fetal lambs, produces significant left-to-right shunting and increases in pulmonary blood flow and pulmonary hypertension. The left ventricular myocardium is stressed by persistent and significant volume loading, while the right ventricle eventually experiences pressure loading. This model is clinically relevant and mimics some congenital heart defects, such as patent ductus arteriosus, large ventricular septal defects, and aorta-pulmonary windows. Furthermore, volume loading of the left ventricle is also clinically seen with valvular heart disease such as aortic and mitral insufficiency. Right ventricular pressure overload is also commonly seen clinically, as in pulmonary hypertension, tetralogy of Fallot, and pulmonary stenosis. Last, the model mimics the surgically created aorta–pulmonary shunts used for the management of single ventricle anomalies with inadequate pulmonary blood flow.
      The central features of the cardiomyocyte hypertrophic response include increases in contractile protein content, induction of contractile protein isoforms, and expression of embryonic markers.
      • Chien K.R.
      • Knowlton K.U.
      • Zhu H.
      • Chien S.
      Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.
      The observed protein biochemical changes in this model are consistent with expected changes in sarcomeric protein expression. There is an upregulation of constitutively expressed genes (myosins), as well as induction of fetal contractile and noncontractile cardiac genes (high-molecular-weight troponin isoforms and atrial natriuretic factor/brain natriuretic peptide [data not shown]). Proto-oncogenes and other members of the immediate-early gene programs, also known to be activated during hypertrophy, were not evaluated in this study, nor were regulators of cell survival and apoptosis.
      • Chien K.R.
      • Knowlton K.U.
      • Zhu H.
      • Chien S.
      Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.
      The finding that TEF-1, NKX2.5, Sp1, and GATA factors are upregulated during the cardiac hypertrophic response is consistent with their chromatin binding and transactivating properties. Likewise, downregulation of Sp3 during pathologic hypertrophy is consistent with its inhibitory effects on cTnT promoter activity. These data suggest that the increase in sarcomeric proteins and induction of embryonic cardiac genes seen during pathologic hypertrophy are preceded and regulated by increases in myocardial transactivators, decreases in cardiac gene repressors, and induction of posttranslational modifications that modulate transcription factor function. These data do not provide direct evidence that TEF-1, MEF2, NKX2.5, GATA-4, and Sp1 upregulation and Sp3 downregulation, in vivo, are mechanistically responsible, in part, for the changes in sarcomeric protein expression that are seen during pathologic cardiac hypertrophy in the ovine model. Further studies are warranted to investigate these important mechanisms.
      The TEF-1 family of transcription factors has been implicated in muscle gene regulation.
      • Farrance I.K.
      • Ordahl C.P.
      The role of transcription enhancer factor-1 (TEF-1) related proteins in the formation of M-CAT binding complexes in muscle and non-muscle tissues.
      We have previously cloned and characterized DTEF-1, a cardiac-enriched family member that regulates cardiac gene expression and is expressed in the early embryo.
      • Azakie A.
      • Lamont L.
      • Fineman J.R.
      • He Y.
      Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.
      • Azakie A.
      • Larkin S.B.
      • Farrance I.K.
      • Grenningloh G.
      • Ordahl C.P.
      DTEF-1, a novel member of the transcription enhancer factor-1 (TEF-1) multigene family.
      During volume loading of the left ventricle and pressure loading of the right ventricle, TEF-1 polypeptides are upregulated to a greater degree in the right side of the heart. TEF-1 upregulation during biomechanical stress may therefore be an asymmetric, chamber-specific response or may act as a pressure-responsive rather than volume-responsive transactivator. Poly-ADP-ribose polymerase, a known coactivator of TEF-1,
      • Butler A.J.
      • Ordahl C.P.
      Poly(ADP-ribose) polymerase binds with transcription enhancer factor 1 to MCAT1 elements to regulate muscle-specific transcription.
      has been implicated in myocardial cell hypoxic-ischemic injury and cardiomyocyte survival. In this model, we have also found that DTEF-1 becomes heavily ADP-ribosylated (data not shown), a process amenable to inhibition and potential target for pharmacotherapy of heart failure.
      • Szabo C.
      Cardioprotective effects of poly(ADP-ribose) polymerase inhibition.
      The Sp family of transcription factors is important in the assembly of the basal transcriptional machinery, potentially acting as a central and widespread regulatory network in pathologic hypertrophy. We have previously shown that Sp1 and Sp3 have counterregulatory functions in avian myocytes.
      • Azakie A.
      • Fineman J.R.
      • He Y.
      Sp3 inhibits Sp1 mediated activation of the cardiac troponin T promoter and is downregulated during pathologic cardiac hypertrophy in vivo.
      Although Sp3 expression is downregulated during pathologic hypertrophy, we have found that Sp3-associated HAT activity is increased and HDAC activity is significantly decreased (Figure E6, Figure E7). Hence, acetylation of Sp3 may convert it from a repressor to a transcriptional activator during the induction of pathologic cardiac hypertrophy and heart failure.
      Figure thumbnail gre6
      Figure E6Histone acetyl transferase (HAT) activity associated with immunoprecipitated Sp3 is upregulated during pathologic cardiac hypertrophy.
      Figure thumbnail gre7
      Figure E7Histone deacetylase activity associated with immunoprecipitated Sp3 is decreased during pathologic hypertrophy.
      Histone acetylation has been implicated in relaxing chromatin and globally exposing genes for transcription. Removal of acetyl groups is presumed to result in chromatin compaction, making some promoters inaccessible for transcriptional activation. Inhibition of HDAC activity, and particularly class I HDACs, has ironically been associated with attenuation of cardiac growth and hypertrophy in vitro and in vivo. The exact mechanism(s) by which HDAC inhibition attenuates hypertrophy is not entirely known. Class II HDACs are antihypertrophic, whereas class I HDACs may stimulate cardiac growth and sarcomeric genes. Selective HDAC or HAT inhibitors are being actively developed and are in trials for the treatment of cancer. As such, identification of the specific HATs or HDACs associated with hypertrophy-responsive transcription factors, such as GATA-4, DTEF-1, or Sp1/3, (1) may allow for the identification of the transcriptional mechanisms of pathologic hypertrophy and (2) may allow for the use of isoform-specific HAT/HDAC inhibitors to target pathologic hypertrophy and contractile dysfunction of the heart.
      We thank Leonard Moon for assistance with the manuscript and figures.

      Appendix E1: Chromatin Immunoprecipitation Supplement

      Method: Chromatin Immunoprecipitation (ChIP)

      All ChIP assays were performed on the basis of the protocols of the chromatin immunoprecipitation assay kit (Upstate, Lake Placid, NY) [Azakie, 2005—AJP]. Five micrograms of primary antibody against DTEF-1, GATA-4, MEF2, NKX2.5, Sp1, Sp3, PARP, or PAR was added to a 500 μL chromatin sample for the immunoprecipitation reaction. ChIP dilution buffer and preimmune rabbit serum were used as controls for nonspecific interactions and DNA contamination. Anti-β-actin and anti-cTnT antibodies were used as negative controls.

      Polymerase Chain Reaction

      After DNA purification, samples were subjected to polymerase chain reaction with primers designed for the chick heart cTnT promoter (cTnT-268) as follows: upper primer: 5′GCTGGCTGGCTTGTGTCA-3′; lower primer: 5′-CTTGGGGGGCAGAGGCTTT-3′.
      The primers used for polymerase chain reaction were designed by a primer analysis software (Oligo 6.8; Molecular Biology Insights, Inc, Cascade, Colo). The amplified polymerase chain reaction product is 265 bp.

      References

        • Hunter J.J.
        • Chien K.R.
        Signaling pathways for cardiac hypertrophy and failure.
        N Engl J Med. 1999; 341: 1276-1283
        • Chien K.R.
        Stress pathways and heart failure.
        Cell. 1999; 98: 555-558
        • McKinsey T.A.
        • Olson E.N.
        Toward transcriptional therapies for the failing heart: chemical screens to modulate genes.
        J Clin Invest. 2005; 115: 538-546
        • Backs J.
        • Olson E.N.
        Control of cardiac growth by histone acetylation/deacetylation.
        Circ Res. 2006; 98: 15-24
        • Charron F.
        • Nemer M.
        GATA transcription factors and cardiac development.
        Semin Cell Dev Biol. 1999; 10: 85-91
        • Jamali M.
        • Rogerson P.J.
        • Wilton S.
        • Skerjanc I.S.
        Nkx2-5 activity is essential for cardiomyogenesis.
        J Biol Chem. 2001; 276: 42252-42258
        • Martin J.F.
        • Schwarz J.J.
        • Olson E.N.
        Myocyte enhancer factor (MEF) 2C: a tissue-restricted member of the MEF-2 family of transcription factors.
        Proc Natl Acad Sci U S A. 1993; 90: 5282-5286
        • McBride K.
        • Nemer M.
        Regulation of the ANF and BNP promoters by GATA factors: lessons learned for cardiac transcription.
        Can J Physiol Pharmacol. 2001; 79: 673-681
        • Azakie A.
        • Lamont L.
        • Fineman J.R.
        • He Y.
        Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.
        Am J Physiol Cell Physiol. 2005; 289: C1522-C1534
        • Azakie A.
        • Larkin S.B.
        • Farrance I.K.
        • Grenningloh G.
        • Ordahl C.P.
        DTEF-1, a novel member of the transcription enhancer factor-1 (TEF-1) multigene family.
        J Biol Chem. 1996; 271: 8260-8265
        • Chien K.R.
        • Knowlton K.U.
        • Zhu H.
        • Chien S.
        Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.
        Faseb J. 1991; 5: 3037-3046
        • McKinsey T.A.
        • Olson E.N.
        Cardiac histone acetylation—therapeutic opportunities abound.
        Trends Genet. 2004; 20: 206-213
        • Yanazume T.
        • Hasegawa K.
        • Morimoto T.
        • Kawamura T.
        • Wada H.
        • Matsumori A.
        • et al.
        Cardiac p300 is involved in myocyte growth with decompensated heart failure.
        Mol Cell Biol. 2003; 23: 3593-3606
        • Zhang C.L.
        • McKinsey T.A.
        • Chang S.
        • Antos C.L.
        • Hill J.A.
        • Olson E.N.
        Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy.
        Cell. 2002; 110: 479-488
        • Kee H.J.
        • Sohn I.S.
        • Nam K.I.
        • Park J.E.
        • Qian Y.R.
        • Yin Z.
        • et al.
        Inhibition of histone deacetylation blocks cardiac hypertrophy induced by angiotensin II infusion and aortic banding.
        Circulation. 2006; 113: 51-59
        • Chen F.
        • Kook H.
        • Milewski R.
        • Gitler A.D.
        • Lu M.M.
        • Li J.
        • et al.
        Hop is an unusual homeobox gene that modulates cardiac development.
        Cell. 2002; 110: 713-723
        • Lau O.D.
        • Kundu T.K.
        • Soccio R.E.
        • Ait-Si-Ali S.
        • Khalil E.M.
        • Vassilev A.
        • et al.
        HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF.
        Mol Cell. 2000; 5: 589-595
        • de Ruijter A.J.
        • van Gennip A.H.
        • Caron H.N.
        • Kemp S.
        • van Kuilenburg A.B.
        Histone deacetylases (HDACs): characterization of the classical HDAC family.
        Biochem J. 2003; 370: 737-749
        • Antos C.L.
        • McKinsey T.A.
        • Dreitz M.
        • Hollingsworth L.M.
        • Zhang C.L.
        • Schreiber K.
        • et al.
        Dose-dependent blockade to cardiomyocyte hypertrophy by histone deacetylase inhibitors.
        J Biol Chem. 2003; 278: 28930-28937
        • Kong Y.
        • Tannous P.
        • Lu G.
        • Berenji K.
        • Rothermel B.A.
        • Olson E.N.
        • et al.
        Suppression of class I and II histone deacetylases blunts pressure-overload cardiac hypertrophy.
        Circulation. 2006; 113: 2579-2588
        • Reddy V.M.
        • Meyrick B.
        • Wong J.
        • Khoor A.
        • Liddicoat J.R.
        • Hanley F.L.
        • et al.
        In utero placement of aortopulmonary shunts.
        Circulation. 1995; 92: 606-613
        • Cripps R.M.
        • Zhao B.
        • Olson E.N.
        Transcription of the myogenic regulatory gene Mef2 in cardiac, somatic, and visceral muscle cell lineages is regulated by a Tinman-dependent core enhancer.
        Dev Biol. 1999; 215: 420-430
        • Han Z.
        • Olson E.N.
        Hand is a direct target of Tinman and GATA factors during Drosophila cardiogenesis and hematopoiesis.
        Development. 2005; 132: 3525-3536
        • Harvey R.P.
        NK-2 homeobox genes and heart development.
        Dev Biol. 1996; 178: 203-216
        • Olson E.N.
        • Perry M.
        • Schulz R.A.
        Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.
        Dev Biol. 1995; 172: 2-14
        • Kim M.Y.
        • Mauro S.
        • Gevry N.
        • Lis J.T.
        • Kraus W.L.
        NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1.
        Cell. 2004; 119: 803-814
        • Farrance I.K.
        • Ordahl C.P.
        The role of transcription enhancer factor-1 (TEF-1) related proteins in the formation of M-CAT binding complexes in muscle and non-muscle tissues.
        J Biol Chem. 1996; 271: 8266-8274
        • Butler A.J.
        • Ordahl C.P.
        Poly(ADP-ribose) polymerase binds with transcription enhancer factor 1 to MCAT1 elements to regulate muscle-specific transcription.
        Mol Cell Biol. 1999; 19: 296-306
        • Szabo C.
        Cardioprotective effects of poly(ADP-ribose) polymerase inhibition.
        Pharmacol Res. 2005; 52: 34-43
        • Azakie A.
        • Fineman J.R.
        • He Y.
        Sp3 inhibits Sp1 mediated activation of the cardiac troponin T promoter and is downregulated during pathologic cardiac hypertrophy in vivo.
        Am J Physiol Heart Circ Physiol. 2006; 291: H600-H611

      Linked Article

      • Discussion
        The Journal of Thoracic and Cardiovascular SurgeryVol. 132Issue 6
        • Preview
          Dr Robert C. Robbins (Stanford, Calif). You went through it very quickly, but this is a tour de force; technically it is a very difficult model and you are to be congratulated for getting the animals through this. Also impressive is the sophisticated use of molecular genetics that you used here to help answer important questions. Certainly heart failure is an important public health issue. Finally, the experimental therapeutic approach to try to identify potential targets for developing small molecules or other strategies to treat heart failure is impressive.
        • Full-Text
        • PDF