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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Serum response factor neutralizes Pur- and Purβ-mediated repression of the fetal vascular smooth muscle -actin gene in stressed adult cardiomyocytes

Aiwen Zhang,¹ Jason J. David,¹ Sukanya V. Subramanian,¹ Xiaoying Liu,¹ Matthew D. Fuerst,¹ Xue Zhao,⁴ Carl V. Leier,² Charles G. Orosz,^3, Robert J. Kelm, Jr.,⁵ and Arthur R. Strauch¹

Departments of ¹Physiology and Cell Biology, ²Internal Medicine, and ³Surgery, the Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, Columbus, Ohio; ⁴Department of Cardiology, Changzheng Hospital, Second Military Medical University, Shanghai, China; and ⁵Department of Medicine, College of Medicine, University of Vermont, Colchester, Vermont

Submitted 25 April 2007 ; accepted in final form 8 January 2008

	ABSTRACT

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Mouse hearts subjected to repeated transplant surgery and ischemia-reperfusion injury develop substantial interstitial and perivascular fibrosis that was spatially associated with dysfunctional activation of fetal smooth muscle -actin (SMA) gene expression in graft ventricular cardiomyocytes. Compared with cardiac fibroblasts in which nuclear levels of the Sp1 and Smad 2/3 transcriptional-activating proteins increased markedly after transplant injury, the most abundant SMA gene-activating protein in cardiomyocyte nuclei was serum response factor (SRF). Additionally, cardiac intercalated discs in heart grafts contained substantial deposits of Pur, an mRNA-binding protein and known negative modulator of SRF-activated SMA gene transcription. Activation of fetal SMA gene expression in perfusion-isolated adult cardiomyocytes was linked to elevated binding of a novel protein complex consisting of SRF and Pur to a purine-rich DNA element in the SMA promoter called SPUR, previously shown to be required for induction of SMA gene transcription in injury-activated myofibroblasts. Increased SRF binding to SPUR DNA plus one of two nearby CArG box consensus elements was observed in SMA-positive cardiomyocytes in parallel with enhanced Pur:SPUR protein:protein interaction. The data suggest that de novo activation of the normally silent SMA gene in reprogrammed adult cardiomyocytes is linked to elevated interaction of SRF with fetal-specific CArG and injury-activated SPUR elements in the SMA promoter as well as the appearance of novel Pur protein complexes in both the nuclear and cytosolic compartments of these cells.

smooth muscle actin; cardiac fibrosis; cardiac transplant; gene transcription

We previously showed that de novo activation of SMA gene transcription was an early indicator of myofibroblast differentiation and cardiomyocyte fetal gene reprogramming in accepted murine heart allografts (47, 48). SMA gene activation in myofibroblasts involved TGF-β1-regulated interplay of the Pur /β transcriptional repressors within a 200-bp region of the promoter that contains essential binding sites for the TEF1, Sp1, serum response factor (SRF), and Smad 2/3 transcriptional activators (10, 11, 30, 49, 57). While reactivation of the developmentally silenced SMA gene in cardiomyocytes has been described in rodent models of heart failure (8), very little is known about its molecular control, particularly in the context of cardiac transplant, fibrosis, and chronic rejection pathobiology. To explore this question, we used a syngeneic mouse heart graft model involving two rounds of transplant surgery and ischemia-reperfusion injury (53) that causes extensive interstitial and perivascular fibrosis and activation of fetal SMA gene expression in cardiomyocytes. Compared with cardiac fibroblasts where retransplant was linked to increased nuclear levels of Sp1, Smad 2, and Smad 3, the most abundant SMA gene-activating protein in cardiomyocytes was SRF. In parallel studies on perfusion-isolated, mechanically unloaded adult rat cardiomyocytes, we observed collaborative interaction between cardiac SRF and the Pur SMA gene repressor protein at the SPUR cis-regulatory promoter element previously shown to mediate TGF-β1 responsiveness in myofibroblasts (49). Moreover, there was notable accumulation of Pur at cardiac intercalated discs that were located proximal to nascent SMA-enriched thin filaments. Dynamic interplay between the SRF activator and Pur repressor proteins at the CArG and SPUR transcriptional regulatory sites represents a new, possibly rate-limiting step for activation of fetal SMA gene expression in stressed cardiomyocytes after cardiac transplant.

	MATERIALS AND METHODS

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Murine syngeneic heart transplantation. Five-week-old, specific pathogen-free female FVB/N mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) or from the Jackson Laboratory (Bar Harbor, ME) and were maintained in a barrier facility for use as heart donors and graft recipients. Donor hearts were heterotopically transplanted into the abdomen of recipient mice using an adaptation of the method of Corry et al. (13) as described in detail in our earlier reports (2–4). Three groups of three syngeneic cardiac grafts (isografts) were harvested 15 days after transplantation, retransplanted into another cohort of FVB/N recipients, and maintained for a second 15-day, posttransplant observation period, during which time grafts were explanted on days 3, 7, 11, and 15 for immunohistochemical and biochemical analyses. Controls were nontransplanted donor hearts from age- and sex-matched FVB/N mice or isografts subjected to only a single round of transplant surgery into FVB/N recipients and harvested at either 15 days or 26 days after transplant. To prepare protein extracts for DNA-binding or immunoblot studies, explanted or nontransplanted control hearts were visualized under a dissection microscope and trimmed to remove the atria and coronary vessels. Trimmed ventricles were frozen in liquid nitrogen, ground to powder while submerged under liquid nitrogen using a mortar and pestle, and extracted as described below. To prepare FVB/N donor mice harboring a SMA promoter:green fluorescent protein (GFP) reporter transgene (VSMP8EGFP), the enhanced GFP gene sequence (BD Biosciences Clonetech, Palo Alto, CA) was ligated to the 1.5-kb mouse VSMP8 promoter (37, 55) and microinjected into mouse embryos using standard protocols used by the Transgenic Mouse Facility in the Neurobiotechnology Center at The Ohio State University. Mice heterozygous for the VSMP8EGFP transgene were identified in litters by PCR analysis of genomic DNA and used as donors for heterotopic cardiac transplant. The investigation was performed using Institutional Laboratory Animal Care and Use Committee-approved protocols and conformed with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Immunohistochemical methods. Antibodies specific for Sp1, SRF, Smad 2/3, and phosphorylated Smad 2 were obtained commercially (Santa Cruz Biotechnology, Santa Cruz, CA, or Upstate Biotechnology, Lake Placid, NY) and the YB-1-, Pur -, and Purβ-specific rabbit polyclonal antibodies (anti-YB1 M85–110, anti-YB1 M276–302, anti-Pur 291–313, and anti-Purβ 302-324) were developed in the laboratory of R. J. Kelm, Jr. as described previously (30–32). Immunohistochemistry was performed on 4-µm-thick, paraffin-embedded sections from 4% paraformaldehyde-fixed isografts or nontransplanted donor hearts (47, 48). Sections were deparaffinized, rehydrated, blocked with 2% goat serum, 0.1% BSA, and 0.05% Tween 20 in PBS (blocking solution), and incubated for 90 min at 37°C with the primary antibodies at a concentration of 2 µg/ml in blocking solution. After being washed, sections were incubated for 30 min with a horseradish peroxidase (HRP)-conjugated, goat anti-rabbit secondary antibody, and a Vectastain protocol was followed for color development using a DAB peroxidase kit (Vector Laboratories, Burlingame, CA). To detect SMA, HRP-conjugated anti-human SMA mouse monoclonal (clone 1A4, Dako Cytomation California, Carpinteria, CA) was used at 1:100 dilution. Dual localization of two antigens was accomplished by incubating tissue sections with a second antibody after the detection protocol was completed for the first antibody and the sections were washed in Tris-buffered saline (TBS), pH 7.4. To distinguish between the two antibody reaction products, a nickel solution was added to the second color development reagent, which produced a blue-black precipitate that contrasted with the red-brown reaction product obtained using the first developing solution without nickel ions. Sections were viewed on a Zeiss Axioscope 40 microscope using x10 and x20 brightfield objectives, and color images were digitally recorded with Zeiss MRGrab 1.0 software.

Isolation, culture, and transfection of mechanically unloaded cardiomyocytes. Highly enriched preparations (>95% pure) of adult rat ventricular cardiomyocytes were isolated from female rats (Sprague-Dawley-Ivanovas, 300–350 g) by retrograde perfusion of hearts with collagenase type 2 (281 U/ml; Worthington Biochemical, Freehold, NJ) according to the method reported by Eppenberger-Eberhardt et al. (18). After perfusion, the tissue was minced and incubated at 37°C for another 10 min with KB medium (26). The enzyme-digested heart tissue suspension was filtered through a single layer of cheesecloth and then centrifuged at 2,500 rpm for 1 min. Cell pellets were resuspended and plated in M199 medium for 4 h at 37°C to allow fibroblast attachment to the substrate and removal. The now enriched, nonadherent, and rhythmically beating cardiomyocytes then were transferred to 1% laminin-coated, 100-mm tissue culture dishes (Upstate) in M199 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 µM cytosine arabinoside. Medium was changed every third day over the course of a 15-day observation period. Isolated cardiomyocytes were transfected with a SMA promoter:chloramphenicol acetyl transferase (CAT) reporter fusion plasmid (VSMP4) plus mammalian transcription factor expression plasmids using the Lipofectamine 2000 (Invitrogen, San Diego, CA) transfection reagent and protocol provided by the manufacturer (11, 12, 49). Plasmid encoding the full-length human SRF protein was kindly provided by Dr. M. Gupta, University of Chicago (15). The His-tagged Pur and Pur β protein expression plasmids have been described previously (10). Plasmids used for transfections were purified using Qiagen preparative resin. Forty-eight hours after transfection, cells were washed three times with cold PBS and then lysed using CAT ELISA lysis buffer (Roche Applied Science, Indianapolis, IN) and clarified at 14,000 g for 10 min at 4°C. Total lysate protein was determined by BCA colorimetric assay (Pierce Chemical, Rockford, IL), and reporter gene activity was determined using a commercial CAT ELISA kit (Roche Applied Science) and expressed on a per µg protein basis. Transfections were performed in triplicate and repeated three to five times. Data sets were subjected to analysis of variance to assess statistical significance set at P < 0.05. Transfection efficiency in isolated cardiomyocytes was assessed using the pmax-GFP plasmid (Amaxa, Gaithersburg, MD). The pmax-GFP (1 µg) was combined with 40 µl Lipofectamine 2000, and after a 20-min incubation period at room temperature, the mixture was added to cardiomyocyte culture preparations. GFP-positive cardiomyocytes were counted using immunofluorescence microscopy, and transfection efficiency was determined to be 40% under the experimental conditions used in our study.

Immunoblotting methods. Isolated cardiomyocytes or frozen, pulverized ventricles were extracted for 30 min on ice using 0.6 ml RIPA buffer containing 1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM dithiothreitol (DTT) and were then centrifuged at 10,000 g for 10 min at 4°C to remove insoluble debris. Aliquots containing 2–4 µg of protein were size-fractionated by SDS-PAGE on 10% polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (Schleicher & Schull, Keene, NH) for 90 min at 300 mA in 25 mM Tris·HCl, 192 mM glycine, and 20% (vol/vol) methanol. After overnight blocking at 4°C in (TBS, 25 mM Tris·HCl, pH 7.5, and 150 mM NaCl) containing 3% (wt/vol) nonfat dry milk and 0.5% bovine serum albumin, blots were incubated with primary antibodies (described above in Immunohistochemical Methods) at 1–2 µg/ml for 90 min at room temperature with gentle rocking. Blots were rinsed four times at room temperature over a 20-min period in TBS containing Tween 20 (0.05% vol/vol; TBST). HRP-conjugated secondary antibody (1:1,500) then was applied for 45 min, and the blots were washed as above and processed for antibody visualization by chemiluminescence (ECL, Amersham Biosciences, Arlington Heights, IL) and imaged onto Biomax film (Eastman Kodak, Rochester, NY). For detection of SMA, membranes were incubated with HRP-conjugated 1A4 (1:100) in TBST. Following a 60–90 min incubation at ambient temperature with gentle mixing, the membrane was washed 2–3 times in TBST and developed using reagents provided in a DAB-peroxidase Vectastain kit (Vector Laboratories).

Preparation of protein extracts for DNA binding assays. Cardiomyocytes were washed twice with Dulbecco's PBS, scraped into fresh PBS, sedimented at 3,000 rpm, washed once more in PBS, and resuspended in 8 packed-cell volumes of ice-cold, low-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 20 mM KCl, 0.2 mM EDTA, protease inhibitor cocktail, 0.2 mM PMSF, and 0.5 mM DTT) using a Dounce homogenizer with a type B pestle. Frozen, pulverized heart graft powder was resuspended in the same low-salt buffer and similarly processed using a Dounce homogenizer. High-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT) equal to one-half the total volume was added, and lysates were further extracted with gentle rocking for 30 min at 4°C. After dialysis against 50 volumes of dialysis buffer containing 10 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 0.2 mM PMSF, and 20% glycerol, supernatants were collected by centrifugation at 14,500 rpm for 20 min, assayed for protein concentration using the BCA colorimetric method, aliquoted, and stored at –80°C for use in DNA-binding assays. Synthetic biotinylated oligonucleotide probes used in the present study correspond to various essential regulatory sequences present in the mouse SMA promoter: 1) double-stranded SPUR DNA, 2) SPUR variants harboring mutations in either the GC-rich Sp1-binding domain or GGA Pur protein-binding domain, and 3) double-stranded probes harboring the CArG B binding site for SRF in both native and mutant contexts (12, 49, 50). Protein extract (100 µg protein) and biotinylated oligonucleotides (100 pmol; Integrated DNA Technologies, Coralville, IA) were combined in a binding buffer containing poly(dI-dC), 10 mM Tris, pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 0.2 mM PMSF, and 4% glycerol, and protein-biotin-DNA complexes were captured on streptavidin-immobilized paramagnetic beads (Promega, Madison, WI; 0.6 ml/reaction, 30 min incubation) as previously described (11, 49). After being washed four times with buffer containing 25 mM Tris·HCl, pH 7.5, 1 mM EDTA, and 100 mM NaCl, bound protein was eluted from the beads using 2x protein denaturing buffer and evaluated by SDS-PAGE immunoblot.

Protein immunoprecipitation methods. SRF:Pur protein interaction was evaluated in protein extracts prepared from transfected fibroblasts. Non-human primate COS7 fibroblasts were maintained in Dulbecco's modified Eagle's medium (4.5 g/l D-glucose) supplemented with penicillin-streptomycin and 10% fetal bovine serum, cultivated in a humidified incubator at 37°C at 5% carbon dioxide, and transfected upon reaching 40–50% confluence. Nuclear or whole cell protein extracts were prepared 48 h after transfection with mammalian protein expression plasmids (11, 49). For immunoprecipitation of recombinant His epitope-tagged Pur protein, extracts were combined with 2 µg of a commercial His-tag antibody (Sigma Chemical, St. Louis, MO) in 200 µl of solution D (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, and 0.2 mM PMSF). After a 1-h incubation at 4°C on a rotating-platform mixer, a 20-µl aliquot of protein G-agarose (Sigma), which had been previously washed and suspended in solution D, was added, and the mixture was incubated 16 h at 4°C with rotation. Agarose beads with immobilized protein were collected by centrifugation at 2,500 rpm for 5 min at 4°C, washed 4 times with PBS, suspended in 1x SDS-PAGE sample buffer, and heated at 95°C for 5 min, and released proteins were evaluated by SDS-PAGE and immunoblotting with selected antibodies as noted in the text. Aliquots of whole protein extract were evaluated by immunoblot before immunoprecipitation to verify protein overexpression in transfected fibroblast preparations. To monitor interaction between native forms of SRF and Pur proteins in protein extracts prepared from perfusion-isolated cardiomyocytes, we used a rabbit anti-SRF primary antibody (Santa Cruz) in combination with a secondary antibody detection system and manufacturer's protocol that avoids interference from immunoglobin heavy and light chains that are coprecipitated with the SRF-Pur protein complexes (Rabbit IgG TrueBlot Set, eBioscience, San Diego, CA).

	RESULTS

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Accepted, major histocompatibility complex (MHC) class I and II mismatched FVB/N (H-2^k) or DBA/2 (H-2^d) donor hearts in gallium nitrate-immunosuppressed C57BL6 (H-2^b) recipients developed chronic rejection with substantial fibroplasia within 60 to 90 days after transplant (Fig. 1A) . Of interest, allograft myofibrils that were in close proximity to regions of interstitial fibrosis in the left ventricle free wall and septum often were strongly reactive with a SMA-specific antibody (Fig. 1B). Myofibrils in isografts (FVB/N to FVB/N or DBA/2 to DBA/2) did not exhibit SMA antibody reactivity as described in our previous works (47, 48). The SMA gene encodes a fetal contractile protein isoform that normally is not expressed in adult mammalian cardiomyocytes (36, 54). Cardiomyocytes in allografts from transgenic FVB/N donor mice harboring the tissue-injury responsive mouse SMA promoter (VSMP8) ligated to an enhanced GFP reporter gene showed robust fluorescence (Fig. 1D) that was most pronounced in regions of substantial interstitial fibrosis (Fig. 1C). The behavior of the reporter transgene in cardiomyocytes following allotransplant essentially recapitulated the histopathologic response of the native SMA gene and was not active in donor hearts or nonfibrotic isografts (data not shown). Although very little is known about the mechanism that governs fetal actin gene reactivation in the heart, the reporter gene data shown in Fig. 1D suggested that transplant chronic rejection activated the same SMA promoter elements previously shown to be required for TGF-β1-dependent myofibroblast differentiation during wound healing (6, 11, 49, 51).

View larger version (96K): [in this window] [in a new window]   Fig. 1. DBA/2 to C57BL6 heterotopic cardiac allografts developed interstitial fibrosis within 60 days after transplant (blue-stained material in A; Masson's trichrome stain). Cardiac myofibrils proximal to allograft fibroplasia (blue-stained material; A) were reactive with a smooth muscle -actin (SMA)-specific antibody (red/brown-stained material; B) indicative of fetal actin gene expression. Donor hearts from transgenic FVB/N mice harboring a SMA promoter:green fluorescent protein (GFP) reporter fusion gene also developed allograft fibroplasia when transplanted into C57BL6 recipients (Masson's trichrome stain; C). Cardiac myofibrils located in fibrotic areas of transgenic donor hearts expressed GFP indicative of fetal SMA gene transcriptional reactivation (D). Magnification, x20 objective.

View larger version (74K): [in this window] [in a new window]   Fig. 2. Retransplanted FVB/N to FVB/N heterotopic cardiac isografts exhibited interstitial and perivascular fibrosis (Masson's trichrome blue-stained material; A) and acquired SMA-positive cardiac myofibrils (red/brown-stained material; B) within 15 days after second transplant. Magnification, x20 objective.

View larger version (93K): [in this window] [in a new window]   Fig. 3. Immunohistochemical evaluation of SMA gene transcriptional activators in retransplanted cardiac isografts. A, C, E, and G: isografts at 7 days (C and E) and 15 days (A and G) after second transplant. B, D, and H: nontransplanted control hearts. An isograft 3 days after second transplant (F) also is presented to illustrate accumulation of phosphorylated Smad 2 relative to the 7-day time point shown in E. Nontransplanted donor hearts exhibited very little Smad 2 and Smad 3 (D), whereas these proteins were observed in the nuclei of interstitial cells within 7 days after retransplant (C). Sp1 (A and B) and serum response factor (SRF; G and H) were both present in isografts 15 days after second transplant but appeared to reside in different cellular compartments. Sp1 was localized in small-sized interstitial cell nuclei (A) compared with SRF that was more prevalent in larger cardiomyocyte nuclei (G). Sp1 appeared de novo after second transplant (compare A with the nontransplanted control shown in B), but SRF was present in donor hearts before retransplant (compare G and H). Magnification, x20 objective.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Abundance and DNA-binding activity of SMA gene transcriptional regulatory proteins after cardiac retransplant. Relative to the level observed in extracts prepared from nontransplanted control hearts (NT), the binding of Pur protein repressor to SPUR DNA increased substantially after cardiac retransplant, as did the net amount of Pur protein detected in heart extracts (Ext.). SRF levels also increased after retransplant relative to nontransplanted control hearts, and binding of SRF to CArG B DNA increased substantially within 7 days after retransplant. During the posttransplant observation period, the level of SMA protein in heart graft extracts increased, with peak expression noted between 7 and 11 days after retransplant. Levels of the GAPDH control protein remained unchanged over this same period.

View larger version (98K): [in this window] [in a new window]   Fig. 5. Pur protein was localized to cardiac intercalated discs after cardiac retransplant. Nontransplanted FVB/N donor hearts did not react with a Pur-specific antibody (A). However, intercalated discs in two-hit isografts were intensely reactive with this antibody within 15 days after retransplant (red/brown-stained material; B). In double-label immunohistologic preparations (C), many Pur antibody-reactive intercalated discs (denoted by arrows) also were associated with short filaments that reacted with the SMA-specific antibody (red/brown-stained material). A few Pur antibody-reactive intercalated discs (gray/black-stained material) lacking SMA filament associations also were evident (boxed region of C). A and B magnification, x10 objective; C magnification, x20 objective.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Morphology and SMA gene transcriptional reprogramming in perfusion-isolated rat cardiomyocytes. Highly purified, perfusion-isolated cardiomyocytes beat spontaneously and exhibited characteristic rod-shape morphology (A). SMA protein levels on immunoblot (B) and transcriptional activity of a SMA promoter:chloramphenicol acetyl transferase (CAT) reporter fusion gene (C) both increased substantially in cultured cardiomyocytes within 10 days after isolation.

View larger version (7K): [in this window] [in a new window]   Fig. 7. Immunoblot analysis of Sp1 protein levels in isolated rat cardiac fibroblasts and cardiomyocytes. Whereas cardiac fibroblasts (CFB) were highly enriched in Sp1 after a 3-day culture period, Sp1 expression in cardiomyocytes was not detected at this time point and was only modestly elevated by the end of the 14-day observation period. Numbers below the blot refer to days after isolation.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Pur protein expression and DNA-binding activity in isolated rat cardiomyocytes. Pur was the dominant Pur protein isoform detected on immunoblots prepared from cardiomyocytes, and its level of expression did not change during the 14-day observation period (A). However, the DNA-binding activity of Pur decreased within the first 10 days after isolation (B). Numbers below each blot refer to days after isolation.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Expression and DNA-binding activity of SRF in isolated rat cardiomyocytes (CMC). Approximately equal amounts of SRF were detected on immunoblots prepared from freshly isolated (labeled 0) and cultured (labeled 14) cardiomyocytes (A). Binding of full-length SRF (FL) and the SRF splice variants (5, 4,5, and 3,4,5) to the CArG B element in the SMA promoter increased markedly in cardiomyocytes over the 14-day culture period (B). SRF binding to DNA was mediated by a 10-bp CC(A/T-rich)GG consensus sequence (CArG B) because point mutation of this motif (mut CArG B) eliminated all SRF binding (C).

View larger version (7K): [in this window] [in a new window]   Fig. 10. Effect of transcriptional regulatory protein expression on SMA promoter activity in transfected rat cardiomyocytes. A: CAT reporter gene activity in cardiomyocytes transfected 5 days after isolation with either a promoterless CAT expression vector (pCDNA3), the VSMP4CAT SMA promoter:CAT reporter fusion gene (P4), or VSMP4CAT in combination with a mammalian SRF expression plasmid (P4+SRF). SMA promoter activity was evident within 48 h after transfection, and output of P4 was nearly threefold higher in preparations cotransfected with the SRF expression plasmid. B: dose-dependent repression of VSMP4CAT activity by coexpression of Pur or Purβ protein in transfected cardiomyocytes. Shown are transfected cardiomyocyte preparations containing the promoterless CAT expression vector alone (sample 1) and the VSMP4CAT SMA promoter:CAT reporter fusion gene either alone (sample 2) or in combination with 0.3-, 0.5-, or 1.0-µg aliquots of a mammalian Pur expression plasmid (samples 3, 4, and 5, respectively) or with 0.3-, 0.5-, or 1.0-µg of mammalian Purβ expression plasmid (samples 6, 7, and 8, respectively).

View larger version (17K): [in this window] [in a new window]   Fig. 11. SRF formed a complex with Pur proteins and neutralized Pur protein-mediated repression of the SMA promoter in COS7 cells. A: immunoblot (IB) analysis of anti-his antibody immunoprecipitates (IP) collected from COS7 cells transfected with expression plasmids encoding SRF plus his-tagged forms of either Pur or Purβ. SRF protein was detected in Pur protein immunoprecipitates. B: whereas either Pur (P4+Pur a) or Purβ (P4+Pur b) significantly repressed basal expression of VSMP4CAT (P4) in transfected COS7 cells by 50% (P < 0.05), repression was statistically insignificant if the SRF expression plasmid was cotransfected with either of the Pur protein expression plasmids.

View larger version (25K): [in this window] [in a new window]   Fig. 12. Pur proteins were required for mediating SRF binding to the SPUR transcriptional activation element in the SMA promoter. A: immunoblot on the left shows that SRF acquired the ability to bind SPUR DNA within 14 days after cardiomyocyte isolation. The immunoblot on the right shows that an intact GGA Pur protein-binding motif (native) was essential for SRF interaction with SPUR DNA because a mutation within this element (Pur-mut) eliminated binding. B: immunoblot analysis of transfected COS7 cells suggested that physical interaction of SRF with SPUR DNA was possible if both Pur and Purβ were coexpressed along with SRF in the transfected cells. C: immunoblot shows that native SRF was coimmunoprecipitated with native Pur proteins from day 14 but not day 0 cardiomyocyte extracts using a pan-specific anti-Pur protein antibody (anti-Pur /β) but not a nonspecific anti-IgG antibody (anti-IgG).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

We presented immunohistologic evidence showing that TGF-β1 receptor-regulated Smad proteins accumulated in cardiac stromal cells within 3 days after isograft retransplant and before peak expression of the SMA gene. This observation is consistent with a recent report showing accumulation of TGF-β1 mRNA in chronically rejected murine heart grafts and extends those findings by showing peritransplant nuclear translocation of Smads in the cardiac interstitium need not necessarily require an alloimmune stimulus (14). Transcription of the SMA and type I collagen subunit genes, two major indicators of myofibroblast differentiation and wound healing activity, is coordinately activated by TGF-β1 receptor-regulated Smad protein signaling (11, 17, 25, 40, 49). In studies on isolated fibroblasts, Sp1 and Smad transcriptional activators bind and neutralize the Pur and Purβ repressor proteins at the SPUR transcriptional control site in the SMA promoter. SPUR DNA contains tandem GC-rich and GGA motifs that mediate Sp1 and Pur protein binding, respectively. Protein:protein interactions at SPUR DNA are regulated by TGF-β1 and appear to be essential for myofibroblast differentiation in vitro (49) and efficient dermal wound healing in the mouse (51). Colocalization of Sp1 and Smads in two-hit cardiac fibroblasts provides new in vivo evidence for the dynamic interplay model for SMA gene activation during myofibroblast differentiation (49, 57).

As a secondary response to cardiac retransplant, the dormant fetal SMA gene in cardiomyocytes becomes stress-activated as fibroplasia emerges in the left ventricle. The SMA gene is one of several fetal-type genes that are coordinately regulated by SRF, a key transcriptional mediator of cardiac hypertrophic responses and progression to heart failure (7, 15, 56). Cardiomyocytes isolated from SRF null mice exhibit impaired expression of contractile protein genes and grossly disrupted sarcomeres and polysomes (5). SRF was the predominant SMA gene-activating protein detected in cardiomyocyte nuclei from two-hit hearts. In normally developing myogenic cells, SRF binds to a canonical CC(A/T-rich)GG consensus motif present in virtually all contractile protein gene promoters including the SMA promoter where it appears twice within the first 150 bp of the 5'-flanking region (CArG A and CArG B). The more upstream element (CArG B) is functionally more important for SMA gene transcription in murine smooth muscle cells and myofibroblasts (12, 50). Importantly, SRF binding to CArG B was acquired only after retransplant and was not observed in nontransplanted adult mouse donor hearts or freshly isolated adult rat cardiomyocytes. However, SRF acquired CArG B-binding activity when cardiomyocytes were maintained in extended culture and thus may represent a new indicator for fetal gene reprogramming in cardiomyocytes during heart graft failure. The relative paucity of Sp1 expression in both two-hit and perfusion-isolated cardiomyocytes further suggested that SRF might play a major role in de novo activation of fetal SMA gene transcription in these cells. In this regard, we discovered that cardiomyocyte SRF binds to the same activating site in the proximal SMA promoter, referred to as SPUR, that was shown in myofibroblasts to be occupied by a TGF-β1-regulated complex consisting of the Sp1 activator and Pur/β protein repressors. However, Sp1 was not detected in freshly isolated cardiomyocytes and only modestly upregulated in reprogrammed cardiomyocytes after a 2-wk culture period. In contrast, the level and SPUR-binding activity of both SRF and Pur were highly elevated after transplant, although DNA-binding activity of Pur decreased somewhat after retransplant. We speculate that Pur protein binding to SPUR in cardiomyocytes might be governed by physical interaction with SRF rather than Sp1. Increased SRF binding to CArG B in stressed cardiomyocytes may influence Pur affinity for SPUR since both sites are located approximately on the same side of the DNA helix within nucleosome structural context (37). Coimmunoprecipitation data suggests that SRF may also form off-DNA complexes with Pur proteins that theoretically could modulate their ability to bind and/or repress transcription from SPUR DNA. Native SRF splice variants in isolated cardiomyocytes did not bind Pur proteins but were able to bind both CArG B and SPUR DNA, supportive of their possible role as dominant-negative transcriptional regulators in the heart (7). By itself, native Pur in cardiomyocyte or whole heart extracts does not seem to bind the SMA gene-specific CArG B motif (X. Liu and A. R. Strauch, unpublished observations), although in vitro synthesized Purβ has been reported to exhibit some binding affinity for certain skeletal muscle actin-type CArG elements (23). SRF has many protein partners that generally fall into two cofactor families (56), the ets domain-containing ternary complex factors (TCF) and the myocardin-related transcription factors (MRTF). Pur proteins, however, are less well studied and are seemingly unique as SRF coactors, although they do bind an ets-like GGA motif favored by TCF cofactors and may indirectly bind actin filaments in a manner resembling MRTF family members (29, 41, 44).

Pur proteins function as SMA gene repressors in transfected cardiomyocytes, much like their role in myofibroblasts. Curiously, formation of a SRF:Pur protein complex in transfected COS7 cells was associated with enhanced activation of the SMA promoter. We hypothesize that SRF binds the NH₂ or COOH termini of SPUR-immobilized Pur protein, causing the repressor's central DNA-binding domain to alter its position on or affinity for the GGA motif within SPUR. "Assisted removal" of Pur probably does not improve SRF interaction at SPUR since this DNA element lacks a CArG box motif needed to stabilize SRF binding. However, removal of Pur protein may improve physical access of SRF to the CArG B element located nearby on an adjacent nucleosomal DNA loop. Alternatively, the SRF:Pur protein complex may have unique activation properties that could stimulate transcription at SPUR in a manner resembling the SRF:myocardin or SRF:Elk-1 heteromeric complexes that operate CArG-dependent promoters in muscle cells (56). In this regard, Pur has been implicated as a transcriptional activator in certain cell types (27, 33). Increased loading of hypothetical SRF:Pur "superactivation" complexes onto SPUR DNA may be a beneficial response if stimulation of fetal SMA gene transcription is an absolute requirement for cardiomyocyte survival and function after cardiac transplant injury. On the other hand, the observed increase in Pur protein binding to SPUR in two-hit heart grafts might represent an exuberant compensatory response to injury whereby stressed cardiomyocytes are attempting to silence a fetal gene that has become inappropriately activated. Finally, it is important to consider that the observed increase in the size of the Pur protein pool after retransplant as well as the ability of Pur to bind single-strand DNA and RNA (41) may reflect a novel role for this protein in packaging and transporting newly synthesized mRNA molecules needed for cardiomyocyte adaptation to stress and/or transplant injury (see below). Future studies should be directed toward identifying essential peptide determinants and biochemical signals needed for SRF:Pur and SRF:SPUR:Pur physical interplay during activation of the SMA promoter in stressed cardiomyocytes and whether this interplay is specifically designed to override developmental restrictions that prevent fetal gene reactivation in the normal heart. We also are planning to examine the kinetics of SRF binding to SPUR DNA in long-term two-hit isografts. As currently implemented, the 15-day posttransplant end point used in the current study mainly captures early aspects of fibroplasia where myofibroblast Sp1 interactions with SPUR DNA may interfere with study of the SRF:SPUR interaction. More abundant cardiac myofibroblast-derived Sp1 in protein extracts prepared from two-hit grafts may dilute or compete with less abundant SRF for SPUR binding because cardiomyocyte remodeling is just emerging at this time and is often highly localized to specific regions of the left ventricle. We speculate that Sp1 may have a competitive advantage over SRF in binding SPUR DNA in pull-down assays because it binds directly to a 10-bp GC-rich consensus sequence, whereas SRF interaction with SPUR is indirect and seems to be mediated by a Pur protein bridge attached to the 3'-end of this site.

During reprogramming of SMA gene expression in cardiomyocytes, the nuclear SRF activator accumulates at SPUR and CArG B DNA while Pur protein repressors are deployed to actin filament anchorage points at the cardiac intercalated discs. Pur proteins have been identified as important linker proteins for tethering mRNA to microtubules, microfilaments, and motor proteins as well as intracellular transport of ribonucleoprotein particles to sites of mRNA translation (29, 41, 44). The YB-1 binding partner of Pur proteins in fibroblasts and muscle cells (10, 30) is a well-known cardiac stress-response protein that has dual function as a SMA gene corepressor in the nucleus and the ability to stimulate actin bundle formation in the cytosol (44) as well as bind components of the intercalated disc such as cardiac ankyrin repeat protein (58). In myofibroblasts, Pur proteins also avidly bind to a sequence motif in exon 3 of SMA mRNA that blocks translation (31). Taken together, these findings suggest that Pur proteins may function posttranscriptionally as RNA chaperones to package and transport mRNAs necessary for expression of wound healing- and/or stress-associated gene products in the injured heart. The localization of Pur proteins at cardiac intercalated discs in two-hit isografts was especially pronounced and may be relevant to reconfiguration of cardiac -actin thin filament ends to accept addition of newly synthesized SMA monomers. Delivery of Pur protein:SMA mRNA ribonucleoprotein complexes to polysomes located near actin thin filament ends anchored at intercalated discs may provide a means for coupling de novo translation of SMA G-actin subunits with F-actin polymerization in stressed cardiomyocytes. The barbed end of F-actin is proximal to the intercalated disc and represents the fast site for addition of new actin monomers. While quite preliminary, the presence of short SMA filamentous bundles emerging from Pur protein-enriched intercalated discs in two-hit isografts seems to provide some credence for this hypothesis. The specific involvement of Pur proteins in linking the mRNA- and actin filament-based aspects of posttransplant cardiac remodeling is an area of active investigation in our laboratory. In this regard, Pur protein appears to be less tightly bound to SMA mRNA in SMA-positive cultured cardiomyocytes compared with freshly isolated cells that do not express this -actin polypeptide (A. Zhang, J. J. David, and A. R. Strauch, unpublished observations). These emerging data are consistent with our earlier reports showing that dissociation of Pur protein from SMA mRNA enhances its overall translational efficiency (31, 57).

In summary, we have shown that posttransplant cardiomyocyte remodeling seems to involve conversion of an adult transcriptional program into one that more closely resembles the scheme used by injury-activated myofibroblasts. SRF may have evolved a unique role in stressed cardiomyocytes to de-repress the fetal SMA gene and provide new types of actin subunits that are functionally more compatible with wound healing activity in the heart compared with cardiac -actin that may be better specialized for high efficiency activation of sarcomeric myosin ATPase (35). In this scheme, SRF appears to neutralize Pur protein transcriptional repressors by forming a novel complex that may reduce repressor potency and/or DNA-binding affinity within the SMA gene promoter. Redirection of Pur protein function after cardiac injury also may provide the means to export newly transcribed SMA mRNA from the nucleus to sites of protein translation and de novo thin filament remodeling at cardiac intercalated discs.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The study was supported by National Heart, Lung, and Blood Institute Grants HL-70294 and HL-85109 (to A. R. Strauch) and HL-54281 (to R. J. Kelm).

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. R. Strauch, Dept. of Physiology and Cell Biology, Davis Heart and Lung Research, Institute Biomedical Research Tower, Rm. 314, 460 West 12th Ave., The Ohio State Univ. College of Medicine, Columbus, OH 43210-1252 (e-mail: strauch.1{at}osu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Charles G. Orosz is deceased.

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