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CARP: Fishing for Novel Mechanisms of Neovascularization

  • Susan E. Samaras
    Affiliations
    Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
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  • Yubin Shi
    Affiliations
    Department of Surgery, Division of Plastic Surgery, Pediatric Surgery Research Laboratory, Stanford University, Stanford, California, USA
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  • Jeffrey M. Davidson
    Correspondence
    Department of Pathology C-3321 MCN, Vanderbilt University School of Medicine, 21st and Garland Sts., Nashville, Tennessee 37232-2561, USA
    Affiliations
    Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Research Service, VA Tennessee Valley Healthcare System, Nashville, Tennessee, USA
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      Gene expression profiling of mouse skin wounds has led to the discovery of numerous target genes that may have therapeutic or diagnostic value. Among these, cardiac ankyrin repeat protein (CARP, ankrd1) expression was markedly and persistently elevated in several cutaneous compartments. This review summarizes the current state of knowledge of CARP and its regulation in biological systems. In addition to its role as a nuclear transcription cofactor in many cell types including vascular endothelium, CARP is also a structural component of the sarcomere. CARP transcripts are prominent in cardiogenesis and muscle injury, and they are under complex regulation by cytokines, hypoxia, doxorubicin, and other forms of stress. CARP overexpression in wounds by adenoviral gene transfer leads to a high vascular density, and CARP exerts effects on endothelial behavior. The unusual cellular distribution and actions of CARP make it a novel candidate gene in tissue repair.
      ANF
      atrial natriuretic factor
      CARP
      cardiac ankyrin repeat protein
      CASQ-2
      cardiac calsequestrin-2
      DOX
      doxorubicin
      EGF
      epidermal growth factor
      HMVEC
      human microvascular endothelial cell
      MARP
      muscle ankyrin repeat protein
      TNC
      troponin C
      TGF-β
      transforming growth factor-beta
      VEC
      vascular endothelial cell
      VSMC
      vascular smooth muscle cell
      3′-UTR
      3′ untranslated region

      Introduction

      Healing of wounds involves an orchestrated cascade of events, and it requires a complex interplay between multiple cell types that must respond to the initial insult with a progressive change in their genetic programming. Beginning with the activation of platelets and their release of clotting factors and cytokines and ending with re-epithelization and the final maturation and remodeling of the wound area, gene expression profiles of all involved cell types are dynamic and responsive to the ever-changing wound milieu. To “fish” for novel genes involved in this process, cDNA subtractive hybridization was performed in collaboration with Switch Biotech AG, comparing intact skin and 1 day full-thickness wounds in the mouse (
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      ). Using this approach, the expression of ankrd1, a gene which encodes the transcriptional cofactor, cardiac ankyrin repeat protein (CARP) was among several hundred genes that were found to be highly induced in wounds. The time course of CARP mRNA and protein expression, determined by quantitative, real-time RT-PCR and Western blot analysis, showed that they were significantly increased within hours, reaching peak levels by 15 hours and remaining elevated for 2 weeks during the wound healing process (Figure 1). Furthermore, immunohistochemistry and in situ hybridization for CARP protein and mRNA in day 1 wounds revealed that monocytes, cells of the epidermis and the vasculature, and skeletal muscle had increased expression (
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      ) (Figure 2). Thus, changes in the abundance of this transcriptional cofactor may modulate the gene expression profiles in these cells. This review will focus on what is known about CARP and its actions, and how it affects its role in neovascularization.
      Figure thumbnail gr1
      Figure 1Comparison of CARP mRNA and protein levels in intact skin and excisional wounds in mice. (a) Time course of CARP mRNA levels in normal mice after wounding. Quantitative real-time RT-PCR showed that CARP transcripts were dramatically upregulated during the first day after wounding and increased expression was maintained between 25- and 30-fold from day 3 to day 14 after wounding. PCR reactions were performed three times. (b) Western blot analysis of CARP protein after wounding. Fifty micrograms of lysate protein from mouse skin, prepared at indicated times after wounding, were fractionated by SDS-PAGE. (c) CARP protein (top row) was normalized to β-actin (bottom row) and band intensities were plotted.
      Reproduced from
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      , Am J Pathol166: 303–12) with permission from the American Society for Investigative Dermatology.
      Figure thumbnail gr2
      Figure 2Immunostaining and in situ hybridization of CARP in mouse skin wounds. (a) Expression of CARP in a day 3 mouse skin excisional wound. The red dashed line indicates the boundary between wound and normal tissue. Higher magnification images of CARP-positive cells are shown in (b) hair follicles (HF, and (c) inflammatory cells in 1 day wounds; and (d) epidermis (EP and (e) skeletal muscle (MU in 3 day wounds. (f) In situ hybridization for CARP mRNA in mouse 1 day skin wounds showed positive signals (dark color) in the skeletal muscle (red arrow), blood vessels (black arrow), and epidermis (yellow arrow). The red dashed line indicates the basement membrane. High magnification of the inflammatory cells (red arrow) and the vessel are shown.
      Reproduced from
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      , Am J Pathol166: 303–12) with permission from the American Society for Investigative Dermatology.

      Background

      The gene encoding CARP was initially identified in a screen for cytokine-inducible factors in microvascular endothelial cells (
      • Chu W.
      • Burns D.K.
      • Swerlick R.A.
      • Presky D.H.
      Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
      ). Found on chromosome 10 in humans and 19 in mouse, the ankrd1 gene structure is highly conserved, with nine exons and a canonical TATA box in the proximal promoter which, to date, appears to produce a single transcript (
      • Chu W.
      • Burns D.K.
      • Swerlick R.A.
      • Presky D.H.
      Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
      ;
      • Jeyaseelan R.
      • Poizat C.
      • Baker R.K.
      • Abdishoo S.
      • Isterabadi L.B.
      • Lyons G.E.
      • et al.
      A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.
      ). No alternative splicing of the RNA has been described. Other canonical response elements identified in the 5′ flanking sequence of CARP include GATA sites, E-box elements, a CCAC box, a CAGA box, and M-CAT, activator protein-1 and SP-1 binding sites (
      • Baumeister A.
      • Arber S.
      • Caroni P.
      Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube endcompartments during muscle morphogenesis.
      ;
      • Kuo H.
      • Chen J.
      • Ruiz-Lozano P.
      • Zou Y.
      • Nemer M.
      • Chien K.R.
      Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis.
      ) (Figure 3). These sites exert both positive and negative regulation of CARP. A short 5′ untranslated region (UTR) and a longer 3′ UTR, which contains a conserved degradation motif, characterize the 10.6 kb CARP pre-mRNA. The primary transcript containing nine exons is processed to encode a 319 amino-acid protein with a molecular weight of 36 kDa. CARP protein sequence and domain organization is highly conserved among mammalian species. Analysis of the peptide sequence identifies a bipartite nuclear localization signal, a PEST-like sequence (a four amino acid sequence associated with rapidly degraded proteins), four highly conserved ankyrin-like repeats and another less conserved half repeat, and numerous potential modification sites for phosphorylation, glycosylation, and myristilation (
      • Chu W.
      • Burns D.K.
      • Swerlick R.A.
      • Presky D.H.
      Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
      ;
      • Jeyaseelan R.
      • Poizat C.
      • Baker R.K.
      • Abdishoo S.
      • Isterabadi L.B.
      • Lyons G.E.
      • et al.
      A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.
      ) (Figure 3). Ankyrin-repeats have been found in proteins that are involved in protein–protein interactions, including those characterized as cyclin-dependent kinase inhibitors (p16, p18, and p19), developmental regulators (Notch and IκBα), and transcription factors (Swi6 and GABPα, β) (reviewed in
      • Sedgwick S.G.
      • Smerdon S.J.
      The ankyrin repeat: a diversity of interactions on a common structural framework.
      ). The family of ankyrins is involved in binding and organization of many cytoplasmic proteins (
      • Mohler P.J.
      • Bennett V.
      Ankyrin-based cardiac arrhythmias: a new class of channelopathies due to loss of cellular targeting.
      ,
      • Mohler P.J.
      • Bennett V.
      Defects in ankyrin-based cellular pathways in metazoan physiology.
      ). CARP belongs to a three-member family of muscle ankyrin repeat proteins or MARPs that also includes AARP/ankrd2 and the diabetes-associated diabetic ankyrin repeat protein (DARP) (
      • Ihara Y.
      • Suzuki Y.J.
      • Kitta K.
      • Jones L.R.
      • Ikeda T.
      Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin.
      ;
      • Miller M.K.
      • Bang M.L.
      • Witt C.C.
      • Labeit D.
      • Trombitas C.
      • Watanabe K.
      • et al.
      The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.
      ). The degradation motif located in the 3′-UTR of the mRNA (
      • Baumeister A.
      • Arber S.
      • Caroni P.
      Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube endcompartments during muscle morphogenesis.
      ;
      • Jeyaseelan R.
      • Poizat C.
      • Baker R.K.
      • Abdishoo S.
      • Isterabadi L.B.
      • Lyons G.E.
      • et al.
      A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.
      ), the PEST-like protein degradation sequence (
      • Chu W.
      • Burns D.K.
      • Swerlick R.A.
      • Presky D.H.
      Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
      ;
      • Jeyaseelan R.
      • Poizat C.
      • Baker R.K.
      • Abdishoo S.
      • Isterabadi L.B.
      • Lyons G.E.
      • et al.
      A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.
      ), and the potential for multiple forms of protein modification suggest that the expression and activity of this protein is highly regulated at the post-transcriptional level. Indeed, the steady-state levels of both CARP protein and mRNA are significantly increased by β-adrenergic agonists (
      • Zolk O.
      • Marx M.
      • Jackel E.
      • El-Armouche A.
      • Eschenhagen T.
      Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase.
      ), but little is known about the mechanisms involved.
      Figure thumbnail gr3
      Figure 3Organization of the CARP gene, mRNA, and protein. The CARP gene (ankrd1) is located on chromosome 10 in the human and 19 in the mouse. The bulk of the identified regulatory sites in the CARP promoter lie within ∼600 bp of the transcription start site (top line). The site labeled as MCAT1 has been identified by one group as a constitutively active promoter element and by another laboratory as a response element for the transcription factor GADD153. The primary transcript of the gene encoded by nine exons is a 10.6 kb mRNA with a short 5′UTR, open reading frame, and a long 3′UTR with a canonical RNA degradation sequence (middle line). Exons 5–8 encode four ankyrin repeat elements (bottom line), the second of which includes a sequence reported to bind to the giant muscle protein, titin (shaded horizontal bar). NH2-terminal to the ankyrin repeats, the diagram illustrates the location of two putative nuclear localization signals (NLS, solid bars) and the PEST sequence (diagonal stripes) that targets ubiquitinated proteins for degradation. The structures of the mRNA and protein are consistent with molecules that undergo high turnover rates. Putative phosphorylation, myristylation, and glycosylation sites have not been confirmed and are thus not indicated.

      Expression Studies

      CARP is sharply upregulated during cardiomyogenesis (
      • Zou Y.
      • Evans S.
      • Chen J.
      • Kuo H.C.
      • Harvey R.P.
      • Chien K.R.
      CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2–5 homeobox gene pathway.
      ;
      • Kuo H.
      • Chen J.
      • Ruiz-Lozano P.
      • Zou Y.
      • Nemer M.
      • Chien K.R.
      Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis.
      ). Lower, constitutive CARP expression in adult mice is most prominent in the heart, whereas lung and skeletal muscle transcripts are also detected by Northern blot analysis. CARP mRNA is also found in the highly vascularized placenta. Using a 2.5 kb CARP promoter-driven lacZ transgene, embryonic expression appears to be primarily localized in the developing heart, where CARP expression has been studied most extensively. Removal of the cardiogenic homeobox gene Nkx2.5 using knockout strategies results in selectively reduced CARP expression (
      • Zou Y.
      • Evans S.
      • Chen J.
      • Kuo H.C.
      • Harvey R.P.
      • Chien K.R.
      CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2–5 homeobox gene pathway.
      ). This result suggests that, at least in cardiac muscle, CARP is downstream of the Nkx2.5 pathway and that Nkx2.5 either directly or indirectly regulates CARP expression. GATA 4, a direct target of Nkx2.5, is also involved in cardiac development and has been shown to regulate CARP expression through direct binding to canonical response elements in the CARP proximal promoter (
      • Kuo H.
      • Chen J.
      • Ruiz-Lozano P.
      • Zou Y.
      • Nemer M.
      • Chien K.R.
      Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis.
      ;
      • Maeda T.
      • Sepulveda J.
      • Chen H.H.
      • Stewart A.F.
      Alpha(1)-adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes.
      ).
      CARP expression is increased in several cardiomyopathies and in cardiovascular injury. Left ventricular dilated cardiomyopathy and ischemic cardiomyopathy in humans exhibit increased CARP expression as compared to non-affected human donor hearts (
      • Zolk O.
      • Frohme M.
      • Maurer A.
      • Kluxen F.W.
      • Hentsch B.
      • Zubakov D.
      • et al.
      Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure.
      ). In an acute model of cardiac hypertrophy induced by ventricular pressure overload in rats, CARP mRNA was increased 8 days after banding of the descending aorta (
      • Aihara Y.
      • Kurabayashi M.
      • Saito Y.
      • Ohyama Y.
      • Tanaka T.
      • Takeda S.
      • et al.
      Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter.
      ). In a well-characterized, in vitro model of cardiac hypertrophy that involves activation of fetal genes in cardiac myocytes by α1-adrenergic stimulation, α1-adrenergic agonists increase endogenous CARP expression whereas antagonists lower expression in myocytes. Analysis of the CARP proximal promoter has identified TEF-1 and GATA 4 binding to MCAT and GATA elements, respectively, as the transcriptional mediators of the α1-adrenergic stimulation (
      • Maeda T.
      • Sepulveda J.
      • Chen H.H.
      • Stewart A.F.
      Alpha(1)-adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes.
      ). Vascular smooth muscle cells (VSMCs) in rat atherosclerotic lesions exhibit increased CARP expression, and similar induction is observed in both VSMCs and vascular endothelial cells (VECs) of collateral arteries that form following femoral artery occlusion (
      • Boengler K.
      • Pipp F.
      • Fernandez B.
      • Ziegelhoeffer T.
      • Schaper W.
      • Deindl E.
      Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp).
      ;
      • de Waard V.
      • van Achterberg T.A.
      • Beauchamp N.J.
      • Pannekoek H.
      • de Vries C.J.
      Cardiac ankyrin repeat protein (CARP) expression in human and murine atherosclerotic lesions: activin induces CARP in smooth muscle cells.
      ). Balloon injury of the aorta in rats also increases CARP expression (
      • Kanai H.
      • Tanaka T.
      • Aihara Y.
      • Takeda S.
      • Kawabata M.
      • Miyazono K.
      • et al.
      Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.
      ). Transforming growth factor beta (TGF-β) signaling induces CARP mRNA, and evidence implicates binding of the Smads to the CAGA element in the CARP promoter as the transduction mediator (
      • Kanai H.
      • Tanaka T.
      • Aihara Y.
      • Takeda S.
      • Kawabata M.
      • Miyazono K.
      • et al.
      Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.
      ). However, our own data employing selective inhibitors in the MS-1 vascular endothelial cell line suggest dominant signaling through the p38 mitogen-activated protein kinase pathway and a lesser contribution of Smad signaling (Y Shi, unpublished).
      CARP is also an acronym for cardiac adriamycin-responsive protein. Adriamycin (doxorubicin; DOX) is a chemotherapeutic agent that can produce collateral damage to the myocardium of patients undergoing chemotherapy, and extravasation of the drug during administration produces severe chemical injury to the skin and adjacent tissues. The mechanism of DOX toxicity is primarily by the hyper-induction of oxygen radicals, leading to oxidative stress and programmed cell death/apoptosis (
      • Singal P.K.
      • Li T.
      • Kumar D.
      • Danelisen I.
      • Iliskovic N.
      Adriamycin-induced heart failure: mechanism and modulation.
      ). Paradoxically, DOX treatment can cause both induction (in vivo) and repression (in vitro) of CARP expression. In piglets, DOX treatment results in left ventricular dilated cardiomyopathy that is associated with increased CARP mRNA and protein 20 days after a single intravenous injection of the drug (
      • Torrado M.
      • Lopez E.
      • Centeno A.
      • Castro-Beiras A.
      • Mikhailov A.T.
      Left-right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure.
      ). A more recent microarray analysis of DOX-sensitive transcription products in the heart compares the effects of a single, acute intraperitoneal injection of DOX to weekly intraperitoneal injections of smaller doses (
      • Yi X.
      • Bekeredjian R.
      • Defilippis N.J.
      • Siddiquee Z.
      • Fernandez E.
      • Shohet R.V.
      Transcriptional analysis of doxorubicin-induced cardiotoxicity.
      ). Acute DOX increases CARP mRNA significantly, and quantitative, real-time RT-PCR confirms a 4.53-fold induction. However, chronic, low-dose DOX exposure does not result in a significant change in cardiac CARP mRNA. In contrast, cultured cardiomyocytes treated with DOX show a sensitive, dose-dependent repression of CARP that is associated with acute oxidative stress. This effect has been attributed to the action of hydrogen peroxide on a H7-sensitive serine/threonine kinase pathway that interacts with a proximal M-CAT element in the human CARP promoter (
      • Aihara Y.
      • Kurabayashi M.
      • Tanaka T.
      • Takeda S.I.
      • Tomaru K.
      • Sekiguchi K.I.
      • et al.
      Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways.
      ). In a separate study, DOX-treated embryonic heart-derived H9c2 cells developed oxidative stress followed by apoptosis that correlated with the level of GADD153 (CHOP-10), a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors (
      • Han X.J.
      • Chae J.K.
      • Lee M.J.
      • You K.R.
      • Lee B.H.
      • Kim D.G.
      Involvement of GADD153 and cardiac ankyrin repeat protein in hypoxia-induced apoptosis of H9c2 cells.
      ). The level of CARP mRNA was found to be inversely proportional to that of GADD153. Furthermore, co-transfection of a GADD153 expression vector with a CARP promoter (-206)-driven luciferase reporter construct resulted in a dose-dependent decrease in luciferase activity. These in vitro data suggest direct regulation of CARP transcription by a GADD153 response element in the proximal promoter.
      The opposing DOX regulation of myocardial CARP expression in vivo and in vitro could be related to the timing of analysis. Acute downregulation of CARP by oxidative stress and cardiomyocyte apoptosis could lead to increased mechanical stress and a rebound in CARP expression in surviving cardiomyocytes during the reparative phase. In addition, many forms of myocardial injury can elicit expression of TGF-β and tumor necrosis factor alpha-α, both of which induce CARP expression ((
      • Kanai H.
      • Tanaka T.
      • Aihara Y.
      • Takeda S.
      • Kawabata M.
      • Miyazono K.
      • et al.
      Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.
      ) and Shi, unpublished)). The in vivo induction of CARP may be cytoprotective, as suggested by the ability of CARP overexpression to protect H9c2 cells from hypoxia-induced apoptosis (
      • Han X.J.
      • Chae J.K.
      • Lee M.J.
      • You K.R.
      • Lee B.H.
      • Kim D.G.
      Involvement of GADD153 and cardiac ankyrin repeat protein in hypoxia-induced apoptosis of H9c2 cells.
      ) and by our preliminary evidence that increased CARP also protects VEC from DOX toxicity (Shi et al., unpublished) and serum starvation (
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      ).

      Cytoplasmic Carp

      CARP is localized in the nuclei of striated muscle, and it has also been colocalized with the giant filamentous polypeptide, titin in the I-band of the sarcomere (
      • Miller M.K.
      • Bang M.L.
      • Witt C.C.
      • Labeit D.
      • Trombitas C.
      • Watanabe K.
      • et al.
      The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.
      ;
      • Kojic S.
      • Medeot E.
      • Guccione E.
      • Krmac H.
      • Zara I.
      • Martinelli V.
      • et al.
      The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle.
      ). All three MARP proteins have been shown to associate to the N2A epitope of titin through binding of their ankyrin repeats to form the N2A signaling complex, which is also composed of myopalladin (
      • Bang M.L.
      • Mudry R.E.
      • McElhinny A.S.
      • Trombitas K.
      • Geach A.J.
      • Yamasaki R.
      • et al.
      Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.
      ) and the calpain protease, p94 (Figure 4). Passive muscle stretch induces increased immunostaining for the MARPs both in the nucleus and the I-band, with DARP staining also increasing in the intercalated discs (
      • Miller M.K.
      • Bang M.L.
      • Witt C.C.
      • Labeit D.
      • Trombitas C.
      • Watanabe K.
      • et al.
      The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.
      ). The concept that CARP is responsive to mechanical stress is consistent with its cytoplasmic distribution in striated muscle (cardiac and skeletal). There is still much to learn about the role of MARPs in striated muscle, and we speculate that these ankyrin repeat proteins may link myofibrillar stretch signals to the transcriptional regulation of muscle gene expression.
      Figure thumbnail gr4
      Figure 4Schematic representation of identified regulatory pathways in CARP expressing cells. CARP is expressed in striated muscle (blue), vascular smooth muscle (green) and VECs (yellow). Although the signaling pathways that regulate CARP expression are not completely defined, signaling molecules that are known to be active are shown. Question marks indicate unknown pathways and/or signaling molecules. Transcription factors that positively (+) and negatively (-) regulate transcription of the CARP gene are shown in the nucleus. CARP is located in the I-band portion of the sarcomere Z-disk in striated muscle, in direct association with myopalladin and the N2A region of titin (enlarged inset). Cardiac calsequestrin-2 (CASQ-2) has also been reported to directly associate with CARP. Mechanical stress increases CARP protein levels in the I-band (+) and in the nucleus (+) of the myofiber by mechanisms that implicate stretch-activated channels and cell adhesion molecules such as the integrins. In cardiac muscle, α-adrenergic agonist-stimulated CARP transcription is, in part, GATA 4 dependent. In contrast, β-adrenergic agonists do not stimulate CARP transcription but increase cellular CARP by stabilization of the protein and the mRNA (+). Doxorubicin and hypoxia-induced reactive oxygen species act via H7-sensitive serine/threonine kinase and p38, respectively, leading to induction of CHOP-10 (GADD153) and direct inhibition of CARP gene transcription. TGF-β, activin A and tumor necrosis factor alpha-α increase CARP transcription. The signaling pathways in cardiac muscles have not been described. Activin A and TGF-β use a Smad-mediated pathway to increase CARP transcription in vascular smooth muscle. In contrast, TGF-β signals via a p38-mediated pathway in VECs. CARP has been characterized as a negative transcriptional cofactor. In cardiac myocytes, CARP overexpression results in decreased ANF, TNC and MLC 2v gene expression at least in part owing to association with the YB-1 transcriptional activator. Transcriptional activation by CARP has not yet been described.
      Cytoplasmic CARP may also participate in regulating cardiac muscle contractility by its interaction with cardiac calsequestrin-2 (CASQ-2), a protein that stores calcium for muscle function (
      • Torrado M.
      • Nespereira B.
      • Lopez E.
      • Centeno A.
      • Castro-Beiras A.
      • Mikhailov A.T.
      ANKRD1 specifically binds CASQ2 in heart extracts and both proteins are co-enriched in piglet cardiac Purkinje cells.
      ). Five distinct CASQ-2 binding regions have been identified in CARP: three in the NH2-terminus and two within the ankyrin repeat region. None of the CASQ-2 binding regions overlap the CARP–titin interaction domains (see Figure 3). Without more functional data, the significance of this interaction can only be conjectured; however, overexpression of CARP in engineered heart tissue results in a significant decrease in the force of contraction stimulated by increasing doses of calcium (
      • Zolk O.
      • Marx M.
      • Jackel E.
      • El-Armouche A.
      • Eschenhagen T.
      Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase.
      ). This attenuation in calcium-stimulated contraction could involve more extensive CARP binding to CASQ-2, thereby sequestering calcium in the sarcoplasm and making it unavailable for muscle contraction.

      Overexpression Studies

      To investigate its actions in the wound-healing process, CARP was transduced into both full-thickness excisional wounds and an experimental sponge granulation tissue model by adenovirus-CARP infection (
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      ). In these models, CARP significantly increased neovascularization, blood perfusion, and the abundance and organization of migrating VEC. In both rabbit ear excisional wounds and ischemic rat skin wounds, exogenous expression of CARP increased neovascularization and blood perfusion, as assessed by histological analysis of vessel size and numbers as well as laser Doppler perfusion imaging. In an experimental granulation tissue model in which polyvinyl alcohol sponges are implanted subcutaneously in rats, sponge injection with adenovirus-CARP resulted in increased vascularization as assessed by CD34 staining of vessels. In flk-1/lacZ knock-in mice 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside staining showed a significant increase in lacZ-positive endothelial cells in the advenovirus-CARP injected sponges relative to those injected with a control virus.

      Potential Actions Of Carp

      As observed in striated muscle, CARP protein has been identified in both the cytoplasm and the nucleus of VSMC and VEC. Although CARP-protein associations within these cells have not been determined, the identification of titin-like proteins in smooth muscle and non-muscle cell types suggest a possible commonality with the CARP associations seen in striated muscle. Smitin, a 700 kDa titin-like protein, is associated with the contractile apparatus in smooth muscle (
      • Kim K.
      • Keller III, T.C.
      Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.
      ;
      • Chi R.J.
      • Olenych S.G.
      • Kim K.
      • Keller III, T.C.
      Smooth muscle alpha-actinin interaction with smitin.
      ). In non-muscle cell types, transfected, labeled z-repeats of titin have been localized to the dense bodies of stress fibers (
      • Ayoob J.C.
      • Turnacioglu K.K.
      • Mittal B.
      • Sanger J.M.
      • Sanger J.W.
      Targeting of cardiac muscle titin fragments to the Z-bands and dense bodies of living muscle and non-muscle cells.
      ). Overexpression of the z-repeat constructs results in the loss of stress fibers, suggesting the presence of a titin-like protein in non-muscle that acts to organize the stress fibers, similar to the sarcomere organizing function of titin in which CARP appears to play a role (
      • Bang M.L.
      • Mudry R.E.
      • McElhinny A.S.
      • Trombitas K.
      • Geach A.J.
      • Yamasaki R.
      • et al.
      Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.
      ). In non-muscle cells, titin-like proteins have also been identified in the nucleus, in association with spindle fibers during mitosis (
      • Wernyj R.P.
      • Ewing C.M.
      • Isaacs W.B.
      Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery.
      ). As yet, the association of CARP with the non-muscle titin-like proteins or any other proteins and the effects of protein–protein interactions on CARP activity have not been explored.
      CARP expression is increased in VSMC and VEC following wounding, but its effects are not certain. CARP overexpression studies in vivo and in vitro have provided some insights into its function. As reported above, overexpression in wounds and experimental granulation tissue increases angiogenesis and vasculogenesis, leading to increased blood perfusion, which appears to be due to the enhanced infiltration of vascular endothelium and endothelial precursor cells (
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      ). Support for this concept is provided by the effects of CARP overexpression in human microvascular endothelial cells (HMVEC). CARP overexpression does not affect proliferation in tissue, as assessed by proliferating cell nuclear antigen staining (Shi, unpublished). Preliminary findings show that overexpression of CARP in vitro results in the same or lower proliferation than HMVEC treated with either a control virus or nothing. Cell migration is CARP-responsive. Electrical cell-substrate impedance sensing was used to examine migration of CARP-transduced cells after ablation of a portion of the cell monolayer with a brief electrical pulse (
      • Shi Y.
      • Reitmaier B.
      • Regenbogen J.
      • Slowey R.M.
      • Opalenik S.R.
      • Wolf E.
      • et al.
      CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
      ). The rate and extent of monolayer restoration (i.e., cell migration) as measured by impedance, was greater in the CARP-overexpressing cells. Additionally, the haptotactic activity of the HMVEC in response to EGF is also increased, indicating that CARP increases the migration of VEC in response to cytokine signaling. Furthermore, CARP is also capable of inducing HMVECs tube formation, a measure of capillary endothelial morphogenesis, in a Matrigel™ assay. This response is partially abrogated with CARP small interfering RNA, further supporting a direct role for CARP in endothelial cell morphogenesis (Shi, unpublished).
      Previous studies indicated that CARP is protective against both hypoxia and oxidative stress-induced programmed cell death or apoptosis (
      • Han X.J.
      • Chae J.K.
      • Lee M.J.
      • You K.R.
      • Lee B.H.
      • Kim D.G.
      Involvement of GADD153 and cardiac ankyrin repeat protein in hypoxia-induced apoptosis of H9c2 cells.
      ). When human umbilical vein endothelial cells are subjected to serum starvation, cells transduced with CARP are able to survive better than those transduced with either a control virus or nothing. Treatment of human umbilical vein endothelial cells with DOX causes oxidative stress and initiation of the apoptotic pathway by inducing caspase 3. CARP transduction increases cell survival that correlates with a decrease in basal- and DOX-induced caspase 3 activity (Shi et al., unpublished). Thus, CARP may increase vascular cell abundance by amplification of the migratory stimulus while reducing the rate of apoptosis due to oxidative stress or physiologic attrition.
      Inasmuch as CARP is implicated in neovascularization, understanding the signaling mechanisms that regulate its expression becomes important. As mentioned above, CARP mRNA and protein are upregulated by TGF-β signaling in VSMC via a pathway that involves Smad factors (
      • Kanai H.
      • Tanaka T.
      • Aihara Y.
      • Takeda S.
      • Kawabata M.
      • Miyazono K.
      • et al.
      Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.
      ). Smad 3 potently increases CAGA binding element-dependent activity of a CARP promoter (-1828)-driven luciferase reporter whereas Smad 6, an inhibitory Smad, inhibits TGF-β stimulated activity. Our unpublished data suggest that both TGF-β and tumor necrosis factor alpha-α, but not vascular endothelial growth factor or basic fibroblast growth factor induce CARP expression in VEC. Though the tumor necrosis factor alpha-α signal transduction pathway has not yet been determined in these cells, we have found that the TGF-β signaling induces CARP mRNA and protein in VEC via the mitogen-activated protein kinase/p38 pathway rather than the Smad pathway. The p38 inhibitor SB203580 and a dominant-negative form of p38 attenuate the TGF-β induction of CARP mRNA but Smad 7, another inhibitory Smad, has no effect (Shi et al., unpublished). Similarly, CARP expression in cardiac myocytes is reported to be specifically upregulated by p38 and a constitutively active form of RAC 1 (
      • Aihara Y.
      • Kurabayashi M.
      • Saito Y.
      • Ohyama Y.
      • Tanaka T.
      • Takeda S.
      • et al.
      Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter.
      ). Activation by both factors is reportedly dependent on an M-CAT element within the CARP proximal promoter. Thus, CARP appears to be induced in endothelial cells by inflammatory cytokines and not angiogenic factors. Furthermore, the same inducers activate alternative signaling pathways in different cell types (Table 1 and Figure 4).
      Table 1Summary of CARP gene regulation
      StimulusTranscription factorMediatorsPromoter target siteEffect on CARPEffect on gene expressionReference
      α-AdrenergicGATA4GATAUpUp
      • Maeda T.
      • Sepulveda J.
      • Chen H.H.
      • Stewart A.F.
      Alpha(1)-adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes.
      GATAUpDown
      TGF-βSMADCAGAUpUp
      • Kanai H.
      • Tanaka T.
      • Aihara Y.
      • Takeda S.
      • Kawabata M.
      • Miyazono K.
      • et al.
      Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.
      TEF-1MCAT(1/3)UpUp
      p38, Rac1MCAT2UpUp
      • Aihara Y.
      • Kurabayashi M.
      • Saito Y.
      • Ohyama Y.
      • Tanaka T.
      • Takeda S.
      • et al.
      Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter.
      IL-1βUp
      • Chu W.
      • Burns D.K.
      • Swerlick R.A.
      • Presky D.H.
      Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
      HypoxiaGADD153P38C/EBP?DownApoptosis
      • Han X.J.
      • Chae J.K.
      • Lee M.J.
      • You K.R.
      • Lee B.H.
      • Kim D.G.
      Involvement of GADD153 and cardiac ankyrin repeat protein in hypoxia-induced apoptosis of H9c2 cells.
      H2O2S/T kinaseMCAT2Down
      • Aihara Y.
      • Kurabayashi M.
      • Tanaka T.
      • Takeda S.I.
      • Tomaru K.
      • Sekiguchi K.I.
      • et al.
      Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways.
      β-AdrenergicPKA, CaMKUp
      • Zolk O.
      • Marx M.
      • Jackel E.
      • El-Armouche A.
      • Eschenhagen T.
      Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase.
      TNF-αUpShi, unpublished
      TNF-αUp
      • Chu W.
      • Burns D.K.
      • Swerlick R.A.
      • Presky D.H.
      Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
      TGF-βp38UpShi, unpublished
      Activin AUp
      • de Waard V.
      • van Achterberg T.A.
      • Beauchamp N.J.
      • Pannekoek H.
      • de Vries C.J.
      Cardiac ankyrin repeat protein (CARP) expression in human and murine atherosclerotic lesions: activin induces CARP in smooth muscle cells.
      TGF-βUp
      • Boengler K.
      • Pipp F.
      • Fernandez B.
      • Ziegelhoeffer T.
      • Schaper W.
      • Deindl E.
      Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp).
      CaMK, calmodulin-dependent protein kinase; CARP, cardiac ankyrin repeat protein; PKA, protein kinase A; TNF-α, tumor necrosis factor-alpha; TGF-β, transforming growth factor-beta.
      Another serious gap in our knowledge of CARP is its downstream effects. Although CARPs function in the cytoplasm of VEC and VSMC remains to be determined, analysis of its nuclear function in cardiomyocytes suggests that it acts as a transcriptional cofactor that regulates expression of heart-enriched genes (Figure 4). CARP is reported to interact with the ubiquitous transcription factor YB-1 (
      • Zou Y.
      • Evans S.
      • Chen J.
      • Kuo H.C.
      • Harvey R.P.
      • Chien K.R.
      CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2–5 homeobox gene pathway.
      ). Studies to date have emphasized the role of this pairing in the downregulation of transcriptional targets such as myosin light chain-2v via the HF-1a element present in its promoter. Other cardiac-enriched genes such as antinuclear factor, have shown regulation by CARP overexpression (
      • Jeyaseelan R.
      • Poizat C.
      • Baker R.K.
      • Abdishoo S.
      • Isterabadi L.B.
      • Lyons G.E.
      • et al.
      A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.
      ), possibly by a similar mechanism. Thus, CARP-YB-1 interactions may serve as a prototype for targets of the Nkx2.5 homeobox protein in ventricular cardiomyocytes. Additionally, CARP expression is also associated with circumstances of muscle regeneration, such as bupavicaine injury and Duchenne muscular dystrophy (
      • Nakada C.
      • Oka A.
      • Nonaka I.
      • Sato K.
      • Mori S.
      • Ito H.
      • et al.
      Cardiac ankyrin repeat protein is preferentially induced in atrophic myofibers of congenital myopathy and spinal muscular atrophy.
      ) suggesting a transcriptional regulatory function in skeletal muscle. Our preliminary gene expression profiling of VSMC and HMVEC after CARP overexpression gives credence to the idea that CARP or its downstream targets are also responsible for upregulation of a number of relevant genes in these cells.
      Although the studies that have been discussed herein describe CARP expression in several systems, these approaches do not define the role of endogenous CARP. As the protein is massively upregulated during cardiomyogenesis (
      • Kuo H.
      • Chen J.
      • Ruiz-Lozano P.
      • Zou Y.
      • Nemer M.
      • Chien K.R.
      Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis.
      ), it may play a central role in the organization of the sarcomere and/or the transcriptome of the developing cardiomyocyte. In this respect, targeted deletion of CARP might be expected to lead to a fatal disruption of the cardiogenic program. Although lethality of global CARP deletion needs to be confirmed, the use of a conditional deletion in selected tissues or at selected sites should greatly improve our understanding of the intrinsic roles of the molecule. Currently, small interfering RNA reagents in this laboratory appear to be able to abrogate CARP-dependent effects such as endothelial morphogenesis. These tools can be used to fill the serious gap in our knowledge of CARPs downstream effects.

      Summary

      Based on associations with elements of the sarcomere and the transcriptional machinery, CARP appears to have both cytoplasmic and nuclear roles. The presence of titin-like proteins in smooth muscle and non-muscle cells suggests that CARP or a related member of the MARP family could have analogous functions in striated, smooth muscle and non-muscle cell types. At the nuclear level, CARP is strongly implicated as a transcriptional cofactor in regulation of cardiovascular development and response to injury in a variety of tissues. A number of studies show that oxidative stress and several stress-induced signaling molecules modulate CARP in the context of its transcriptional activity, and its own expression through promoter elements that are partially characterized. The mode of action of CARP on target genes is not well understood. The relationship between the cytoplasmic and nuclear sites of CARP interaction may have parallels with the dual role of molecules such as β-catenin (
      • Brembeck F.H.
      • Rosario M.
      • Birchmeier W.
      Balancing cell adhesion and Wnt signaling, the key role of beta-catenin.
      ), in which a structural element of the cytoplasm transforms into a regulatory element when shuttled to the nucleus. In the case of striated muscle, CARP is a key element of a complex containing titin, CASQ-2 and other molecules, which could be capable of relaying mechanical signals. It remains to be determined whether nuclear translocation of CARP contributes to mechanical or other stress responses. Overexpression of CARP in cells and tissues suggests that it could significantly contribute to the quality and extent of tissue repair, particularly with respect to neovascularization. It is not known whether these effects of excess CARP are a direct reflection of its intrinsic role, and targeted downregulation or deletion of CARP will be necessary to define its function. Regarding (cutaneous) wound repair, the pharmacologic action of CARP underscores the potential of gene transfer to reveal heretofore unknown activities of genes, the ability of adenoviral gene delivery to expand the wound healing pharmacopeia to nuclear transcription factors, and the possible presence of a novel mechanism for neovascularization.

      Conflict Of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      This work was supported by the NIDDK, the Department of Veterans Affairs, and Switch Biotech AG. We thank Susan R. Opalenik for critical reading of the manuscript and R. Michael Slowey for graphic illustration. Birgit Reitmaier, Michaela Bittner, and Andreas Goppelt contributed significantly to the development of the concepts presented.

      REFERENCES

        • Aihara Y.
        • Kurabayashi M.
        • Saito Y.
        • Ohyama Y.
        • Tanaka T.
        • Takeda S.
        • et al.
        Cardiac ankyrin repeat protein is a novel marker of cardiac hypertrophy: role of M-CAT element within the promoter.
        Hypertension. 2000; 36: 48-53
        • Aihara Y.
        • Kurabayashi M.
        • Tanaka T.
        • Takeda S.I.
        • Tomaru K.
        • Sekiguchi K.I.
        • et al.
        Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways.
        J Mol Cell Cardiol. 2000; 32: 1401-1414
        • Ayoob J.C.
        • Turnacioglu K.K.
        • Mittal B.
        • Sanger J.M.
        • Sanger J.W.
        Targeting of cardiac muscle titin fragments to the Z-bands and dense bodies of living muscle and non-muscle cells.
        Cell Motil Cytoskeleton. 2000; 45: 67-82
        • Bang M.L.
        • Mudry R.E.
        • McElhinny A.S.
        • Trombitas K.
        • Geach A.J.
        • Yamasaki R.
        • et al.
        Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.
        J Cell Biol. 2001; 153: 413-427
        • Baumeister A.
        • Arber S.
        • Caroni P.
        Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube endcompartments during muscle morphogenesis.
        J Cell Biol. 1997; 139: 1231-1242
        • Boengler K.
        • Pipp F.
        • Fernandez B.
        • Ziegelhoeffer T.
        • Schaper W.
        • Deindl E.
        Arteriogenesis is associated with an induction of the cardiac ankyrin repeat protein (carp).
        Cardiovasc Res. 2003; 59: 573-581
        • Brembeck F.H.
        • Rosario M.
        • Birchmeier W.
        Balancing cell adhesion and Wnt signaling, the key role of beta-catenin.
        Curr Opin Genet Dev. 2006; 16: 51-59
        • Chi R.J.
        • Olenych S.G.
        • Kim K.
        • Keller III, T.C.
        Smooth muscle alpha-actinin interaction with smitin.
        Int J Biochem Cell Biol. 2005; 37: 1470-1482
        • Chu W.
        • Burns D.K.
        • Swerlick R.A.
        • Presky D.H.
        Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
        J Biol Chem. 1995; 270: 10236-10245
        • de Waard V.
        • van Achterberg T.A.
        • Beauchamp N.J.
        • Pannekoek H.
        • de Vries C.J.
        Cardiac ankyrin repeat protein (CARP) expression in human and murine atherosclerotic lesions: activin induces CARP in smooth muscle cells.
        Arterioscler Thromb Vasc Biol. 2003; 23: 64-68
        • Han X.J.
        • Chae J.K.
        • Lee M.J.
        • You K.R.
        • Lee B.H.
        • Kim D.G.
        Involvement of GADD153 and cardiac ankyrin repeat protein in hypoxia-induced apoptosis of H9c2 cells.
        J Biol Chem. 2005; 280: 23122-23129
        • Ihara Y.
        • Suzuki Y.J.
        • Kitta K.
        • Jones L.R.
        • Ikeda T.
        Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin.
        Cell Calcium. 2002; 32: 21-29
        • Jeyaseelan R.
        • Poizat C.
        • Baker R.K.
        • Abdishoo S.
        • Isterabadi L.B.
        • Lyons G.E.
        • et al.
        A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.
        J Biol Chem. 1997; 272: 22800-22808
        • Kanai H.
        • Tanaka T.
        • Aihara Y.
        • Takeda S.
        • Kawabata M.
        • Miyazono K.
        • et al.
        Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.
        Circ Res. 2001; 88: 30-36
        • Kim K.
        • Keller III, T.C.
        Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.
        J Cell Biol. 2002; 156: 101-111
        • Kojic S.
        • Medeot E.
        • Guccione E.
        • Krmac H.
        • Zara I.
        • Martinelli V.
        • et al.
        The Ankrd2 protein, a link between the sarcomere and the nucleus in skeletal muscle.
        J Mol Biol. 2004; 339: 313-325
        • Kuo H.
        • Chen J.
        • Ruiz-Lozano P.
        • Zou Y.
        • Nemer M.
        • Chien K.R.
        Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis.
        Development. 1999; 126: 4223-4234
        • Maeda T.
        • Sepulveda J.
        • Chen H.H.
        • Stewart A.F.
        Alpha(1)-adrenergic activation of the cardiac ankyrin repeat protein gene in cardiac myocytes.
        Gene. 2002; 297: 1-9
        • Miller M.K.
        • Bang M.L.
        • Witt C.C.
        • Labeit D.
        • Trombitas C.
        • Watanabe K.
        • et al.
        The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.
        J Mol Biol. 2003; 333: 951-964
        • Mohler P.J.
        • Bennett V.
        Ankyrin-based cardiac arrhythmias: a new class of channelopathies due to loss of cellular targeting.
        Curr Opin Cardiol. 2005; 20: 189-193
        • Mohler P.J.
        • Bennett V.
        Defects in ankyrin-based cellular pathways in metazoan physiology.
        Front Biosci. 2005; 10: 2832-2840
        • Nakada C.
        • Oka A.
        • Nonaka I.
        • Sato K.
        • Mori S.
        • Ito H.
        • et al.
        Cardiac ankyrin repeat protein is preferentially induced in atrophic myofibers of congenital myopathy and spinal muscular atrophy.
        Pathol Int. 2003; 53: 653-658
        • Sedgwick S.G.
        • Smerdon S.J.
        The ankyrin repeat: a diversity of interactions on a common structural framework.
        Trends Biochem Sci. 1999; 24: 311-316
        • Shi Y.
        • Reitmaier B.
        • Regenbogen J.
        • Slowey R.M.
        • Opalenik S.R.
        • Wolf E.
        • et al.
        CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.
        Am J Pathol. 2005; 166: 303-312
        • Singal P.K.
        • Li T.
        • Kumar D.
        • Danelisen I.
        • Iliskovic N.
        Adriamycin-induced heart failure: mechanism and modulation.
        Mol Cell Biochem. 2000; 207: 77-86
        • Torrado M.
        • Lopez E.
        • Centeno A.
        • Castro-Beiras A.
        • Mikhailov A.T.
        Left-right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure.
        Eur J Heart Fail. 2004; 6: 161-172
        • Torrado M.
        • Nespereira B.
        • Lopez E.
        • Centeno A.
        • Castro-Beiras A.
        • Mikhailov A.T.
        ANKRD1 specifically binds CASQ2 in heart extracts and both proteins are co-enriched in piglet cardiac Purkinje cells.
        J Mol Cell Cardiol. 2005; 38: 353-365
        • Wernyj R.P.
        • Ewing C.M.
        • Isaacs W.B.
        Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery.
        Cell Motil Cytoskeleton. 2001; 50: 101-113
        • Yi X.
        • Bekeredjian R.
        • Defilippis N.J.
        • Siddiquee Z.
        • Fernandez E.
        • Shohet R.V.
        Transcriptional analysis of doxorubicin-induced cardiotoxicity.
        Am J Physiol Heart Circ Physiol. 2006; 290: H1098-H1102
        • Zolk O.
        • Frohme M.
        • Maurer A.
        • Kluxen F.W.
        • Hentsch B.
        • Zubakov D.
        • et al.
        Cardiac ankyrin repeat protein, a negative regulator of cardiac gene expression, is augmented in human heart failure.
        Biochem Biophys Res Commun. 2002; 293: 1377-1382
        • Zolk O.
        • Marx M.
        • Jackel E.
        • El-Armouche A.
        • Eschenhagen T.
        Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase.
        Cardiovasc Res. 2003; 59: 563-572
        • Zou Y.
        • Evans S.
        • Chen J.
        • Kuo H.C.
        • Harvey R.P.
        • Chien K.R.
        CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2–5 homeobox gene pathway.
        Development. 1997; 124: 793-804