This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kong, Y. Articles by Williams, R. S. Search for Related Content PubMed PubMed Citation Articles by Kong, Y. Articles by Williams, R. S. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH Related Collections Genetically altered mice

(Circulation. 2001;103:2731.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Cardiac-Specific LIM Protein FHL2 Modifies the Hypertrophic Response to ß-Adrenergic Stimulation

Yanfeng Kong, PhD; John M. Shelton, BS; Beverly Rothermel, PhD; Xiangqing Li, PhD; James A. Richardson, PhD; Rhonda Bassel-Duby, PhD; R. Sanders Williams, MD

From the Departments of Internal Medicine, Molecular Biology, and Pathology (J.A.R.), University of Texas Southwestern Medical Center, Dallas, Tex.

Correspondence to R. Sanders Williams, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, NB11.200, Dallas, TX 75390-8573. E-mail williams{at}ryburn.swmed.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—A deficiency of muscle LIM protein results in dilated cardiomyopathy, but the function of other LIM proteins in the heart has not been assessed previously. We have characterized the expression and function of FHL2, a heart-specific member of the LIM domain gene family.

Methods and Results—Expression of FHL2 mRNA and protein was examined by Northern blot, in situ hybridization, and Western blot analyses of fetal and adult mice. FHL2 transcripts are present at embryonic day (E) 7.5 within the cardiac crescent in a pattern that resembles that of Nkx2.5 mRNA. During later stages of cardiac development and in adult animals, FHL2 expression is localized to the myocardium and absent from endocardium, cardiac cushion, outflow tract, or coronary vasculature. The gene encoding FHL2 was disrupted by homologous recombination, and knockout mice devoid of FHL2 were found to undergo normal cardiovascular development. In the absence of FHL2, however, cardiac hypertrophy resulting from chronic infusion of isoproterenol is exaggerated (59% versus 20% increase in heart weight/body weight in FHL null versus wild-type mice; P<0.01).

Conclusions—FHL2 is an early marker of cardiogenic cells and a cardiac-specific LIM protein in the adult. FHL2 is not required for normal cardiac development but modifies the hypertrophic response to ß-adrenergic stimulation.

Key Words: hypertrophy • genetics • molecular biology • myocardium

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The LIM protein superfamily comprises transcription factors, protein kinases, and cytoskeletal proteins that are involved in a wide range of cellular functions, including cell lineage determination, differentiation, focal adhesion, and signal transduction.^{1 2} The signature LIM motif is defined by the presence of 1 or multiple consensus sequence motifs (CX₂CX_17–19HX₂C₂X₂CX_16–20CX₂C), and LIM proteins can be subdivided into several subclasses based on the number of LIM motifs and the presence or absence of additional motifs in the protein. LIM-homeodomain proteins include both LIM motifs and a homeobox region, and several are known to be essential for development of skeletal, endocrine, or neuronal structures in vertebrates.^{3 4 5} LIM-only (LMO) proteins lack a homeodomain and contain variable numbers of LIM motifs. An LMO protein called muscle LIM protein (MLP) is a positive regulator of skeletal muscle differentiation^{6 7} and is required for maintenance of normal structure and function of the myocardium. Mice deficient in this LMO protein develop dilated cardiomyopathy during postnatal life.⁸

A specific subclass of LMO proteins contain four and a half LIM motifs and are designated as FHL proteins. Human FHL2, originally known as DRAL (Down-Regulated in Rhabdomyosarcoma LIM protein), was first identified by subtractive hybridization of normal myoblasts versus a rhabdomyosarcoma cell line.⁹ We isolated a murine homologue of FHL2 from a cardiac cDNA library in a screen for proteins interacting with myocyte nuclear factor (MNF), a forkhead/winged helix transcription factor expressed in developing heart and skeletal muscles and in myogenic progenitor cells (satellite cells) of adult mice.^{10 11 12} In this report, we describe the temporal and spatial pattern of FHL2 expression pattern throughout mouse development. FHL2 expression is evident in the earliest identifiable cardiac precursor cells of the embryo and is restricted primarily to cardiomyocytes during fetal and adult life. Analysis of homozygous knockout mice shows that FHL2 is not required for normal cardiac development. Cardiac mass is increased more, however, in adult mice devoid of FHL2 than in wild-type littermates after administration of the ß-adrenergic agonist isoproterenol. We conclude that the FHL2 is an early and persistent marker of the cardiomyocyte lineage in the mouse and can modify responses to certain stresses in the adult heart.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cloning of FHL2 Gene and Construction of the Targeting Vector
FHL2 cDNA was isolated from a murine cardiac library in a yeast 2-hybrid screen using MNF^{10 11 12} as bait. Mouse genomic DNA encoding FHL2 was isolated from a 129SVJ genomic P1 library (Stratagene) by use of probes based on the cDNA sequence. The FHL2^LacZ/neo targeting vector was constructed in pBluescript (Stratagene) to include 1.3 kb and 8 kb of homologous sequence regions located 5' and 3', respectively, to the region of the FHL2 gene altered by homologous recombination. In the mutant FHL2 allele, protein coding sequences from exon 2 (E2) of the native gene, including the translation initiation codon ATG, the N-terminal half LIM motif, part of the LIM1 domain, and 0.5 kb of intronic sequence downstream of E2 are replaced with a LacZ reporter gene and a neo resistance cassette. LoxP sites flank the neo cassette. The final construct was verified by restriction analysis and partial sequencing.

Generation of FHL2 Knockout Mice
Southern blot analysis performed on DNA isolated from neomycin-resistant ES clones showed that 8 of 500 ES clones had undergone homologous recombination of the targeting vector into the FHL2 locus. Three targeted ES clones transmitted the mutant allele through the germ line and generated FHL2^LacZ/neo heterozygote (FHL2^+/-) animals, which were crossed to generate FHL2^-/- mice. The phenotypic consequences of FHL2 deficiency were equivalent in offspring of the 3 separately derived lines of transgenic mice.

Mouse Embryo Analysis
Embryonic stage (days postcoitum) was estimated by timed pregnancies and somite counts. The embryos and decidua were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Expression of ß-galactosidase in murine embryos was assessed as previously described.¹³

Expression of FHL2 mRNA and Protein
RNA was isolated from adult or fetal tissues, and Northern blots were prepared and probed as previously described.¹⁴ Proteins (10 µg/lane) were separated by electrophoresis through a 12% SDS-polyacrylamide gel, transferred to nylon filters, and probed with a rabbit polyclonal antibody against FHL2. In situ hybridizations were performed as previously described.¹⁵

Chronic Infusion of Isoproterenol
Seven-day osmotic minipumps (Alzet, model 2001) were loaded with 0.2 mL of isoproterenol (28 mg/mL per 25 g body weight) and implanted into the subcutaneous space of 6- to 7-month-old mice. After 10 days, hearts were harvested, weighed, and analyzed histologically.

Statistical Analysis
The hypertrophic responses to chronic isoproterenol administration in FHL2 knockout and wild-type mice were compared by the Wilcoxon rank sum test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Conservation of Human and Mouse FHL2 cDNA Sequences
The cloned FHL2 cDNA encodes a 279-amino-acid protein that has 91% homology to the human FHL2 protein^{9 16 17} (Figure 1A). The FHL motifs are arrayed in tandem and separated by 8 amino acid linkers that are conserved between human and mouse FHL2 proteins, as well as in FHL1/SLIM1 and FHL3/SLIM2.^{18 19} A list of currently known FHL and cysteine-rich proteins (CRP proteins) is provided in Figure 1B.

View larger version (49K): [in this window] [in a new window]   Figure 1. A, Nucleotide sequence and predicted amino acid sequence of mouse and human FHL2 cDNA. Open reading frame codes for a protein of 279 amino acids. Amino acids comprising LIM domains are bracketed. B, Schematic representations of secondary structures of FHL and CRP proteins and summaries of their expression patterns and functions.1 2

Expression of FHL2 in Adult Tissues
Expression of FHL2 mRNA in adult mice is detected by Northern blot analysis only in heart, and not in skeletal muscle, brain, spleen, liver, kidney, or testis (Figure 2A). A rabbit polyclonal antibody raised against recombinant murine FHL2 protein detects a 33-kDa protein, approximately the size predicted from the cDNA sequence, in extracts of adult mouse heart (Figure 2B), with trace levels also evident in stomach. In situ hybridization performed with a ³⁵S-labeled antisense FHL2 probe on transverse sections of adult cardiac ventricles (Figure 2C and 2D) demonstrates the presence of FHL2 transcripts within cardiomyocytes throughout the ventricular free walls and prominently in the septum. In contrast, no FHL2 mRNA is present within cells of the coronary vasculature (arrows in Figure 2D).

View larger version (113K): [in this window] [in a new window]   Figure 2. Expression of FHL2 mRNA and protein in adult tissues. A, Distribution of FHL2 mRNA among tissues of adult mouse. A 1.4-kb FHL2 transcript was detected by Northern blot analysis only in heart. B, Distribution of FHL2 protein among tissues of adult mouse. A 33-kDa FHL2 protein, as detected by immunoblot analysis, is most abundant in heart, and trace amounts are present in stomach. Recombinant FHL2 (rFHL2) produced in Escherichia coli provides a positive control. C, In situ hybridization analysis of FHL2 mRNA in adult mouse heart. D, Inset of C shown at higher power. Bar=200 µm. FHL2 transcript is present in cardiac myocytes but not within coronary vasculature (arrow).

FHL2 Is an Early and Persistent Marker of the Cardiomyocyte Lineage
FHL2 mRNA can be detected by Northern blot analysis as early as embryonic day 7 (Figure 3A) and continues to be detected in later-stage embryos. In situ hybridization (Figure 3B through 3E) showed that FHL2 transcripts are present during heart development; in the common atrium, primitive ventricle and bulbus cordis of the looping heart tube at E9.5; in the common atrium, the developing septal primordium, and compact and trabecular components of the bulbus cordis at E10.5; in the atria and ventricular free walls but not the atrioventricular cushion at E12.5; and in the myocardium, including the intraventricular septum, but not the outflow tract or atrioventricular cushions at E14.5.

View larger version (45K): [in this window] [in a new window]   Figure 3. Expression of FHL2 mRNA in fetal tissues. A, Northern analysis of RNA isolated from mouse embryos at 7, 11, 15, and 17 days postcoitum (p.c.). FHL2 mRNA is detected as early as day 7 p.c. B through E, In situ hybridization analysis of FHL2 mRNA in mouse embryos. Bar=400 µm. High levels of expression of FHL2 are evident within myocardium at all stages from 9.5 to 14.5 days postcoitum (E9.5 to E14.5). Septal primordium has particularly high levels of FHL2 expression at E14.5. No FHL2 transcript is detected in cardiac cushion or in outflow tracts (E). Low to moderate levels of FHL2 expression are detected in gut and in epithelium around neural tube.

FHL2 expression also can be detected in extracardiac tissues at certain stages of development (Figure 3B through 3E). From E9.5 to E12.5, FHL2 is expressed in undifferentiated cells of the head and gut mesenchyme, within the endothelium of the dorsal aorta and branchial arch arteries, and in a narrow subepithelial zone of the developing mid gut and bronchi. At E14.5, FHL2 expression is evident in the wall of the gut, in the urogenital sinus and associated umbilical artery, and in mesenchyme surrounding the spinal cord.

The earliest stages of cardiac development in vertebrates can be defined by the expression of the homeodomain protein Nkx2.5/Csx^{20 21} in precardiac mesoderm. At E7.5, Nkx2.5 is expressed in cardiac progenitor cells located at the anterior and lateral regions of the embryo, which fuse in the midline to form the cardiac crescent.^{13 22 23 24} Whole-mount in situ hybridizations performed on littermate embryos (Figure 4) show that FHL2 and Nkx2.5 transcripts are colocalized within precardiac mesoderm of embryos at the early head-fold stage (E7.5) and at subsequent stages of heart development.

View larger version (28K): [in this window] [in a new window]   Figure 4. Whole-mount in situ hybridization analysis of FHL2 and Nkx-2.5 mRNA in early mouse embryos. Expression of cardiac lineage marker Nkx-2.5 at E7.5 to E7.75 is localized to anterior and lateral mesoderm of embryo in a structure known as cardiac crescent. Nkx2.5 continues to be expressed predominantly within myocardium at later stages of heart development (E9.5). FHL2 mRNA is expressed in a similar pattern.

Expression of a ß-Galactosidase (LacZ) Reporter Gene Inserted Into the Mouse FHL2 Gene Locus
Heterozygous FHL2^LacZ/neo embryos were generated by homologous recombination of a targeting vector into the FHL2 locus (Figure 5) and were analyzed for ß-galactosidase activity at stages from E3.5 through E9.5 (Figure 6). In early embryos before heart development (E3.5 to E6.5), transcription of the LacZ transgene is evident in extraembryonic tissues, such as polar trophectoderm around the blastocoelic cavity of the intact blastocyst (Figure 6A), and in extraembryonic ectoderm at the onset of gastrulation (Figure 6B). FHL2^LacZ/neo expression is observed in the chorion (part of the future placenta) of the extraembryonic tissue and in the chorioallantoic placenta at E9.5 (Figure 6A and 6B). At E7.5, ß-galactosidase activity is detected in the cardiac crescent, corroborating results obtained with whole-mount in situ hybridizations (Figure 4, E7.5). At later stages of heart development, strong FHL2^LacZ/neo expression continues in developing cardiomyocytes, but no expression is seen in the endocardium or the bulbotruncal junction region of the outflow tract of the primitive heart. Weak FHL2^LacZ/neo expression can be detected in the stomach, fat pads, and spleen, but no detectable levels of LacZ expression are observed in brain, lung, liver, and skeletal muscle (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 5. Targeted replacement of FHL2 gene with a LacZ reporter gene. A, Schematic of FHL2 locus, targeting vector, and mutant allele. FHL2LacZ/neo targeting vector includes 1.3 kb and 8 kb of homologous sequence regions located 5' and 3', respectively, to region of FHL2 gene altered by homologous recombination. In mutant FHL2 allele, sequences from E2 of native gene are replaced with LacZ reporter and neo resistance cassette. Arrow indicates direction of transcription. LoxP sites flanking neo cassette are indicated with open arrows. Correctly recombined locus is transcribed to produce an mRNA that fuses noncoding FHL2 sequences to LacZ coding region. B, Southern blot analysis of FHL2LacZ/neo and wild-type FHL2 alleles. By use of 5' external probe and EcoRI digestion, wild-type and targeted alleles generate 3.6-kb and 5.6-kb fragments, respectively. C, Northern blot from heart or lung of wild-type (+/+), heterozygous (+/-), and homozygous null (-/-) mice. Ethidium bromide stain of 18S rRNA provides a loading control. D, Western blots prepared from hearts of wild-type (+/+) and FHL2 null (-/-) mice. Instaview stain of protein (BDH Laboratories) provides a loading control.

View larger version (62K): [in this window] [in a new window]   Figure 6. FHL2LacZ/neo expression in early mouse embryos. LacZ staining of whole-mount preparations from embryos harvested 3.5 to 9.5 days postcoitum. A, At E3.5, embryo, LacZ-positive cells are seen around blastocoelic cavity. B, Expression was localized to extraembryonic tissue at E6.5. C and D, As gastrulation proceeds, FHL2LacZ/neo expression is localized to chorion of extraembryonic tissue and to cardiac crescent. E and F, LacZ staining is evident predominantly in developing heart, with some LacZ-positive cells seen within allantois. G, H, and I, Histological sections of embryos in C, D, and E. Bar=200 µm.

Normal Cardiovascular Development in Mice Lacking FHL2
The gene-targeting strategy used to insert a LacZ reporter gene into the FHL2 locus was designed also to produce a null allele by eliminating E2 of the FHL2 gene (Figure 5). Genotyping revealed wild-type (FHL2^+/+), heterozygous (FHL2^+/-), and homozygous (FHL2^-/-) offspring close to the mendelian prediction, indicating no survival disadvantage for embryos lacking FHL2. The null state was confirmed by the absence of FHL2 mRNA and protein as assessed by Northern (Figure 5C) and Western (Figure 5D) blot analyses of hearts from FHL2^-/- mice. Gross and microscopic anatomy of hearts from adult mice without FHL2 were normal.

Hypertrophic Responses to ß-Adrenergic Stimulation
Osmotic minipumps loaded with 0.2 mL of isoproterenol (28 mg/mL per 25 g body weight) were implanted into mice to subject these animals to the stress of sustained ß-adrenergic stimulation. This stimulus is known to provoke cardiac hypertrophy in wild-type mice,²⁵ and we confirmed this finding in wild-type littermates of FHL2^-/- animals on harvesting the hearts 10 days after implantation. We noted, however, that the hypertrophic response of hearts from mice lacking FHL2 was exaggerated above that in wild-type animals (Figure 7A). As shown in Figure 7B, in the absence of drug treatment, there was no apparent difference in heart-weight/body-weight ratios among 12 FHL2^-/- mice (5.5±0.3 mg/g) compared with an equal number of FHL2^+/+ littermates (5.7±0.3 mg/g). After isoproterenol, however, heart-weight/body-weight ratios in 12 FHL2^-/- mice (8.7±0.4 mg/g) were greater than those of FHL2^+/+ littermates (6.9±0.2 mg/g) (P<0. 01). The increase in mean cardiac mass of FHL2^-/- mice was 58%, compared with 20% for wild-type animals. This exaggerated hypertrophic response in FHL2^-/- mice was associated with a greater activation in expression of atrial natriuretic factor, a well-characterized molecular marker of cardiac hypertrophy (Figure 7C).

View larger version (42K): [in this window] [in a new window]   Figure 7. Hypertrophic response to ß-adrenergic stimulation is augmented in mice lacking FHL2. A, Trichrome-stained histological sections of adult heart from untreated or isoproterenol-treated FHL2+/+ and FLH2-/- mice. Multifocal areas of myocardial fibrosis (blue) are evident within left ventricular free wall of isoproterenol-treated FHL2-/- animal. B, Heart-weight–to–body-weight ratios are increased by infusion of isoproterenol from osmotic minipumps in both wild-type (FHL2+/+) and mutant (FHL2-/-) mice. Magnitude of increase in FHL2-/- animals (59%), however, was greater than that observed in FHL2+/+ mice (20%). C, Northern blot analysis showing increased expression of hypertrophic marker atrial natriuretic factor in mice lacking FHL2 compared with wild-type mice after isoproterenol treatment.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
There are 2 principal findings of this study. First, the LIM protein FHL2 is an early and persistent marker of cells within the cardiomyocyte lineage. FHL2 is expressed in the earliest identifiable myocardial progenitor cells of the cardiac crescent in a pattern that resembles that of the homeodomain protein Nkx2.5. FHL2 continues to be expressed selectively in cells of the atrial and ventricular myocardium at later stages of cardiac development and in the adult heart. Like Nkx2.5, FHL2 is not restricted entirely to the heart, but expression in other fetal and adult tissues is transient and/or minimal compared with the strong and persistent expression within the myocardium. The second major finding is that although FHL2 is not required for normal cardiovascular development, FHL2 modifies responses of the adult myocardium to certain stresses. Specifically, we observed an exaggerated hypertrophic response to the ß-adrenergic receptor agonist isoproterenol in FHL2^-/- mice.

Other LIM proteins are expressed in the heart, but none exhibit the temporal and spatial pattern of expression we report here for FHL2. The LMO protein MLP is highly enriched in adult cardiomyocytes, but the earliest reported onset of MLP expression during embryonic life (E9)²⁶ follows that of FHL2 (E7.5). In addition, MLP is expressed prominently in somites and developing skeletal muscles,⁶ whereas FHL2 expression is not detected in these tissues. The LMO proteins CRP1 and CRP2, which are more closely related to MLP than to FHL2 (see Figure 1B), are expressed in the developing heart, but in a pattern that differs from that of FHL2. CRP1 expression is detected in both atria and ventricles at E9.5 but, unlike FHL2, is also abundant in vascular and nonvascular smooth muscle cells of the outflow tract.²⁶ This pattern differs from both FHL2 and MLP, the expression of which within the heart is restricted to cardiomyocytes. FHL1/SLIM1 and FHL3/SLIM2 are closely related structurally to FHL2 and are expressed in skeletal muscles and heart.^{16 18 19 27 28 29 30} Although FHL2 exhibits a greater degree of cardiac specificity than other known LIM proteins, it is possible that related FHL proteins can substitute for developmental functions of FHL2 within in the developing heart.

The present study did not address biochemical functions of FHL2, but the literature provides several interesting leads for future investigations. The FHL protein ACT functions as a transcriptional coactivator with CREM and CREB proteins.³¹ FHL2 itself was recently shown to function as a coactivator in association with androgen receptors.³² It will be interesting to determine whether FHL2 collaborates with nuclear receptors or CREM/CREB proteins in the myocardium, particularly because transgenic mice expressing a dominant negative form of CREB develop dilated cardiomyopathy.³³ Our own preliminary data showing a physical association between FHL2- and DNA-binding MNF proteins 10^-12 also lend credence to the speculation that FHL2 may be a transcriptional modulator in the heart.

Among the set of transcriptional regulators known to have an important role in cardiac development, FHL2 expression most closely parallels that of the homeodomain protein Nkx2.5. The colocalized expression of FHL2 and Nkx2.5 suggests either that these 2 genes are responding to a common set of upstream regulators or that one may serve to control the other. In either case, detailed examination of promoter and enhancer elements from the FHL2 gene, in parallel with similar studies of Nkx2.5 gene regulation, may shed light on transcriptional events pertinent to the earliest steps of cardiomyocyte determination.

Finally, the observation that FHL2^-/- mice have an exaggerated response to the hypertrophic effects of isoproterenol suggests that FHL2 interacts with downstream effectors of ß-adrenergic receptor signaling in the myocardium or with proteins shared among different hypertrophic signaling pathways. As a gene that is not required for normal development but is capable of modifying cardiac responses to environmental stress, it is possible that allelic variants of the FHL2 gene may be present within human populations and influence the natural history of cardiovascular diseases in a clinical setting.

	Acknowledgments

 
This work was supported by grants from the NIH (AR-40849, HL-61624, GM-62114, and HL-07360) and the D.W. Reynolds Foundation. We thank S. Clay Williams for assistance with gross dissections and transcardial perfusions and Jeffrey M. Stark for preparation of histological sections.

Received October 19, 2000; revision received December 31, 2000; accepted February 1, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Jurata LW, Gill GN. Structure and function of LIM domains. Curr Top Microbiol Immunol. 1998;228:75–113.[Medline] [Order article via Infotrieve]
   
2. Bach I. The LIM domain: regulation by association. Mech Dev. 2000;91:5–17.[Medline] [Order article via Infotrieve]
   
3. Shawlot W, Behringer RR. Requirement for Lim1 in head-organizer function. Nature. 1995;374:425–430.[Medline] [Order article via Infotrieve]
   
4. Sheng HZ, Moriyama K, Yamashita T, et al. Multistep control of pituitary organogenesis. Science. 1997;278:1809–1812.[Abstract/Free Full Text]
   
5. Pfaff SL, Mendelsohn M, Stewart CL, et al. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell. 1996;84:309–320.[Medline] [Order article via Infotrieve]
   
6. Arber S, Halder G, Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell. 1994;79:221–231.[Medline] [Order article via Infotrieve]
   
7. Kong YF, Flick MJ, Kudla AJ, et al. Muscle LIM protein promotes myogenesis by enhancing the activity of myoD. Mol Cell Biol. 1997;17:4750–4760.[Abstract]
   
8. Arber S, Hunter JJ, Ross J, et al. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell. 1997;88:393–403.[Medline] [Order article via Infotrieve]
   
9. Genini M, Schwalbe P, Scholl FA, et al. Subtractive cloning and characterization of DRAL, a novel LIM-domain protein down-regulated in rhabdomyosarcoma. DNA Cell Biol. 1997;16:433–442.[Medline] [Order article via Infotrieve]
   
10. Garry DJ, Yang Q, Bassel-Duby R, et al. Persistent expression of MNF identifies myogenic stem cells in postnatal muscles. Dev Biol. 1997;188:280–294.[Medline] [Order article via Infotrieve]
    
11. Yang Q, Bassel-Duby R, Williams RS. Transient expression of a winged-helix protein, MNF-beta, during myogenesis. Mol Cell Biol. 1997;17:5236–5243.[Abstract]
    
12. Yang Q, Kong Y, Rothermel B, et al. The winged-helix/forkhead protein myocyte nuclear factor beta (MNF-beta) forms a co-repressor complex with mammalian sin3B. Biochem J. 2000;345:335–343.
    
13. Reecy JM, Li X, Yamada M, et al. Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5. Development. 1999;126:839–849.[Abstract]
    
14. Garry DJ, Bassel-Duby R, Richardson JA, et al. Postnatal development and plasticity of specialized muscle fiber characteristics in the hindlimb. Dev Genetics. 1996;19:146–156.[Medline] [Order article via Infotrieve]
    
15. Shelton JM, Lee MH, Richardson JA, et al. Microsomal triglyceride transfer protein expression during mouse development. J Lipid Res. 2000;41:532–537.[Abstract/Free Full Text]
    
16. Morgan MJ, Madgwick AJ. Slim defines a novel family of LIM-proteins expressed in skeletal muscle. Biochem Biophys Res Commun. 1996;225:632–638.[Medline] [Order article via Infotrieve]
    
17. Chan KK, Tsui SK, Lee SM, et al. Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart. Gene. 1998;210:345–350.[Medline] [Order article via Infotrieve]
    
18. Morgan MJ, Madgwick AJ. The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle. Biochem Biophys Res Commun. 1999;255:245–250.[Medline] [Order article via Infotrieve]
    
19. Brown S, Biben C, Ooms LM, et al. The cardiac expression of striated muscle LIM protein 1 (SLIM1) is restricted to the outflow tract of the developing heart. J Mol Cell Cardiol. 1999;31:837–843.[Medline] [Order article via Infotrieve]
    
20. Lints TJ, Parsons LM, Hartley L, et al. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development. 1993;119:419–431.[Abstract]
    
21. Komuro I, Izumo S. Csx: a murine homeobox-containing gene specifically expressed in the developing heart. Proc Natl Acad Sci U S A. 1993;90:8145–8149.[Abstract/Free Full Text]
    
22. Tanaka M, Wechsler SB, Lee IW, et al. Complex modular cis-acting elements regulate expression of the cardiac specifying homeobox gene Csx/Nkx2.5. Development. 1999;126:1439–1450.[Abstract]
    
23. Searcy RD, Vincent EB, Liberatore CM, et al. A GATA-dependent nkx-2.5 regulatory element activates early cardiac gene expression in transgenic mice. Development. 1998;125:4461–4470.[Abstract]
    
24. Lien CL, Wu C, Mercer B, et al. Control of early cardiac-specific transcription of Nkx2-5 by a GATA-dependent enhancer. Development. 1999;126:75–84.[Abstract]
    
25. Kudej RK, Iwase M, Uechi M, et al. Effects of chronic beta-adrenergic receptor stimulation in mice. J Mol Cell Cardiol. 1997;29:2735–2746.[Medline] [Order article via Infotrieve]
    
26. Jain MK, Kashiki S, Hsieh CM, et al. Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system. Circ Res. 1998;83:980–985.[Abstract/Free Full Text]
    
27. Chu P, Ruiz-Lozano P, Zhou Q, et al. Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system. Mech Dev. 2000;95:259–265.[Medline] [Order article via Infotrieve]
    
28. Brown S, McGrath MJ, Ooms LM, et al. Characterization of two isoforms of the skeletal muscle LIM protein 1, SLIM1: localization of SLIM1 at focal adhesions and the isoform slimmer in the nucleus of myoblasts and cytoplasm of myotubes suggests distinct roles in the cytoskeleton and in nuclear-cytoplasmic communication. J Biol Chem. 1999;274:27083–27091.[Abstract/Free Full Text]
    
29. Greene WK, Baker E, Rabbitts TH, et al. Genomic structure, tissue expression and chromosomal location of the LIM-only gene, SLIM1. Gene. 1999;232:203–207.[Medline] [Order article via Infotrieve]
    
30. Morgan MJ, Madgwick AJA. The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle. Biochem Biophys Res Commun. 1999;255:245–250.
    
31. Fimia GM, De Cesare D, Sassone-Corsi P. CBP-independent activation of CREM and CREB by the LIM-only protein ACT. Nature. 1999;398:165–169.[Medline] [Order article via Infotrieve]
    
32. Muller JM, Isele U, Metzger E, et al. FHL2, a novel tissue-specific coactivator of the androgen receptor. EMBO J. 2000;19:359–369.[Medline] [Order article via Infotrieve]
    
33. Fentzke RC, Korcarz CE, Lang RM, et al. Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J Clin Invest. 1998;101:2415–2426. [Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				J. Park, C. Will, B. Martin, L. Gullotti, N. Friedrichs, R. Buettner, H. Schneider, S. Ludwig, and V. Wixler Deficiency in the LIM-only protein FHL2 impairs assembly of extracellular matrix proteins FASEB J, July 1, 2008; 22(7): 2508 - 2520. [Abstract] [Full Text] [PDF]

				C. Labalette, Y. Nouet, J. Sobczak-Thepot, C. Armengol, F. Levillayer, M.-C. Gendron, C.-A. Renard, B. Regnault, J. Chen, M.-A. Buendia, et al. The LIM-only Protein FHL2 Regulates Cyclin D1 Expression and Cell Proliferation J. Biol. Chem., May 30, 2008; 283(22): 15201 - 15208. [Abstract] [Full Text] [PDF]

				D. W. Donker, J. G. Maessen, F. Verheyen, F. C. Ramaekers, R. L. H. M. G. Spatjens, H. Kuijpers, C. Ramakers, P. M. H. Schiffers, M. A. Vos, H. J. G. M. Crijns, et al. Impact of acute and enduring volume overload on mechanotransduction and cytoskeletal integrity of canine left ventricular myocardium Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2324 - H2332. [Abstract] [Full Text] [PDF]

				D. L. Cottle, M. J. McGrath, B. S. Cowling, I. D. Coghill, S. Brown, and C. A. Mitchell FHL3 binds MyoD and negatively regulates myotube formation J. Cell Sci., April 15, 2007; 120(8): 1423 - 1435. [Abstract] [Full Text] [PDF]

				V. Wixler, S. Hirner, J. M. Muller, L. Gullotti, C. Will, J. Kirfel, T. Gunther, H. Schneider, A. Bosserhoff, H. Schorle, et al. Deficiency in the LIM-only protein Fhl2 impairs skin wound healing J. Cell Biol., April 9, 2007; 177(1): 163 - 172. [Abstract] [Full Text] [PDF]

				J. Peng, K. Raddatz, J. D. Molkentin, Y. Wu, S. Labeit, H. Granzier, and M. Gotthardt Cardiac Hypertrophy and Reduced Contractility in Hearts Deficient in the Titin Kinase Region Circulation, February 13, 2007; 115(6): 743 - 751. [Abstract] [Full Text] [PDF]

				J. Sun, G. Yan, A. Ren, B. You, and J. K. Liao FHL2/SLIM3 Decreases Cardiomyocyte Survival by Inhibitory Interaction With Sphingosine Kinase-1 Circ. Res., September 1, 2006; 99(5): 468 - 476. [Abstract] [Full Text] [PDF]

				M. Hoshijima Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1313 - H1325. [Abstract] [Full Text] [PDF]

				C. Labalette, C.-A. Renard, C. Neuveut, M.-A. Buendia, and Y. Wei Interaction and Functional Cooperation between the LIM Protein FHL2, CBP/p300, and {beta}-Catenin Mol. Cell. Biol., December 15, 2004; 24(24): 10689 - 10702. [Abstract] [Full Text] [PDF]

				A. A. Hill and P. R. Riley Differential Regulation of Hand1 Homodimer and Hand1-E12 Heterodimer Activity by the Cofactor FHL2 Mol. Cell. Biol., November 15, 2004; 24(22): 9835 - 9847. [Abstract] [Full Text] [PDF]

				T. Samson, N. Smyth, S. Janetzky, O. Wendler, J. M. Muller, R. Schule, H. von der Mark, K. von der Mark, and V. Wixler The LIM-only Proteins FHL2 and FHL3 Interact with {alpha}- and {beta}-Subunits of the Muscle {alpha}7{beta}1 Integrin Receptor J. Biol. Chem., July 2, 2004; 279(27): 28641 - 28652. [Abstract] [Full Text] [PDF]

				N. H. Purcell, D. Darwis, O. F. Bueno, J. M. Muller, R. Schule, and J. D. Molkentin Extracellular Signal-Regulated Kinase 2 Interacts with and Is Negatively Regulated by the LIM-Only Protein FHL2 in Cardiomyocytes Mol. Cell. Biol., February 1, 2004; 24(3): 1081 - 1095. [Abstract] [Full Text] [PDF]

				C.-L. Hsu, Y.-L. Chen, S. Yeh, H.-J. Ting, Y.-C. Hu, H. Lin, X. Wang, and C. Chang The Use of Phage Display Technique for the Isolation of Androgen Receptor Interacting Peptides with (F/W)XXL(F/W) and FXXLY New Signature Motifs J. Biol. Chem., June 20, 2003; 278(26): 23691 - 23698. [Abstract] [Full Text] [PDF]

				I. D. Coghill, S. Brown, D. L. Cottle, M. J. McGrath, P. A. Robinson, H. H. Nandurkar, J. M. Dyson, and C. A. Mitchell FHL3 Is an Actin-binding Protein That Regulates {alpha}-Actinin-mediated Actin Bundling: FHL3 LOCALIZES TO ACTIN STRESS FIBERS AND ENHANCES CELL SPREADING AND STRESS FIBER DISASSEMBLY J. Biol. Chem., June 20, 2003; 278(26): 24139 - 24152. [Abstract] [Full Text] [PDF]

				S. Lange, D. Auerbach, P. McLoughlin, E. Perriard, B. W. Schafer, J.-C. Perriard, and E. Ehler Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2 J. Cell Sci., March 14, 2003; 115(24): 4925 - 4936. [Abstract] [Full Text] [PDF]

				P. A. Robinson, S. Brown, M. J. McGrath, I. D. Coghill, R. Gurung, and C. A. Mitchell Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion, spreading, and migration Am J Physiol Cell Physiol, March 1, 2003; 284(3): C681 - C695. [Abstract] [Full Text] [PDF]

				K. Schafer, P. Neuhaus, J. Kruse, and T. Braun The Homeobox Gene Lbx1 Specifies a Subpopulation of Cardiac Neural Crest Necessary for Normal Heart Development Circ. Res., January 10, 2003; 92(1): 73 - 80. [Abstract] [Full Text] [PDF]

				R. Knoll, M. Hoshijima, and K.R. Chien Z-line proteins: implications for additional functions Eur. Heart J. Suppl., December 1, 2002; 4(suppl_I): I13 - I17. [Abstract] [PDF]

				B. Martin, R. Schneider, S. Janetzky, Z. Waibler, P. Pandur, M. Kuhl, J. Behrens, K. von der Mark, A. Starzinski-Powitz, and V. Wixler The LIM-only protein FHL2 interacts with {beta}-catenin and promotes differentiation of mouse myoblasts J. Cell Biol., October 14, 2002; 159(1): 113 - 122. [Abstract] [Full Text] [PDF]

				S. Kupershmidt, I. C.-H. Yang, M. Sutherland, K.S. Wells, T. Yang, P. Yang, J. R. Balser, and D. M. Roden Cardiac-enriched LIM domain protein fhl2 is required to generate IKs in a heterologous system Cardiovasc Res, October 1, 2002; 56(1): 93 - 103. [Abstract] [Full Text] [PDF]

				P. McLoughlin, E. Ehler, G. Carlile, J. D. Licht, and B. W. Schafer The LIM-only Protein DRAL/FHL2 Interacts with and Is a Corepressor for the Promyelocytic Leukemia Zinc Finger Protein J. Biol. Chem., September 27, 2002; 277(40): 37045 - 37053. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager