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BMC Developmental Biology Biomed Central BMC Developmental Biology BioMed Central Research article Open Access Targeted disruption of the mouse Csrp2 gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure Julia F Sagave1, Markus Moser2, Elisabeth Ehler3, Sabine Weiskirchen1, Doris Stoll1, Kalle Günther4, Reinhard Büttner5 and Ralf Weiskirchen*1 Address: 1Institute of Clinical Chemistry and Pathobiochemistry, RWTH- University Hospital Aachen, Germany, 2Max Planck-Institute for Biochemistry, Martinsried, Germany, 3The Randall Division of Cell and Molecular Biophysics and The Cardiovascular Division, King's College London, UK, 4Qiagen, Hilden, Germany and 5Institute of Pathology, University Hospital Bonn, Germany Email: Julia F Sagave - [email protected]; Markus Moser - [email protected]; Elisabeth Ehler - [email protected]; Sabine Weiskirchen - [email protected]; Doris Stoll - [email protected]; Kalle Günther - [email protected]; Reinhard Büttner - [email protected]; Ralf Weiskirchen* - [email protected] * Corresponding author Published: 19 August 2008 Received: 19 March 2008 Accepted: 19 August 2008 BMC Developmental Biology 2008, 8:80 doi:10.1186/1471-213X-8-80 This article is available from: http://www.biomedcentral.com/1471-213X/8/80 © 2008 Sagave et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype. Results: A ~17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2- deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as β- catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue. Conclusion: We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy. Background served proteins that define a subset of zinc-binding LIM In vertebrates, the cysteine- and glycine-rich proteins domain proteins. As structural hallmarks, these proteins (CRPs) encoded by the Csrp genes are evolutionarily con- exhibit two LIM domains with a characteristic spacing, Page 1 of 12 (page number not for citation purposes) BMC Developmental Biology 2008, 8:80 http://www.biomedcentral.com/1471-213X/8/80 adjacent glycine-rich repeats, and a potential nuclear Furthermore, during embryogenesis and in adult tissue, localization signal [1]. Originally, this family of LIM Csrp2 gene expression is also prominently associated with domain proteins included three members (CRP1, CRP2, mesenchyme and epithelia [18,19]. Interestingly, com- CRP3/MLP) that were independently isolated in the pared to other CRP family members, CRP2 expression course of different experimental strategies [2-4]. Subse- begins early in gestation and has a distinct pattern of tis- quently, based on structural and sequence similarities, the sue distribution during cardiovascular development. [18]. thymus LIM protein (TLP) was grouped into this subclass CRP2 is expressed transiently in early embryonic cardio- of LIM domain proteins [5]. The four CRPs possess signif- myocytes similar to smooth muscle cell markers like α- icant differences in their temporal and spatial patterns of smooth muscle actin, calponin, and SM22α [18] but its expression raising interesting questions regarding the expression is downregulated in adult cardiomyocytes. physiological and biological significance of the CRP mul- Additionally, it was demonstrated that the expression of tigene family [6]. For example, it is uncertain if these pro- CRP2 is downregulated with cellular dedifferentiation teins perform unique functions or substitute for each induced by oncogenic transformation, injury, or wound other within a living organism. The cell types and organs healing [3,16,10]. that express the different CRPs suggest several hypotheti- cal functions for this group of LIM domain proteins, Recently, it was demonstrated that the loss of CRP2 did including possible roles in organization and stabilization not result in any apparent gross vascular defects or altered of the contractile myofibrillar/cytoskeletal apparatus [6- expression of smooth muscle cell markers [20]. Moreover, 8], maintenance of cellular functions [3], differentiation vascular development, morphology, cell proliferation, [5], transcriptional regulation [9], and in the establish- endothelial regeneration and the expression of several ment of fibrogenic responses [10]. In addition to this characteristic smooth muscle specific genes were similar potential functional versatility, there is growing evidence between WT and Csrp2 nulls. However, the loss of CRP2 supporting the notion that the two LIM domains of CRPs is correlated with increased neointima formation in serve as protein interfaces mediating specific protein-pro- response to vascular injury. Furthermore, vascular smooth tein interactions thereby arranging two or more protein muscle cells isolated from mice lacking CRP2 migrated constituents into nuclear transcription or cytoskeletal more rapidly in response to PDGF-BB with an increased complexes [11,12]. In this regard, the CRP3 protein (also activation of the Rho GTPase Rac1 suggesting that Csrp2 termed MLP for muscle LIM protein) is best characterized. and its protein product CRP2 are functionally linked to It is a positive regulator of myogenic differentiation that cell migration [20]. was first identified in a screen for genes that become tran- scriptionally upregulated as a result of skeletal muscle We here report about the generation and characterization denervation [4]. In accordance, the overexpression of MLP of a similar Csrp2 null mouse model. We demonstrate that in C2 myoblasts potentiates myogenic differentiation [4] these deficient mice are viable and fertile, exhibiting a and the absence of the Csrp3/Mlp gene product causes a mild cardiac phenotype in which the cardiomyocytes dis- phenotype of dilated cardiomyopathy underscoring the play a slight increase in their thickness, indicating moder- hypothesis that CRP3/MLP is an essential regulator of car- ate hypertrophy at the cellular level. In line with these diac muscle development [7]. In line with this hypothesis, findings, the peculiarity of heart architecture reflected by the morphological and clinical picture of dilated cardio- the typical arrangement of intercalated disc-associated myopathy in humans is associated with altered Csrp3/Mlp proteins (i.e. β-catenin, N-RAP, connexin-43) was altered expression [13] and Csrp3/Mlp mutations were found in suggesting that CRP2 is involved in the organization of families suffering from dilated as well as from hyper- the cytoskeleton in cardiac muscle cells. trophic cardiomyopathy [14,15]. Results Together, CRP1 and CRP2 were shown to be potent Chromosomal localization of the murine Csrp2 gene smooth muscle differentiation cofactors triggering the By use of the T31 mouse/hamster radiation hybrid (RH) conversion of pluripotent 10T1/2 fibroblasts into smooth panel [21,22] containing a set of 100 different DNAs from muscle cells when overexpressed together with serum somatic cell hybrids, we localized the murine Csrp2 gene response factor (SRF) and GATA proteins [9]. Compatible to Chromosome 10. In this analysis with the highest with this presumed function is the finding that CRP2 is anchor LOD of 23.2 was assigned to D10Mit150 with a present at highest levels in arterial samples [16,6]. Moreo- best-fit location between markers D10Mit203 proximal ver, a recent report demonstrated that CRP2 can effec- and D10Mit150 central, confirming the gene position tively switch on smooth muscle gene activity in adult that was recently launched by the Mouse Genome cardiac myocytes [17] suggesting that CRP2 has essential Sequencing Consortium (NT_09500). Noteworthy, this functions in controlling smooth muscle gene activity. region is syntenic to human chromosomal region Page 2 of 12 (page number not for citation purposes) BMC Developmental Biology 2008, 8:80 http://www.biomedcentral.com/1471-213X/8/80 12q21.1 (Fig. 1), essentially the region to which the human orthologue was previously assigned [23]. Targeted disruption of the Csrp2 gene For generation of the targeting vector we isolated
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