Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles
et al. (2014) Imbalanced Expression of Vcan mRNA Splice Form Proteins
Alters Heart Morphology and Cellular Protein Profiles. PLoS ONE 9(2): e89133. doi:10.1371/journal.pone.0089133
Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles
Tara A. Burns 0
Maria T. Dours-Zimmermann 0
Dieter R. Zimmermann 0
Edward L. Krug 0
Susana Comte- 0
Walters 0
Leticia Reyes 0
Monica A. Davis 0
Kevin L. Schey 0
John H. Schwacke 0
Christine B. Kern 0
Corey H. Mjaatvedt 0
Thomas Brand, Heart Science Centre, Imperial College London, United Kingdom
0 1 Departments of Regenerative Medicine & Cell Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America, 2 Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina, United States of America, 3 Institute of Surgical Pathology, University Hospital Zurich , Zurich , Switzerland , 4 Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee , United States of America
The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.
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Funding: This work was supported by NIH NHLBI 66231 and American Heart Association Grant-in Aid to CHM; NIH NHLBI Cardiovascular Proteomics Center
(contract N01-HV-28282; to ELK and KLS); NIH NCRR C06 RR018823 (to ELK); and grants from the Swiss National Science Foundation and Velux Foundation (to
MTD-Z and DRZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
During embryonic development, the extracellular matrix plays a
central role in restructuring the single heart tube into a mature
multi-chambered organ. To date, only two structural components
of the extracellular matrix, versican and its binding partner
hyaluronan, have been shown to be indispensably required for
successful completion of this morphogenetic transition. Complete
loss of either of these matrix molecules results in early embryonic
lethality with a failure of endocardial cushion formation and a
highly dilated myocardium of the primitive heart tube [1,2].
Versican is a chondroitin sulfate proteoglycan that was first
identified in fibroblastic extracts [3]. Versican is abundantly
expressed within the extracellular matrix compartment of the
developing and mature cardiovascular system. Alterations in its
expression have been associated with vascular disease [4] and its
expression is required early for normal early heart development
[5]. The versican gene (Vcan; formerly Cspg2) consists of 15 exons
and spans a region of approximately 100 kb [6,7]. The exons
encode modular protein domains whose presence in the protein
core is regulated by alternative splicing of the mRNA [8,9]. At
least 4 different mRNA splice forms (V0, V1, V2 and V3) have
been identified in adult tissue and are produced by the alternative
splicing of exons 7 and 8 (Fig. 1). Several studies of expression have
shown that these alternatively spliced mRNA forms are
differentially expressed in specific tissues types, e.g. brain, smooth muscle,
tumors, suggesting that the regulation of functional domains
within versican are required for differentiation and persistence of a
tissues phenotype [915]. Additionally, the activity of versicans
protein do (...truncated)