The development of pathological hypertrophy in the myocardium has been frequently associated with abnormal accumulation of extracellular matrix proteins (ECM) (mostly collagens) in the interstitium. The abnormal accumulation of fibrillar collagens results in an altered ventricular stiffness and can ultimately contribute to the development of myocardial failure. Cardiac collagens are comprised of types I and III and to a lesser extent of types IV and V. It has been demonstrated that during the early phases of cardiac pressure overload hypertrophy the ratio of type III to type I collagen increases. Results obtained from in vitro experiments performed with cardiac fibroblasts also suggest that III collagen gene expression is more responsive (vs. that of type I) to challenges derived from treating cells with either growth factors or mechanical stimulation. However, the molecular mechanisms by which collagen type III is regulated in the cardiac fibroblast are currently unknown. The overall aim of this proposal is to examine the regulation of the collagen type III gene by factors that are postulated to play an important role in directing extracellular matrix protein production in the myocardium. We propose to address the following questions: 1) Does transforming growth factor beta-1, angiotensin II (ANG II) and mechanical deformation result in an increase in type III collagen production and deposition in a culture of mouse cardiac fibroblasts? If so through what mechanisms? For this purpose, we would determine if stimulation of mouse cardiac fibroblast with these factors results in an increased expression of collagen type III mRNA and protein production using Northern blot and ELISA techniques. In order to determine the nature of the molecular mechanisms responsible for the increase in ECM protein production nuclear runoffs, mRNA stability and transcriptional (CAT-transfection) assays will be performed; 2) Does increased expression of collagen type III depend on the interaction between the angiotensin II receptor, ANG II, TGFbeta-1 and/or stretch? (a) Using binding assays we would explore if stimulation of mouse cardiac fibroblasts using the above described manipulations regulate the expression (number) of angiotensin II receptors. (b) Biochemical techniques would be used to examine if ANG II and/or stretch increases the levels of TGFbeta-1 gene expression and release. (c) Using the techniques described in Aim #1, we will examine if the simultaneous mechanical and growth factor stimulation of cardiac fibroblasts results in enhanced levels of collagen type III, and; 3) Does regulation of collagen type III gene expression by these factors depend on specific cis-acting elements contained within the promoter region of the gene? Utilizing transfection assays based on a CAT-collagen type III mouse promoter construct (and selective deletions) we would like to determine what are the specific cis-acting elements contained within the gene that might mediate the transcriptional activation by each of these different factors (TGFbeta-1, ANG II and stretch).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Minority School Faculty Development Awards (K14)
Project #
5K14HL003160-05
Application #
2734931
Study Section
Special Emphasis Panel (ZHL1-CCT-L (F1))
Project Start
1994-07-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
2000-06-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Chen, Yinghong; Epperson, Sara; Makhsudova, Lala et al. (2004) Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts. Am J Physiol Heart Circ Physiol 287:H2478-86
Villarreal, Francisco J; Griffin, Michael; Omens, Jeffrey et al. (2003) Early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling. Circulation 108:1487-92
Villarreal, Francisco; Zimmermann, Scott; Makhsudova, Lala et al. (2003) Modulation of cardiac remodeling by adenosine: in vitro and in vivo effects. Mol Cell Biochem 251:17-26
Lee, A A; Delhaas, T; McCulloch, A D et al. (1999) Differential responses of adult cardiac fibroblasts to in vitro biaxial strain patterns. J Mol Cell Cardiol 31:1833-43
Villarreal, F J; Hong, D; Omens, J (1999) Nicotine-modified postinfarction left ventricular remodeling. Am J Physiol 276:H1103-6
Kim, N N; Villegas, S; Summerour, S R et al. (1999) Regulation of cardiac fibroblast extracellular matrix production by bradykinin and nitric oxide. J Mol Cell Cardiol 31:457-66
Villarreal, F J; Bahnson, T; Kim, N N (1998) Human cardiac fibroblasts and receptors for angiotensin II and bradykinin: a potential role for bradykinin in the modulation of cardiac extracellular matrix. Basic Res Cardiol 93 Suppl 3:4-7
Lee, A A; Delhaas, T; Waldman, L K et al. (1996) An equibiaxial strain system for cultured cells. Am J Physiol 271:C1400-8