© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Serum depletion induces cell loss of rat cardiac fibroblasts and increased expression of extracellular matrix proteins in surviving cells
Carl-Ludwig-Institute of Physiology, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany
* Corresponding author. Tel.: +49-0341-971-5500; fax: +49-0341-971-5509 leicht{at}medizin.uni-leipzig.de
Objective: Since reduced nutrient supply is one component of ischemia, we have studied the effect of serum depletion on the survival of fibroblasts isolated from adult rat hearts and on the expression and degradation of extracellular matrix (ECM) proteins. Furthermore, we measured the role of the cAMP-dependent pathway in these processes. Methods: Isolated cardiac fibroblasts were grown to confluency in 10% serum containing medium. Serum was then removed and cell number was measured by use of a Coulter Counter. The activity of the cAMP response element binding protein (CREB) was investigated by Western blotting and subsequent use of the specific antibody which binds to the active form of the protein. The expression of colligin, collagen I and III, matrix metalloproteinases 2 (MMP-2), and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) was examined by ribonuclease protection assay (RPA) and Western blotting. Zymographic measurements were done to investigate gelatinase activity of MMP-2. Results: Serum withdrawal caused the death of 36% of the cells during the first 8 h. CREB was strongly phosphorylated 5 min after serum removal. Activation persisted up to 8 h and decreased thereafter. The mRNA abundance of colligin, collagen I and III, MMP-2, and TIMP-2 started to increase after 5 and 10 h, respectively, reaching a maximum after 20–30 h and decreasing thereafter. Protein levels of collagen I, collagen III, colligin and TIMP-2 were higher after 24 h until up to 96 h. MMP-2 zymographic activity was elevated 15-fold after 72 h. Application of the protein kinase A (PKA) blocker RpcAMPS suppressed the increase in phosphorylation of CREB. The increase in collagen III and MMP-2 mRNA abundance and elevation of collagen I and III, and TIMP-2 protein was diminished by RpcAMPS. The rise of colligin protein was completely suppressed. The increase in MMP-2 zymographic activity was also attenuated. RpcAMPS improved survival rate from 56 to 84%. Conclusions: Serum depletion led to cell death of isolated cardiac fibroblasts. Survival was associated with the increase in the expression of various ECM proteins. The transcription factor CREB was activated after serum removal. Inhibition of PKA improved the serum depletion induced decrease in the survival rate. The increase in collagen I, collagen III, MMP-2, TIMP-2, and colligin evoked by serum depletion was also diminished by PKA inhibition.
KEYWORDS Cell culture/isolation; Extracellular matrix; Gene expression; Ischemia; Signal transduction; Apoptosis; Protein kinases
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