Effect of protein kinase C isoforms on collagen gel contraction and collagen production in cardiac fibroblasts

The aim of this study was to investigate whether protein kinase C (PKC) isoform inhibitors such as staurosporine, calphostin C, deaqualinium, rottlerin, hispidin, the PKCɛ translocation inhibitor peptide or myristolyated PKCη,θ or ξpseudosubstrate affect the collagen gel contraction or the collagen production in cardiac fibroblasts. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel and incubated for 3 days in serum-free conditions. Soluble collagen has been measured in the conditioned medium and nonsoluble collagen in the cell layer by assaying 3H-Proline incorporation. As compared to control, the collagen gel contraction in cardiac fibroblasts (n = 4) was dose-dependently inhibited by staurosporine, a broad spectrum PKC inhibitor; by calphostin C, an inhibitor of PKCα,β and γ; by deaqualinium a PKCα inhibitor and by rottlerin, a PKCδ inhibitor. However the PKCβ inhibitor hispidin and the PKCɛ,η,θ and ζ inhibitors did not affect the collagen gel contraction in cardiac fibroblasts. At 10−6 M staurosporine, calphostin C, deaqualinium and rottlerin the % inhibition averaged, respectively, 92.5 ± 2.2 (SEM) %, 97.2 ± 1.1 %, 28.8 ± 0.6 % and 73.2 ± 0.6 %. Staurosporine, calphostin C, deaqualinium and rottlerin (10−6 M) also inhibited soluble collagen production by 64.7 ± 2.9 %, 76.9 ± 3 %, 45.3 ± 2.6 % and 65.7 ± 2.2 %, respectively, and the nonsoluble collagen production by 79.3 ± 5.0 %, 95.3 ± 2.1 %, 60.4 ± 0.6 % and 70.2 ± 8.2 %, respectively, in cardiac fibroblasts in culture. Our data suggest that especially the PKC isoforms α, γ and δ are involved in the collagen gel contraction and collagen production in cardiac fibroblasts while PKCβ,ɛ,η,θ and ξ are not.