Figure 1.
Gene expression of collagen I (COL1A1; top) and collagen III (COL3A1; bottom) in intact atrial tissue samples and in 4 day cultured atrial fibroblasts as measured by qPCR.
(A) COL1A1 mRNA levels in atrial tissue. (B) COL3A1 mRNA levels in atrial tissue. (C) COL1A1 mRNA levels in atrial fibroblasts after culture. (D) COL1A1 mRNA levels in atrial fibroblasts after culture. **P<0.01, *P<0.05 by t-test, number of dogs indicated in figure.
Figure 2.
Western blots were used to quantify caveolin 3, vimentin, and smoothelin expression. Caveolin 3 was found in digested whole tissue and also in the cardiomyocyte fraction but was completely absent from the freshly isolated fibroblast fraction (A).
Vimentin showed strong expression in the fibroblast fraction but was not seen in whole digested tissue or the cardiomyocyte fraction (B). To assess smooth muscle cell contamination, smoothelin immunopositivity was verified in the cardiomyocyte and fibroblast fractions, and compared to pulmonary artery as a positive control (C). Smoothelin was strongly expressed in pulmonary artery but was not present in the cardiomyocyte or fibroblast fraction. N = 3 dogs/group.
Figure 3.
Comparative gene expression studies with freshly isolated versus cultured control (CTL) and CHF dog fibroblasts.
(A) COL1A1 (B) COL3A1 (C) Fibronectin (FN1) mRNA expression in freshly isolated CTL and CHF dog fibroblasts and after 48 hour culture. *P<0.05 by ANOVA with Bonferroni post-hoc tests, number of dogs indicated in figure.
Figure 4.
Dependence of fibroblast expression of various ECM proteins on time in culture. Results are shown for (A) COL1A1 mRNA, (B) COL3A1 mRNA, (C) FN1 mRNA, (D) COL1A1 and FN1 protein.
Control fibroblasts were cultured for 1, 2, 4, 7, and 12 days. For the last time point, cells were passaged (P1) after 7 days of culture and then grown to confluence (∼5 days). Representative Western blot images for COL1A1 and FN1 are shown in the upper panels of (D), with mean±SEM data beneath. Protein levels were measured from culture supernatant for the 24 hours preceding cell collection. *P<0.05, **P<0.01, ***P<0.001 by ANOVA with Bonferroni post-hoc tests, number of dogs indicated in figure.
Figure 5.
Immunofluorescent images of freshly isolated and cultured fibroblasts.
(A) Freshly isolated control (CTL) dog and (B) freshly isolated CHF dog fibroblasts. Expanded images of freshly isolated CTL and CHF fibroblasts are shown in (C) and (D) respectively. Staining shown is DAPI (blue, nuclear), vimentin (green, stress fibers), and α-SMA (red). Shape factor was used to quantify cell spreading in freshly isolated fibroblasts and the distribution is shown in (E). The average shape factor for both CHF and CTL is shown in (F). *P<0.05 by t-test. N/N = Number of cells/number of dogs. (G) and (H), CTL dog (G) and CHF dog (H) fibroblasts after 48 hrs of culture.
Figure 6.
Representative recordings of K+-current at baseline and after TEA treatment, along with TEA-sensitive K+-current-voltage relations for control and CHF fibroblasts.
Results are shown for freshly isolated fibroblasts (top), 48-hour cultured cells (middle) and 7-day cultured cells (bottom). Each set of data shows currents before and after TEA in representative fibroblasts from a control and a CHF dog, followed by mean current-voltage relations. (A,D,G) Recordings from CTL fibroblasts freshly isolated, after 48 hr culture, and after 7 day culture at baseline and after 30 mM TEA. (B,E,H) Recordings from CHF fibroblasts freshly isolated, after 48 hr culture, and after 7 day culture. (C,F,I) The current voltage relationship for control and CHF fibroblasts that were freshly isolated (C), cultured for 48 hours (F), and after culture for 7 days (I). ***P<0.001, *P<0.05 by 2-way ANOVA followed by Bonferroni post-tests. Number of cells studied is indicated first, followed by the number of dogs.
Figure 7.
Control fibroblasts were either freshly isolated or grown in culture for 48 hours on standard plastic culture dishes or on plastic culture dishes coated with collagen-I.
Gene expression was measured for COL1A1 (A) and FN1 (B). The current-voltage relationship for TEA-sensitive K+-current is shown for control fibroblasts grown on plastic and collagen I plates (C). The number of dogs is indicated in the figure, with the patch clamp data showing the number of cells studied/number of animals. *P<0.05 by ANOVA with Bonferroni post-hoc tests.