Lithium Reduces Migration and Collagen Synthesis Activity in Human Cardiac Fibroblasts by Inhibiting Store-Operated Ca2+ Entry

Figures

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  Figure 1. Cell migration, proliferation, and collagen synthesis abilities of human cardiac fibroblasts with and without LiCl treatment for 24 h. (A) Photograph and graph presenting mean data of the migration assay in human cardiac fibroblasts (n = 7 from four independent experiments). (B) Photograph and bar graph presenting average data of the pro-collagen type IA1 (n = 5 from three independent experiments) and soluble collagen type I (n = 5 from three independent experiments) production in human cardiac fibroblasts. (C) Average data of the proliferation rate in the cardiac fibroblasts (n = 5 from three independent experiments). (D) Photograph of immunofluorescence microscopy imaging and graph presenting average data of the α-smooth muscle actin expression on Western blot (n = 5 from three independent experiments). GAPDH was used as a loading control. One-way repeated measures analysis of variance (ANOVA) test with Tukey’s post hoc test was used to compare the human cardiac fibroblasts under different treatment conditions. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. Cell migration, proliferation, and collagen synthesis abilities of human cardiac fibroblasts with and without LiCl/angiotensin II treatment for 24 h. (A) Photograph and graph presenting mean data of the migration assay in human cardiac fibroblasts (n = 6 from three independent experiments). (B) Photograph and bar graph presenting average data of the pro-collagen type IA1 (n = 5 from three independent experiments) and soluble collagen type I (n = 5 from three independent experiments) production in human cardiac fibroblasts. (C) Average data of the proliferation rate in the cardiac fibroblasts (n = 5 from three independent experiments). (D) Photograph of immunofluorescence microscopy imaging and graph presenting average data of the α-smooth muscle actin expression on Western blot (n = 5 from three independent experiments). GAPDH was used as a loading control. One-way repeated measures analysis of variance (ANOVA) test with Tukey’s post hoc test was used to compare the human cardiac fibroblasts under different treatment conditions. * p < 0.05, *** p < 0.001.

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  Figure 3. Store-operated Ca2+ entry in human cardiac fibroblasts with and without LiCl/angiotensin II treatment for 24 h. Representative tracings and average data of the maximum amplitude and AUC of the intracellular Ca2+ concentration in Fura-2 AM-loaded human cardiac fibroblasts following the removal of extracellular Ca2+, the addition of the sarco/ER Ca2+-ATPase inhibitor thapsigargin (2.5 μM), and the re-addition of extracellular Ca2+ (2 mM). Panel (A) shows that LiCl (0.1 mM)-treated cells had a similar maximum amplitude and AUC of ER release and a lower maximum amplitude and AUC of Ca2+ entry than the control cells (n = 3 independent experiments). Panel (B) shows that LiCl (1.0 mM) and control cells had a similar maximum amplitude and AUC of ER release and Ca2+ entry (n = 3 independent experiments). Panel (C) shows that angiotensin II (50 nM)-treated cells had a larger maximum amplitude of Ca2+ entry than the control cells. Combined lithium (0.1 mM) and angiotensin II (50 nM)-treated cells had a smaller maximum amplitude and AUC of Ca2+ entry compared to the angiotensin II (50 nM)-treated cells (n = 4 independent experiments). Unpaired t test, Mann–Whitney rank-sum test, and one-way…

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  Figure 4. Expression of store-operated Ca2+ channel proteins in human cardiac fibroblasts with and without LiCl treatment for 24 h. (A) Photograph showing the Western blot of Orai1 in human cardiac fibroblasts with and without siRNA treatment for 48 h. (B) Photograph and graph showing the mean data of Orai1 protein (n = 5 from four independent experiments) and mRNA (n = 5 from three independent experiments) expression in human cardiac fibroblasts. GAPDH was used as a loading control. (C) Photograph and graph showing the mean data of STIM1, TRPC3, and TRPC6 protein expression in human cardiac fibroblasts (n = 5 from three independent experiments). GAPDH was used as a loading control. One-way repeated measures analysis of variance (ANOVA) test with Tukey’s post hoc test was used to compare the expression levels of proteins or mRNAs under different treatment conditions. * p < 0.05.

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  Figure 5. Cell migration, collagen synthesis ability, and store-operated Ca2+ entry in human cardiac fibroblasts treated with LiCl (0.1 mM) and 2-APB (10 μM) for 24 h. (A) Photographs and average data of the migration assay in human cardiac fibroblasts (n = 7 from four independent experiments). (B) Photographs and average data of the pro-collagen type IA1 production (n = 5 from three independent experiments) in human cardiac fibroblasts. (C) Representative tracings and average data of the maximum amplitude and AUC of intracellular Ca2+ concentration following the re-addition of extracellular Ca2+ (2 mM) in control, LiCl (0.1 mM), 2-APB (10 μM), or combined lithium (0.1 mM) and 2-APB (10 μM)-treated human cardiac fibroblasts (n = 3 independent experiments). One-way repeated measures analysis of variance (ANOVA) test with Tukey’s post hoc test was used to compare the human cardiac fibroblasts under different treatment conditions. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. Expression levels of pro-fibrotic signaling proteins in human cardiac fibroblasts with and without LiCl treatment for 24 h. Photograph and graph showing the mean data of expression levels and ratios of Akt, mTOR, GSK-3β, and β-catenin in human cardiac fibroblasts (n = 5 from three independent experiments). GAPDH was used as a loading control. One-way repeated measures analysis of variance (ANOVA) test with Tukey’s post hoc test was used to compare the expression levels of proteins under different treatment conditions. * p < 0.05.