Figure 3.
NAC abolished the ET1 stimulated increase in TGFβ expression and miR-29a suppression in neonatal rat cardiac myocyte cultures. (A1) Representative images of cardiac myocytes labeled with DCF and Mitosox, obtained by 2-photon microscopy. ET1 (100 nM) increased H2O2 (DCF fluorescence) and superoxide (MitoSOX fluorescence) in cardiac myocytes, which is prevented by NAC (3.5 mM). (A2) Summary of fluorescence data representing H2O2 (DCF) and superoxide (Mitosox) reveals increase in H2O2 and superoxide following ET1 (100 nM) treatment, which is abrogated by NAC (3.5 mM). DCF and Mitosox fluorescence was calculated from 10 randomly selected regions from three experiments (~300 cardiac myocytes). (B1) Heat map summarizing expression of miR-29a, select pro-fibrotic and redox genes in cardiac myocytes, following treatment with ET1 (100 nM) and ET1 + NAC (3.5 mM). ET1 suppressed miR-29a expression and increased expression of TGFB1-3, CTGF, and collagen genes. NAC increased miR-29a and blunted expression of pro-fibrotic genes following ET1 treatment. Differentially expressed genes are shown in graduated red (up-regulated) or blue (down-regulated) according to their log2 fold change compared to control (untreated cells). (B2) TGFB2 was the most up-regulated TGFB gene in cardiac myocytes following ET1. TGFB2 gene expression increased 6-fold following ET1 (100 nM) treatment. NAC antagonized the effect of ET1 and reduced TGFB2 expression back to baseline levels in cardiac myocytes (linear fold change is presented). (B3) ET1 suppressed miR-29a expression, and NAC + ET1 increased miR-29a expression in cardiac myocytes (linear fold change is presented). (C1) Representative western blot image for TGFβ expression in cardiac myocytes (from one experiment) demonstrates that ET1 increases TGFβ, which is antagonized by NAC. (C2) Bar graph summarizing band intensity data from three experiments (biological replicates) following treatment with ET1 (100 nM) and ET1 + NAC (3.5 mM) shows that ET1 increases TGFβ protein expression in cardiac myocytes, which is blunted by NAC (data are expressed as mean ± S.D.; n = 3; one-way ANOVA and Tukey's test were used to compare ET1, ET1 + NAC, and control; *P < 0.05). (D) TGFβ2 secretion by cardiac myocytes was measured by ELISA. ET1 (100 nM) stimulated TGFβ2 secretion by cardiac myocytes, which was abrogated by NAC (3.5 mM). [(A1,A2,B1–B3,D) Data are expressed as mean ± S.D.; n = 3 for each condition; all experiments were repeated three times. One-way ANOVA and Tukey's test were used to compare ET1, ET1 + NAC, and control; **P < 0.01, *P < 0.05].