Differences in microRNA-29 and Pro-fibrotic Gene Expression in Mouse and Human Hypertrophic Cardiomyopathy

Figures

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  Figure 1. Schematic for overall study design.

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  Figure 2. ET1 induced TGF-beta expression in neonatal rat cardiac myocyte cultures. (A) Real-time RT-PCR indicated 5-fold higher expression of miR-29a in cultured rat cardiac fibroblasts, when compared to rat cardiac myocytes. (B) ET1 increased TGFB1/2 expression in cardiac myocytes. (C) ET1 increased TGFβ expression in cardiac myocytes. Image shows representative blot from one experiment and bar graph summarizes band intensity data from three experiments (data is expressed as mean ± S.D; n = 3; two tailed Student's t-test was used to determine significance; * P < 0.05). (D) TGFβ2 secretion was estimated by ELISA. ET1 (100 nM) stimulated TGFβ2 secretion by cardiac myocytes. [(A,B,D) CON, control; ET1, Endothelin1 (100 nM) treatment for 24 h. All data are expressed as mean ± S.D; n = 3 for each condition; each experiment was repeated three times. Two tailed Student's t-test was used to determine significance; **P < 0.01, *P < 0.05].

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  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…

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  Figure 4. TGFβ-stimulated collagen expression in neonatal rat cardiac fibroblast cultures is prevented by miR-29a mimic. (A1,A2) Heat maps summarizing effect of TGFβ1 and TGFβ2 on expression of miR-29a and select pro-fibrotic target genes in cardiac fibroblasts. TGFβ1 and TGFβ2 suppress miR-29a expression and increase expression of pro-fibrotic genes, which is antagonized by the miR-29a-mimic, in cardiac fibroblasts. 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); miR-29a in the miR-29a-mimic condition is depicted in gray. (B1,C1) Representative western blot images for collagen type I following TGFβ1 and TGFβ2 treatment of cardiac fibroblasts (from one experiment) demonstrates that TGFβ1 and TGFβ12 stimulate collagen expression in cardiac fibroblasts, which is antagonized by a miR-29a-mimic. (B2,C2) Summary of western blot data for collagen type I following TGFβ1 or TGFβ2 treatment of cardiac fibroblasts from three experiments illustrates that TGFβ1 and TGFβ2 increase collagen type I expression in cardiac fibroblasts, which is inhibited by a miR-29a-mimic (data are…

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  Figure 5. Two photon microscopy to assess redox status in mouse cardiac myocytes at 5 and 24 weeks of age. Cardiac myocytes were labeled with monochlorobimane (MCB; 50 μM, blue λem 480 ± 20 nm) at 37°C, to monitor reduced glutathione (GSH). Data from mutant myocytes is presented as fluorescence units normalized to littermate control myocyte data. NAD(P)H was assessed in non-labeled cells by measuring autofluorescence (total fluorescence collected at <490 nm). Potassium cyanide (KCN; 1 mM) and carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 5 μM) were used to calibrate the NAD(P)H signal, permitting estimation of the NAD(P)H pool. All fluorescence recordings were obtained from non-paced cardiac myocytes. (A) TnT mutant myocytes had lower GSH (MCB signal), and MyHC mutant myocytes had higher GSH when compared to respective littermate control myocytes at 5 weeks of age. (B) NAD(P)H pool was lower in TnT mutant myocytes, and higher in MyHC mutant myocytes, when compared to respective littermate control myocytes at 5 weeks of age. (C) At 24 weeks of age, GSH (MCB signal) was lower in both mutant myocytes when compared to respective littermate control myocytes. (D) At 24 weeks of…

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  Figure 6. Allele-specific differences in expression of miR-29 family and pro-fibrotic genes in left ventricle of HCM mice at 5 and 24 weeks of age. (A1) Heat map summarizing expression data for miR-29a/b/c, select profibrotic and redox genes in the LV of TnT and MyHC mutant mice and respective littermate controls at 5 weeks of age. Expression of miR-29 family is significantly lower and profibrotic genes in significantly higher only in TnT mutant-LV when compared to littermate control-LV at 5 weeks of age. Differentially-expressed genes are shown in graduated red (upregulated) or blue (downregulated) based on their log2 fold-change compared to respective littermate controls. (A2–4) Summary for expression of TGFB2, miR-29a, and Col1A1 in LV of TnT and MyHC mutants, and respective littermate controls at 5 weeks of age shows greater upregulation of TGFB2 in TnT mutant-LV than MyHC mutant-LV, when compared to respective littermate control-LV. miR-29a suppression and increase in COLl1A1 expression was only observed in TnT mutant-LV (linear fold change is presented). (B1) Heat map summarizing expression data for miR-29a/b/c, select pro-fibrotic and redox genes in LV of TnT and MyHC mutant mice…

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  Figure 7. Microarray data from HCM patients undergoing myectomy compared to control (healthy organ donor) hearts. (A) Age and sex distribution of HCM patients and controls. (B) PCA plot represents the results of a principal component analysis of all samples' normalized log2 fluorescence values, wherein each dot represents a single sample, its color indicates HCM (red) or control (blue) status. Each of the plot's three dimensions separates the samples based on a subset of all the microarray's genes, selected by correlation. The X-axis gene set represents the highest percentage of overall variation, followed by Y and Z in that order. One HCM sample on the X-axis (far left) is an outlier and was excluded from normalization of the ANOVA signal values. The Y-axis depicts a good, albeit not perfect, separation of the two biological classes, which ANOVA defines as the genes that distinguish HCM hearts from control hearts.

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  Figure 8. Gene expression in human ventricular septal tissue of HCM patients undergoing myectomy compared with control (healthy organ donor) hearts. (A) Summary of mRNA data (select pro-fibrotic and redox genes) from 105 HCM patients compared to data from 39 controls shows upregulation of TGFB2 and TGFB3 and downregulation of TGFBR2, ELN, αMYHC, SERCA2a, and GLUT1 genes. (B) Summary of data for miR-29a/b/c from 106 HCM patients compared with data from 20 controls shows no difference in expression of miR-29a/b/c between HCM patients and controls. *P < 0.05.

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  Figure 9. Ingenuity pathway analysis of mRNA microarray data obtained from HCM patient myectomy tissue compared to control (healthy donor) heart tissue. (A) This volcano plot represents the ANOVA results of HCM vs. controls, depicting genes' log2 fold changes on the X-axis and their –log10 P-values on the Y-axis. Each dot in the figure represents a gene and is colored according to the standard deviation (SD) of that gene's log fold change from the mean. Genes with greater than 2SD up or down were used in the IPA analysis. (B) IPA identified pathways that are significantly (P < 0.032) up-regulated (Z-score > 0) or down-regulated (Z-score < 0) in HCM patients compared to control subjects. (C) Identification of upstream transcriptional regulators that are upregulated and downregulated in HCM patients compared to control subjects.