FHL-1 is not involved in pressure overload-induced maladaptive right ventricular remodeling and dysfunction

Figures

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  Fig. 1. Changes in FHL-1 expression after PAB. a Real-time PCR analysis of FHL-1 expression in right ventricles of C57/BL6 mice after sham or PAB for 7, 14 or 21 days (d). Data were analyzed by analysis of variance followed by Dunnett’s multiple-to-one comparison post hoc tests and are presented as mean ± standard error of mean (SEM). *Significant differences between sham and PAB; ns not significantly different. b Left: representative Western blot analysis of FHL-1 expression in right ventricles of C57/BL6 mice after sham or PAB. Right: densitometric analysis. Data were normalized to β-tubulin and sham was set to 100%. Data were analyzed by analysis of variance followed by Dunnett’s multiple-to-one comparison post hoc tests and are presented as mean ± SEM. c Immunohistochemical staining of FHL-1 (in red) after sham or PAB in C57/BL6 mice. NC isotype control staining. d Immunofluorescence staining of α-actinin (in red) and FHL-1 (in green) after 7 days of PAB in C57/BL6 mice. Nuclear staining was performed with DAPI (blue)

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  Fig. 2. Hypertrophic signaling following PAB. a Left: representative Western blot analysis of Akt and Erk phosphorylation in right ventricles of C57/BL6 mice after sham or PAB for 7, 14 or 21 days (d). Right: densitometric analysis of Akt and Erk phosphorylation. Data were normalized to β-tubulin and sham was set to 100%. Data were analyzed by analysis of variance followed by Dunnett’s multiple-to-one comparison post hoc tests and are presented as mean ± standard error of mean (SEM). b Immunohistochemical staining of PCNA (in red) in C57/BL6 mice after sham or PAB. NC isotype control staining. Arrows indicate PCNA-positive nuclei

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  Fig. 3. PAB-induced echocardiographic changes in WT Swiss and FHL-1−/− mice. a Western blot analysis of FHL-1 expression in right ventricles derived from WT Swiss and FHL-1−/− mice. b Right ventricular internal diameter (RVID) after sham or PAB for 3 days (d) or 1, 3 or 5 weeks in WT and FHL-1−/− mice. c Right ventricular wall thickness (RVWT). d Tricuspid annular plane systolic excursion (TAPSE). *Significant differences between WT PAB and FHL-1−/− PAB. $Significant differences between sham and PAB. #Interaction. Data were analyzed by 2-way analysis of variance followed by Tukey’s multiple comparison post hoc tests and are presented as mean ± standard error of mean (SEM)

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  Fig. 4. PAB-induced hemodynamic and right ventricular morphological changes in WT Swiss and FHL-1−/− mice. a Right ventricular systolic pressure (RVSP) after sham or PAB for 3 days (d) or 1, 3 or 5 weeks in WT and FHL-1−/− mice. b Systolic arterial pressure (SAP). c Mean arterial pressure (MAP). d Ratio of right ventricle to tibia length. $Significant differences between sham and PAB. #Interaction. Data were analyzed by 2-way analysis of variance followed by Tukey’s multiple comparison post hoc tests and are presented as mean ± standard error of mean (SEM)

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  Fig. 5. PAB-induced hemodynamic changes in WT Swiss and FHL-1−/− mice. a Right ventricular end-diastolic pressure (RVEDP) after sham or PAB for 3 days (d) or 1, 3 or 5 weeks in WT and FHL-1−/− mice. b Contractility index. c Maximal changes in blood pressure during isovolumetric contraction (max dP/dT). d Minimal changes in blood pressure (min dP/dT). e Tau, time constant of RV relaxation. *Significant differences between WT PAB and FHL-1−/− PAB. $Significant differences between sham and PAB. #Interaction. Data were analyzed by 2-way analysis of variance followed by Tukey’s multiple comparison post hoc tests and are presented as mean ± standard error of mean (SEM)

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  Fig. 6. PAB-induced right ventricular remodeling in WT Swiss and FHL-1−/− mice. a Representative pictures of sirius red in picric acid staining to detect fibrotic changes (in red) after sham or PAB for 7 days in WT and FHL-1−/− mice. b Quantification of right ventricular fibrosis after sham or PAB for 3 days (d) or 1, 3 or 5 weeks in WT and FHL-1−/− mice. c Representative pictures of FITC conjugated wheat germ agglutinin (WGA FITC; in green) staining to determine cell size. d Quantification of cardiomyocyte size depicted by cross-sectional area. e Representative pictures of FITC conjugated wheat germ agglutinin (WGA FITC; in green) and IB4-TRITC (in red) staining to determine cell size and number of capillaries. Nuclear staining was performed with DAPI (blue). f Quantification of number of capillaries to cardiomyocytes. *Significant differences between WT PAB and FHL-1−/− PAB. $Significant differences between sham and PAB. #Interaction. Data were analyzed by 2-way analysis of variance followed by Tukey´s multiple comparison post hoc tests and are presented as mean ± standard error of mean (SEM)

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  Fig. 7. Changes in gene expression after PAB in WT Swiss and FHL-1−/− mice. a Ventricular wall stress markers, left: Anp and right: Bnp after sham or PAB for 3 days (d) or 1, 3 or 5 weeks. n = 4–6 each. b Ca2+-handling proteins, left: Plb, middle: Atp2a2 and right: Ryr. n = 4–6 each. c Fetal genes, left: α-Mhc, middle: β-Mhc and right: β-Mhc/α-Mhc ratio. d Collagens, left: collagen 1a1, middle: collagen 1a2 and right: collagen 3a1. e FHL-2. $Significant differences between sham and PAB. Data were analyzed by 2-way analysis of variance followed by Tukey´s multiple comparison post hoc tests and are presented as mean ± standard error of mean (SEM). f Western blot analysis of Erk and Akt phosphorylation in right ventricles of WT and FHL-1−/− mice after 3 weeks of PAB