Protection against pressure overload-induced right heart failure by uncoupling protein 2 silencing

Figures

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  Figure 1. Distribution of UCP2 and UCP3 mRNA expression in left and right ventricle (A), myocytes and non-myocytes (B), and UCP2 protein expression in myocyes and non-myocytes (C). Data are from n = 4 mice and subsequent cell preparations and they are expressed as 25%, 50%, and 75% quartiles with whiskers representing the total range. The dots represent the individual data points. Data were analysed by a Shapiro–Wilk test to check whether data points are normally distributed and Levene test for equal variance. Subsequently, data were tested with an unpaired t-test. In C an original western blot is given with staining for UCP2 and actin.

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  Figure 2. Pressure overload, hypertrophy, and UCP2 expression in mice 3 weeks after pulmonary artery banding (PAB; n = 14 wild type; n = 14 UCP2−/−) or sham surgery (n = 8 wild type; n = 15 UCP2−/−). (A) Right ventricular systolic pressure (RVSP in mmHg), (B) systemic arterial pressure (SAP, in mmHg), (C) right ventricular weight normalized to body weight (RV/BW, in mg/g), and (D) right ventricular weight normalized to the left ventricle and septum weight [RV/(LV + S)]. Data are given for sham surgery and PAB banding of each genotype. A one-way ANOVA with a subsequent Student–Newman–Keuls test for post hoc analysis was performed. Normal distribution was tested with a Shapiro–Wilk test. (E) Original blot of RV samples from wild-type mice 3 weeks after banding (PAB; lane 5–7), sham (lane 1–4), or UCP−/− mice (lane 8). (F) Quantification of UCP2 expression (after normalization to GAPDH). Data are expressed as % of sham (n = 3–4 as indicated in E). They are expressed as 25%, 50%, and 75% quartiles with whiskers representing the total range. Data in F were analysed by a Shapiro–Wilk test to check whether data points are normally distributed and Levene test for equal variance. Subsequently, data…

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  Figure 3. Cardiac function in mice 3 weeks after pulmonary artery banding (PAB). (A) Cardiac output (CO, in mL/min), (B) tricuspid annular plane systolic excursion (TAPSE, in mm); (C) right ventricular wall thickness (RVWT, in mm), (D) right ventricular internal diameter (RVID, in mm), and (E) representative images of echo analysis. Data are given for sham surgery and PAB banding of each genotype. A one-way ANOVA with a subsequent Student–Newman–Keuls test for post hoc analysis was performed. Normal distribution was tested with a Shapiro–Wilk test. The number of mice in the individual groups corresponds to Figure 2A–D. *P<0.05.

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  Figure 4. Analysis of cardiac fibroblasts in vitro. (A) Proliferation of fibroblasts isolated from wild-type mice (WT) or UCP2−/− mice was quantified by Brd-incorporation and subsequent determination of optical densities (O.D.) in the cultures dishes. Data are from n = 24 samples each (six mice). (B) Quantification of collagen-1 mRNA expression in isolated fibroblasts from WT and UCP2−/− mice (n = 4 each). Data were analysed by a Shapiro–Wilk test to check whether data points are normally distributed and Levene test for equal variance. Subsequently, data were tested with an unpaired t-test. *P<0.05.

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  Figure 5. Right ventricular fibrosis in mice 3 weeks after pulmonary artery banding (PAB). (A) Collagen staining quantified as percent PircoSirius Red staining. Data are from n = 7 mice (sham, wild type), n = 7 mice (PAB wild type); n = 5 (sham UCP2−/−), and n = 5 (PAB UCP2−/−). (B) Representative images. Data are given for sham surgery and PAB banding of each genotype. A one-way ANOVA with a subsequent Student–Newman–Keuls test for post hoc analysis was performed. Normal distribution was tested with a Shapiro–Wilk test. *P<0.05.

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  Figure 6. Cell shortening and calcium transients of isolated right ventricular myocytes. (A) Data show the normalized cell shortening (ΔL/L, %), maximal contraction velocity (Con-Vel, µm/s), and the maximal relaxation velocity (Rel-Max, µm/s). Data are from n = 90 to 122 cells (eight mice) and they are expressed as 25%, 50%, and 75% quartiles with whiskers representing the total range. (B) Data show the amplitude of calcium transients (Fura-2 ratio), of the time to reach 50% of the maximal peak (Time-to-Peak 50%), and the time to reach 50% of the basal level of intracellular calcium (Time-to-Basal 50%). Data are from n = 18 to 26 cells and they are expressed as 25%, 50%, and 75% quartiles with whiskers representing the total range. A one-way ANOVA with a subsequent Student–Newman–Keuls test for post hoc analysis was performed. Normal distribution was tested with a Shapiro–Wilk test. *P<0.05.

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  Figure 7. Cellular adaptation to culture conditions. (A) The phalloidin-TRITC stained myocytes indicate the native shape (Day 0) and the adaptation by performing pseudopodia-like structures (spreading, Day 3). (B) Representative time relationship of the ability of mouse myocytes to adapt to the culture dish by spreading. (C) Quantitative analysis of the percent of cells that respond to cultivation by spreading. Data are from n = 4 preparations/mice each and they are expressed as 25%, 50%, and 75% quartiles with whiskers representing the total range. Data in C were analysed by a Shapiro–Wilk test to check whether data points are normally distributed and Levene test for equal variance. Subsequently, data were tested with an unpaired t-test (P > 0.05).