Cardiac mitofusin-1 is reduced in non-responding patients with idiopathic dilated cardiomyopathy

Figure 1. Cardiac Mfn1 expression is suppressed in non-responders together with reduction in the size and number of mitochondria in cardiomyocytes. (A) UCG data of responders (Res) and non-responders (Non-Res) at baseline cardiac biopsy (pre) and 7–15 months later (post). LVEF and LV diastolic dimension (LVDd) (n = 14, 14, 8, and 8). (B, C) Transmission EM of cardiac tissue from Res and Non-Res (B), and quantification of the size, area and number of mitochondria per field (n = 10, 8) (C). Scale bar = 2.0 μm. Case 1–5 in Fig. 1B indicates panels are from different individuals. (D) Immunofluorescence for Mfn1 in cardiac tissue from Res and Non-Res. Right panel shows quantification of Mfn1-positive area (n = 12, 5). Scale bar = 50 μm. For this study, an outlier (n = 1 in Res group) and an abnormal value (n = 1 in Non-Res group) were excluded by boxplot (SPSS) for further statistical analysis. (E) Quantitative PCR of cardiac MFN1 expression in Res and Non-Res (n = 13, 8). For this study, an abnormal value (n = 1 in Res) was excluded by boxplot (SPSS) for further statistical analysis. Data were analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01. Results are shown as mean ± SEM. NS…

Figure 2. Suppression of Mfn1 promotes the decline of cardiomyocytes metabolism (A) Quantitative PCR of Mfn1 expression in NRVMs after introduction of control si-RNA (Con) or si-Mfn1 (n = 6, 6). (B,C) Evaluation of mitochondrial respiration with the Seahorse extracellular flux analyzer (B), basal respiration (n = 5, 5), maximal respiration (n = 4, 5), ATP production (n = 5, 5), and Proton leak (n = 5, 5). (C) In NRVMs after introduction of control si-RNA (Con) or si-Mfn1. For maximal respiration study, an abnormal value (n = 1 in si-Mfn1 group) was excluded by boxplot (SPSS) for further statistical analysis. (D) Staining of functional mitochondria (MitoRed) and total mitochondria (Mito Tracker Green FM (MitoGreen)) in NRVMs after introduction of control si-RNA (Con) or si-Mfn1. Scale bar = 5 μm. The right panel shows quantification of the MitoRed-positive area (n = 3, 3). Data were analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01. Results are shown as mean ± SEM. NS not significant. Small circle indicates outlier, triangle indicates abnormal value.

Figure 3. Cardiac Mfn1 KO mice show reduced cardiac function. (A) Metabolomic study showing pCre/ATP ratio in cardiac tissues of wild type (WT) or MHC-Cre+/−; Mitofusin-1fl/fl (c-Mfn1 KO) mice (n = 7, 8). An abnormal value (n = 1 in the c-Mfn1 KO group) was excluded by boxplot (SPSS) for further statistical analysis. (B) echocardiography data of WT and c-Mfn1 KO mice two weeks after TAC. Fractional shortening (FS) (n = 18, 10) and LV systolic dimension (LVDs) (n = 18, 10) were analyzed. (C) Heart weight/body weight (HW/BW) ratio of indicated groups (n = 18, 10). (D) Transmission EM of cardiac tissues from WT and c-Mfn1 KO mice two weeks after TAC. No 1–4 indicates panels from different mice. Scale bar = 1 μm (E) Picrosirius staining of indicated mice. Right panel indicates relative fibrotic area (area/slice) (n = 5, 6). An abnormal value (n = 1 in c-Mfn1 KO) was excluded by boxplot (SPSS) for further statistical analysis. Scale bar = 500 μm. (F) Quantitative PCR of Col1a1 (n = 10, 11), Col3a1 (n = 10, 11), Tgfb1 (n = 8, 11), Cd68 (n = 8, 10) and Ccl2 (n = 10, 11) in cardiac tissues of indicated mice. An outlier (n = 1 in c-Mfn1 KO (Cd68)) and an abnormal value (n = 1 in WT (Tgfb1)) were…

Figure 4. Activation of adrenergic signaling reduces mitofusin-1 expression. (A,E) Quantitative PCR of Mfn1 in NRVMs treated with PBS (Con) or isoproterenol (Iso, 1 μM, 72 h) (n = 18, 14) (A), PBS (Con) or cAMP analog (30 μM, 24 h) (n = 6,6) (E). (B) Western blot analysis of Mfn1 in NRVMs administrated with PBS (Con) or isoproterenol (Iso, 1 μM, 96 h). (B) Right panel, quantification for Mfn1 relative to Actin loading control (n = 8, 9). (C,D,F) Quantitative PCR of miR-140-5p in NRVMs administrated with isoproterenol (Iso, 1 μM, 72 h). (C) cAMP analog (30 μM, 3 h, n = 6) (D) cAMP analog (30 μM, 3 h) + PKA inhibitor (PKA-I, 2 μM, 30 min pre-treatment) (n = 6, 6) (F) (n = 4, 4). (G) Quantitative PCR of Mfn1 in NRVMs subjected to cAMP analog (30 μM, 24 h) + anti-miR-140-5p (50 nM pretreatment, 1 h) (n = 9, 9) or anti-miR negative control (Anti-miR-NC). (H) In situ hybridization detecting miR-140-5p in cardiac tissue of responders (Res) (n = 4) or non-responders (Non-Res) (n = 4). Right panel indicates miR-140-5p positive (n = 4, 4) (scale bar = 10 μM). (I) Scheme summarizing findings of β-AR/ cAMP/ PKA/ miR-140-5p signaling pathways leading to down-regulation of Mfn1 expression in cardiac…