Fig 2: Rescue cardiac hypertrophy by re-expression Dnm1l and Mfn1. (A) Experimental design for Dnm1l and Mfn1 re-expression in vivo. AAV was injected to induce Dnm1l and Mfn1 re-expression specifically in cardiomyocytes. (B) Echocardiographic measurement of myocardial thickness and contractile function. N = 6 hearts per group. (C) HE staining demonstrated significant reduced hypertrophy on DRP1 and MFN1 re-expressed AAC heart. (D) qPCR showed the significant increased expression of Dnm1l and Mfn1 in cardiac tissue. N = 3 hearts per group. (E) Cross-section with WGA staining of AAC and treated hearts. Box plots demonstrated the individual and combine infection rate of two AAV vectors. (F) In situ optical sections and quantification between AAC and treated hearts indicating reduced cell size on bi-infected cardiomyocytes, revealing a cell autonomous manner on cell size by re-expression of Dnm1l and Mfn1. (G) MitoTracker staining and quantification of mitochondria morphology in YAP1-overexpressing NMVMs upon MFN1 and DRP1 addback by AdVs. N = 3 biological replicates in each group. (H) MitoSox density and quantification of mitochondrial ROS level in YAP1-overexpressing NMVMs upon MFN1 and DRP1 addback by AdVs. N = 3 biological replicates in each group. (I) OCR measurement of NMVMs normalized to cell number. N = 9 hearts per group. Mean ± SD. scale bars, 50 µm. Student's t-test or Mann-Whitney U-test was applied. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. Non-significant P values in parenthesis.

Fig 3: YAP1 overexpression triggers cardiac hypertrophy by impairing mitochondrial gene Dnm1l and Mfn1. (A) Principal component analysis (PCA) of RNA-seq data for control and YAP1 overexpressing hearts treated for 5 days or 1 month. N = 3 hearts per group. (B) GSEA of mitotic nuclear division gene set in the YAP1 overexpression RNA-seq analysis. N = 3 hearts per group. (C) Heatmap of mitochondrial biogenesis related genes based on RNA-seq are shown among three different time points post YAP1 activation, indicating the impairment of mitochondrial biogenesis upon YAP1 activation. N = 3 for each group. (D-E) CellROX staining for adult isolated cardiomyocytes indicated a significant increasing of fluoresces density of CellROX among AAC mice by qualitative analyses. N = 3 biological replicates in each group. (F) Cultured YAP1-overexpression NMVMs were treated with H2O2 (positive control), AdV-LacZ, and AdV-Cre. The fraction of CellROX positive cells was measured by FACS. N = 3 biological replicates in each group. (G) Quantification of CellROX positive cells between H2O2, AdV-LacZ and AdV-Cre treated groups. Since LacZ control cells did not activate the mcherry reporter, we compared total CellROX positive cells among groups. (H-J) Dnm1l and Mfn1 were measured by quantitative PCR and immunofluorescent staining. Dnm1l and Mfn1 had markedly decreased upon YAP1 activation both in heart tissues and cultured NMVMs. Scale bar, 50 µm. N = 3 biological replicates in each group. Mean ± SD. Student's t-test was applied. *P < 0.05. ***P < 0.001. Non-significant P values in parenthesis.

Fig 4: HIF-1a is a transcription factor that regulates HO-1 gene expression, and HO-1 facilitates mitochondrial fusion (Mfn1 and Mfn2) and inhibits mitochondrial fission (Drp1 and Fis1), thus maintaining mitochondrial homeostasis. Under diabetic conditions, kidney tissues are hypoxic, which induces the expression of HIF-1a through an impaired compensatory response. However, tubular-specific deletion of HIF-1a in the kidneys of diabetic mice exacerbated mitochondrial dysfunction and ROS accumulation and then caused tubular damage, thereby leading to diabetic kidney injury

Fig 5: Scutellarin reversed oxidative stress and mitochondrial dysfunction and reduced ATP production induced by TNF-a in HNPCs. (A) HNPCs were cultured with PBS, TNF-a (10 ng/ml), TNF-a (10 ng/ml) +scutellarin (1 µM) for 24 h. The mitochondrial morphology of HNPCs was observed using TEM (scale bar: 5 µm). (B–F) After treatment as indicated for 48 h, total protein was extracted from each group. The protein levels of OPA1, Mfn1, Mfn2, and Drp1 in each group were detected by Western blotting. (G) ATP levels of HNPCs in each indicated group were examined. (H,I) ROS levels in HNPCs in each indicated group were detected with DCFDA (scale bar: 100 µm). (J,K) JC-1 was used to detect the mitochondrial membrane potential of HNPCs in each indicated group (scale bar: 100 µm). (L,M) MitoTracker was used to detect the mitochondrial membrane potential of HNPCs treated as indicated (scale bar: 100 µm). Each experiment was performed three times independently. (*p < 0.05, **p < 0.01, ***p < 0.001).