Fig 2: Protein levels of transforming growth factor beta (TGFB) and interleukin (IL)18 in liver samples from mice fed the experimental diets for 10 weeks. Sample western blots and their quantification are shown. MB, minimum essential medium (MEM) mice fed a barley-based diet; MR, MEM mice fed a rice-based diet; KC, potassium simplex-optimized medium control mice fed a rice-based diet. Data are expressed as means ± SEMs for 8–11 animals and were analyzed using Student's t-test. *P < 0.05 vs. the KC group; #P < 0.05 vs. the MR group.

Fig 3: 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 and respective littermate controls at 24 weeks of age. Only TnT mutant-LV demonstrate downregulation of miR-29 family at 24 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. (B2–4) Summary for expression of TGFB2, miR-29a, and COL1A1 in LV of TnT and MyHC mutant mice, and respective littermate controls at 24 weeks of age shows greater upregulation of TGFB2 in TnT mutant-LV than MyHC mutant-LV, when compared to respective littermate controls. Expression of miR-29a/b/c is lower and COL1A1 gene expression is higher in TnT mutant-LV, but not MyHC mutant-LV when compared to respective littermate controls (linear fold change is presented) (control-T, TnT-littermate control; TnT, TnT-mutant; Control-M, MyHC-littermate control; MyHC, MyHC-mutant). Data are expressed as mean ± S.D.; n = 5 in each group. The two-tailed unpaired Student's t-test was used to compare TnT mutants and MyHC mutants with respective littermate controls; **P < 0.01; *P < 0.05; n.s., non-significant).

Fig 4: Defective autophagy in VSMCs elicits cellular hypertrophy, and increases migration capacity and total collagen amount. (A) VSMCs isolated from Atg7+/+Tagln-Cre+ (+/+) and Atg7F/FTagln-Cre+ (-/-) aorta were labeled with calcein AM and visualized by confocal fluorescence microscopy. Scale bar: 10 µm. Cell size was measured using z-stack images (*, P < 0.05; n = 2 experiments; Student t test). (B) Thoracic aorta of Atg7+/+ and atg7-/- mice were stained with H&E to measure the width of the media (white arrows) (**, P < 0.01; n = 6 regions/aorta; Univariate). Scale bar: 25 µm. Note that the number of VSMC layers between Atg7+/+ and atg7-/- aorta was not different. (C) Migratory capacity of Atg7+/+ and atg7-/- VSMCs was analyzed using an Oris Migration Assay (***, P < 0.001; n = 2 experiments in triplicate; Student t test). Western blot analysis of TGFB and CXCL12 in Atg7+/+ and atg7-/- VSMCs. (D) Atg7+/+ and atg7-/- VSMCs were left untreated or treated with 10 ng/ml TGFB for 48 h and stained with Sirius red to examine total collagen amount (***, P < 0.001 vs. Atg7+/+; ###, P < 0.001 vs. control; n = 4 experiments in triplicate; two-way ANOVA with genotype and treatment as category factors).

Fig 5: Defective VSMC autophagy promotes upregulation of MMP9, TGFB and CXCL12, 5 d after ligation-induced injury. (A) The left common carotid artery (LCCA) of Atg7+/+Tagln-Cre+ (+/+) and Atg7F/FTagln-Cre+ (-/-) mice (n = 3) was ligated for 5 d. Gelatin zymographic analysis of the LCCA to detect MMP9 and MMP2 activity followed by densitometric analysis (***, P < 0.001; NS, not significant; Student t test). (B) Western blot analysis of the LCCA for TGFB, CXCL12 and GAPDH. Relative expression of TGFB/GAPDH and CXCL12/GAPDH was determined by densitometric analysis (*, P < 0.05; ***, P < 0.001; Student t test).