Mib2 Regulates Lipid Metabolism in Heart Failure With Preserved Ejection Fraction via the Runx2–Hmgcs2 Axis

Figures

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  FIGURE 1. DEGs in metabolic HFpEF. (A–F) Volcano plot of DEGs in GSE163665, GSE240171, GSE235934, GSE249409, GSE218733 and GSE249411 datasets. (G) Petal diagrams showing the number of DEGs that overlap among GSE163665, GSE240171, GSE235934, GSE249409, GSE218733 and GSE249411 datasets. (H) TPM expression of Hmgcs2 gene in GSE163665 dataset. (I) TPM expression of Hmgcs2 gene in GSE240171 dataset. (J) Log2 normalised expression of Hmgcs2 gene in GSE235934, GSE249409, GSE218733 and GSE249411 datasets.

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  FIGURE 2. General biological and echocardiography features of HFpEF mice. (A) Representative M‐mode, PW‐mode and Tissue‐mode echocardiographic images of wt and ob/ob mice. (B) Assessments of echocardiographic parameters of left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), ratio between mitral E wave and E' wave (E/E'), E' wave, left ventricular end‐diastolic internal diameter (LVIDd), and left ventricular end‐systolic internal diameter (LVIDs) in wt and ob/ob mice. (C) Body weight (BW) in wt and ob/ob mice. (D) Heart weight (mg) and tibia length (mm) ratio (HW/TL) in wt and ob/ob mice. (E) Representative images of cardiac haematoxylin–eosin (HE) staining and (F) Quantification of cardiomyocyte cross‐sectional area based on HE staining. (G) Cardiac Masson trichrome staining in the interstitial and peripheral area, and (H) Quantification of the left ventricular interstitial collagen volume. (I) The serum random blood glucose concentration in wt and ob/ob mice. (J) The serum insulin concentration in wt and ob/ob mice. (K) Intraperitoneal glucose tolerance test (ipGTT) (left) and bar graphs depicting the area under the curve of the ipGTT experiment…

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  FIGURE 3. Proteomic signature of heart tissue in HFpEF mice. (A) Volcano plot of DEPs in the heart tissue of ob/ob and wt mice. (B) BP of GO pathway enrichment analysis of DEPs in the heart tissue of ob/ob and wt mice. (C) GSEA analysis of fatty acid metabolic process based on GO analysis of BP. (D) CC of GO pathway enrichment analysis of DEPs in the heart tissue of ob/ob and wt mice. (E) KEGG pathway enrichment analysis of DEPs in the heart tissue of ob/ob and wt mice. (F) GSEA analysis of peroxisome based on GO analysis of CC. (G) Wiki pathway enrichment analysis of DEPs in the heart tissue of ob/ob and wt mice. (H) Venn diagrams (upper) displaying the number of MitoDEPs that overlap between DEPs and MitoCarta3.0. Pie chart (lower) showing the proportion of MitoDEPs in the total DEPs. (I) GO pathway enrichment analysis of MitoDEPs. (J) KEGG pathway enrichment analysis of MitoDEPs. (K) Petal chart displaying the number of differential expressions that overlap between GSE163665, GSE240171, GSE235934, GSE249409, GSE218733, GSE249411, MitoCarta3.0 and Proteome. (L) Log2 normalised Hmgcs2 protein expression level of wt and ob/ob mice in proteome.

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  FIGURE 4. Runx2 is the upstream transcriptional regulatory factor of Hmgcs2. (A) Venn diagrams displaying the number of differentially expressed transcription factors overlapped with predicted transcription factors. (B) Heatmap showing candidates of transcription factors in wt and ob/ob hearts. (C) Representative immunoblotting images (left) showing Runx2 and Hmgcs2 protein levels in heart tissues of wt mice and ob/ob mice; and statistical analyses (right) of relative Runx2 and Hmgcs2 protein levels. (D) Relative mRNA levels of Runx2 and Hmgcs2 in NRVMs infected with Ad‐NC or Ad‐Runx2 followed by stimulation with FA for 24 h. (E) Representative immunoblotting images (left) showing protein levels of Runx2 and Hmgcs2 in NRVMs infected with Ad‐NC or Ad‐Runx2 followed by stimulation with FA for 24 h; and statistical analyses (right) of relative Runx2 and Hmgcs2 protein levels. (F) Luciferase activation driven by the Hmgcs2 promoter after normalisation to renilla luciferase in HEK‐293 cells. (J) Relative mRNA expression of Mib2 genes in wt and ob/ob hearts. (H) TPM expression of Runx2 gene in GSE163665 dataset. (I) Log2 normalised expression of Runx2 gene in GSE235934, GSE249409, GSE218733 and…

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  FIGURE 5. Mib2 promotes the ubiquitination and degradation of Runx2. (A) Dot matrix plot showing differentially expressed E3 Ubiquitin ligases in wt and ob/ob hearts. (B) Representative immunoblotting images (left) showing Mib2 protein levels in heart tissues of wt mice and ob/ob mice and statistical analyses (right) of relative Mib2 protein levels. (C) and (D) Endogenous immunoprecipitation analysis of the interaction of Mib2 and Runx2 in heart tissues of mice using Mib2 or Runx2 antibodies. (E, F) Co‐IP assays of the interaction between Mib2 and Runx2 in HEK293T cells transfected with the indicated plasmids. (G) Ubiquitination assays confirming the ubiquitination of Runx2 after overexpression of Mib2 in HEK293T cells. (H, I) Representative immunoblot image (upper) of Runx2 protein levels in HEK293T cells treated with MG132(10 μmol/L) after transfection with Flag‐Mib2 or si‐Mib2, and quantification of Runx2 level (lower). (J, K) Representative immunoblot image (upper) of Runx2 protein levels in HEK293T cells treated with CHX (10 μmol/L) after transfection with Flag‐Mib2 or si‐Mib2, and quantification of Runx2 level (lower). Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p <…

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  FIGURE 6. Mib2 regulates lipid accumulation in a Hmgcs2‐dependent manner. (A) Representative immunoblotting images (left) showing protein levels in NRVMs treated with FA and statistical analyses (right) of relative protein levels. (B) Relative mRNA expression of Runx2 and Hmgcs2 genes in NRVMs treated with FA. (C) Representative immunoblotting images (left) of Mib2 protein levels in NRVMs after transfection with Ad‐Mib2 and statistical analyses (right) of relative Mib2 protein levels. (D) Relative ATP levels in NRVMs after transfection with Ad‐Mib2. (E) Representative Oil Red O staining (left) in NRVMs after transfection with Ad‐Mib2 and quantitative analyses (right) of Oil Red O staining. (F) Representative immunoblotting images (left) of Mib2 protein levels in NRVMs after transfection with si‐Mib2 and statistical analyses (right) of relative Mib2 protein levels. (G) Relative ATP levels in NRVMs after transfection with si‐Mib2. (H) Representative Oil Red O staining (left) in NRVMs after transfection with si‐Mib2 and quantitative analyses (right) of Oil Red O staining. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 7. Cardiac‐specific Mib2 overexpression aggravates diastolic dysfunction and lipid accumulation. (A) Representative immunoblotting images (left) of Mib2 protein levels in heart tissues after infection with AAV9‐Mib2 and statistical analyses (right) of relative Mib2 protein levels. (B) Representative M‐mode, PW‐mode and Tissue‐mode echocardiographic images of the left ventricle after infection with AAV9‐Mib2. (C) Assessments of echocardiographic parameters of left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), ratio between mitral E wave and E' wave (E/E'), E' wave, left ventricular end‐diastolic internal diameter (LVIDd), and left ventricular end‐systolic internal diameter (LVIDs). (D) Cardiac triglyceride content in myocardial tissue from wt and ob/ob mice after infection with AAV9‐Mib2. (E) Representative Oil Red O staining (left) images in heart tissue from wt and ob/ob mice after infection with AAV9‐Mib2 and quantitative analyses (right) of Oil Red O staining. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.