Multi-Omics Analysis of Diabetic Heart Disease in the db/db Model Reveals Potential Targets for Treatment by a Longevity-Associated Gene

Figures

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  Figure 1. RNA sequencing. (A) Cluster analysis and (B) volcano plot in the contrast between type-2 diabetic db/db mice given PBS vehicle (DIAB-V) and untreated ND controls. (C) Cluster analysis and (D) volcano plot in the contrast between LAV-BPIFB4-treated diabetic db/db mice (DIAB-LAV) and ND controls. (E) Principal component analysis emphasising the variation in gene expression between the different groups. (F) Cluster analysis and (G,H) volcano plots in the contrast between type-2 diabetic db/db mice given LAV-BPIFB4 or PBS vehicle at >2.0 FC or >1.5 FC level. n = 3 to 4 mice per group.

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  Figure 2. Validation of sequencing data by RT-qPCR (Study 2). (A) Graphs showing the mRNA levels of 12 genes found differentially modulated by the LAV-BPIFB4 treatment in type-2 diabetic db/db mouse hearts. RT-qPCR was performed on the samples from sequencing, plus additional animals, for an n = 6 animals per group (non-diabetic, ND; PBS vehicle-treated diabetic, V; and LAV-BPIFB4-treated diabetic, LAV). Data (fold changes vs. ND) are expressed as individual values and mean ± SEM. * p < 0.05 and ** p < 0.01. (B) Comparison of fold changes from the two methodologies. (C) Linear regression indicating the correlation of data from the two methodologies.

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  Figure 3. Focused transcriptomic analysis of mitochondrial metabolic targets. Graphs show the mRNA and protein expression levels of components involved in the regulation of mitochondrial pyruvate uptake (PDK4) (A); ketone biosynthesis (HMGCS2) (B); fatty acid metabolism (MCAD, CPT1B, UCP3, & ACOT1) (C–F); the TCA cycle (CS) (G); and ATP synthesis (ATPB). Data (fold changes vs. ND) are expressed as individual values and means ± SEM of n = 6 per group. * p < 0.05 and ** p < 0.01. Representative western blots show n=1 sample per group, presented in the same order as in the graphs. Line within blots indicates non-adjacent lanes on the same membrane (see online Supplementary gels file S1). For clarity, mRNA data for Pdk4, Hmgcs2 and Acot1 is the same as that presented in Figure 2.

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  Figure 4. Focused transcriptomic analysis of mitochondrial biogenesis and dynamics. Graphs show the mRNA and protein expression levels of components involved in mitochondrial protein import (TOMM20) (A); biogenesis (PCG1α and TFAM) (B,C); and fussion/fission (MFN2 and DRP1) (D,E). Data (fold changes vs. ND) are expressed as individual values and means ± SEM of n = 6 per group. * p < 0.05 and ** p < 0.01. Representative western blots show n = 1 sample per group, presented in the same order as in the graphs. Line within blots indicates non-adjacent lanes on the same membrane (see online Supplementary gels file S1).

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  Figure 5. Schematic representation of metabolic protein changes induced by LAV-BPIFB4. LAV-BPIFB4 treatment is associated with a subtle up-regulation in proteins involved in mitochondrial metabolism. Red arrows indicate the enzymes induced by LAV-BPIFB4 treatment.