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. 2019 Dec 2;9(1):18050.
doi: 10.1038/s41598-019-54522-2.

Landscape of Heart Proteome Changes in a Diet-Induced Obesity Model

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Free PMC article

Landscape of Heart Proteome Changes in a Diet-Induced Obesity Model

Danielle F Vileigas et al. Sci Rep. .
Free PMC article

Abstract

Obesity is a pandemic associated with a high incidence of cardiovascular disease; however, the mechanisms are not fully elucidated. Proteomics may provide a more in-depth understanding of the pathophysiological mechanisms and contribute to the identification of potential therapeutic targets. Thus, our study evaluated myocardial protein expression in healthy and obese rats, employing two proteomic approaches. Male Wistar rats were established in two groups (n = 13/group): control diet and Western diet fed for 41 weeks. Obesity was determined by the adipose index, and cardiac function was evaluated in vivo by echocardiogram and in vitro by isolated papillary muscle analysis. Proteomics was based on two-dimensional gel electrophoresis (2-DE) along with mass spectrometry identification, and shotgun proteomics with label-free quantification. The Western diet was efficient in triggering obesity and impaired contractile function in vitro; however, no cardiac dysfunction was observed in vivo. The combination of two proteomic approaches was able to increase the cardiac proteomic map and to identify 82 differentially expressed proteins involved in different biological processes, mainly metabolism. Furthermore, the data also indicated a cardiac alteration in fatty acids transport, antioxidant defence, cytoskeleton, and proteasome complex, which have not previously been associated with obesity. Thus, we define a robust alteration in the myocardial proteome of diet-induced obese rats, even before functional impairment could be detected in vivo by echocardiogram.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Left ventricular papillary muscle study. (A) Baseline condition, (B) post‐rest contraction and (C) effects of increasing extracellular Ca2+ concentration in isolated papillary muscle from control and Western diet (WD) groups (n = 11/group). DT, developed tension (g/mm2); RT, resting tension (g/mm2); +dT/dt, peak of positive tension derivatives (g/mm2/sec); −dT/dt, peak of negative tension derivatives (g/mm2/sec). All parameters normalized per cross-sectional area. Data are means ± SD. Student’s t-test for independent samples in (A) and repeated-measures two-way ANOVA and Bonferroni post hoc test in (B) and (C). *p < 0.05 vs. control.
Figure 2
Figure 2
Proteomics data based on 2-DE of heart tissue from control (C) and Western diet (WD) groups. (A) Correlation analysis between 2-DE gels triplicates of control (C) and Western diet (WD) groups performed by Image Master 2D Platinum software. (B) Representative 2-DE gel image. The position of molecular weight (MW) markers are indicated to the right and the pI (isoelectric point) at the bottom of the gel. The sequence of numbers (1–47) refers to identification spots of the significantly up- (red circle) and down-regulated (blue circle) proteins in the WD group compared to the C group identified by LC-MS/MS. The detailed list of proteins for highlighted spots are shown in Supplementary Table S3.
Figure 3
Figure 3
Label-free proteomics data clustering of heart tissue from control (C) and Western diet (WD) groups. (A) Univariate, significance (p-value) vs. fold change analyses highlighting several significant deregulated proteins of interest. (B) Unsupervised multivariate principal component analysis (PCA). (C) Hierarchical clustering analyses (Heatmap) using unsupervised Euclidean distance of all differentially expressed proteins between the groups. The detailed description of protein names is shown in Supplementary Table S4.
Figure 4
Figure 4
Biological functions of the differentially expressed proteins in the myocardium of obese rats identified by proteomics (A) Protein-protein interaction network. The interaction network analyses were built using the STRING online software with a medium confidence level (0.4). The circles represent proteins, while the straight lines represent the interactions between different proteins. The line thickness indicates the strength of evidence, with thicker connections indicating higher confidence in the protein-protein interaction. Green circles represent the proteins involved in metabolic processes; shaded area delimits the proteins involved in lipid metabolic processes. (B) Gene ontology (GO) enrichment analysis. The analysis was performed using the PANTHER tool (http://www.pantherdb.org), providing the significantly enriched GO terms Molecular Function, Biological Process, and Cellular Component. On the left panel, the horizontal axis indicates the significance (−log10 p-value) of the functional association, which is dependent on the number of proteins in the class. On the right panel, changes are displayed as the number of proteins with increased or decreased levels (horizontal axis). (C) Violin plot of the fold changes for up- and down-regulated proteins in the WD group compared to their controls. The fold changes of up- and down-regulated proteins were further separated into the metabolic processes associated with fatty acid, glucose, amino acid, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (Oxi. Phosp.). The circles inside the plots represent the changed proteins. The detailed description of protein names is shown in Supplementary Tables S3 and S4.
Figure 5
Figure 5
Overview of the obesity effects over cardiomyocyte proteome. The identified proteins are shown according to the magnitude of fold-change; red color for proteins up-regulated and blue color for proteins down-regulated in the myocardium of Western diet-induced obese rats compared to their controls. ROS, reactive oxygen species; AGEs, advanced glycation end products; Val, Ile, and Leu, valine, isoleucine, and leucine; BCAA, branched-chain amino acids; P5C, pyrroline-5-carboxylate; OAA, oxaloacetate; α-KG, α-ketoglutarate; TCA, tricarboxylic acid cycle; SSA, succinate semialdehyde; I, II, III, IV, and V, complexes of mitochondrial oxidative phosphorylation (I-IV: respiratory chain complexes; V: ATP synthase complex); CK, creatine kinase; Cr, creatine; PCr, phosphocreatine; SR, sarcoplasmic reticulum. The detailed description of protein names is shown in Supplementary Tables S3 and S4.
Figure 6
Figure 6
Pathways validation of proteomic data. The protein expression levels of (A) platelet glycoprotein 4 (CD36) and (B) fatty acid-binding protein (FABP3) were measured by Western blot and normalized to beta-actin (internal control) in myocardium from control and Western diet (WD) groups (n = 4–6/group). A representative Western blot and quantification of protein levels are shown. Images of blots have been cropped; the full-length blots are presented in Supplementary Fig. S2. Cardiac levels of (C) malondialdehyde (MDA) and (D) carbonylated protein of control and WD groups (n = 8–13/group). Values are mean ± SD or median (Min-Max). Student’s t‐test (in A, B, and D) or Mann-Whitney U-test (in C) for independent samples.

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