Proteomic and Structural Manifestations of Cardiomyopathy in Rat Models of Obesity and Weight Loss

Figures

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  Figure 1. Characteristics of the obesity phenotype in rats fed with a cafeteria diet for 12 weeks. As a consequence of high-calorie feeding, the animals (n=16) gained weight intensively (A) and developed insulin resistance (n=5) (B) with hyperlipidemia and hypercholesterolemia (n=6) (C). Two-way ANOVA with Sidak’s multiple comparison test (A, B) and the Student’s t-test or Mann-Whitney test (cholesterol) were used for analyzing the data (*p<0.05, **p<0.01, ***p<0.001).

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  Figure 2. Hallmarks of obesity cardiomyopathy in rats. The hearts of obese animals were heavier (n=9) (A) with enlarged myocytes (n=4) (B). The oversized hearts contained higher levels of fibrotic tissue both in the non-vessel (C) and vessel containing (D) areas (n=6). The expression of phospho-mTOR on ser2448 (E) but not on Thr2446 (F) was higher in the LV of obese rats, suggesting the up-regulation of this anabolic pathway (n=5). No significant differences were observed with regard to of AMPK activity (G). The Student’s t-test was used for analyzing the data (*p<0.05, **p<0.01, ***p<0.001).

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  Figure 3. Global proteomic profile of the cardiac muscle of obese rats. The amount of 87 proteins differed in the left ventricle (LV) tissue of obese rats as compared to homogenous samples from control (n=5). Protein expression data are presented as Z-score transformed.

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  Figure 4. Molecular processes changed in the heart muscle of obese rats (n=5). A scaling plot of individual proteomes revealed the relatively homogenous composition in the control group (ContO) and the more dispersed proteomic phenotype in experimental (obese) animals (A).Two biological processes: (B) the directed movement of a phospholipid out of a cell or organelle (p=0.024), and (C) the chemical reactions and pathways involving ATP (p=0.043), were significantly changed in the tissue (Bonferoni correction). By using the INTACT database we determined that the pool of all significantly changed proteins interacted with GLUT-4 (27 proteins, p<0.001, Fisher correction) and ACSL-1 (four proteins, p=0.0035, Fisher correction), suggesting that these proteins may be impacted by obesity. The evaluation of the protein expression by means of WB showed that the cardiac expression of ACSL-1 (D) (p=0.045) but not GLUT-4 (E) was changed (increased) in obese rats. GLUT-1 is the next glucose carrier in cardiomyocytes beyond GLUT-4, and its expression was downregulated in obese rats (F) (p=0.026). The Student’s t-test was used for analyzing the data (*p<0.05, ***p<0.001). ACSL-1, long-chain-fatty-acid-CoA…

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  Figure 5. Global proteomic profile of plasma of obese rats. The amount of 49 proteins differed in the plasma of obese rats as compared to homogenous samples from control (n=8). Protein expression data are presented as Z-score transformed.

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  Figure 6. Clustering of the plasma proteins significantly changed in obese rats. Based on the GO : BP (DAVID functional annotation results with Bonferroni statistics) database, plasma proteins were assigned to their respective particular biological function (n=8). Most of the proteins (36%) were involved in the negative regulation of endopeptidase activity (p<0.001, not shown; refer to Supplemental Data for details). 17% of the proteins were involved in immunological regulation (GO:0006956~complement activation, GO:0006958~complement activation). In addition, 17% of molecules participated in the pro-inflammatory response (GO:0006953~acute-phase response, p<0.001; GO:0045087~innate immune response, p<0.01; GO:0006954~inflammatory response, p<0.05). The next biological process changed in the plasma of obese rats included the abnormal level of proteins involved in the regulation of any process that results in a systemic change as a result of a triglyceride stimulus (GO:0034014~response to triglyceride, p<0.05). The pool of proteins (three molecules) involved in processes that stop, prevent, or reduce the frequency, rate, or extent of fibrinolysis resulting in the removal of small blood clots…

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  Figure 7. Characteristics of rats after weight loss. After 12 weeks of feeding with a cafeteria diet, rats (n=16) intensively gained weight (A), developing the obesity phenotype. Obese animals were subjected to CR for 6 weeks to lose weight. For the next four weeks, animals from the experimental (After Weight Loss, AWL) and control (ContA) groups received an isocaloric amount of calories (stabilization period) to ensure stable body weight. After weight loss, the rats did not show features of insulin resistance which was confirmed by insulin (B) and glucose (C) tolerance tests (n=5). The mass of the heart (n=9) (D) and size of the cardiomyocytes (n=3-4) (E) were restored after weight loss in rats which were previously obese. However, the interstitial (F) and perivascular (G) amount of connective tissue was elevated in these animals (n=6). Two-way ANOVA with Sidak’s multiple comparison test (A–C) and Student’s t-test was used for analyzing the data (*p<0.05, **p<0.01).