Altered Cellular Protein Quality Control System Modulates Cardiomyocyte Function in Volume Overload-Induced Hypertrophy

Figures

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  Figure 1. Protocol figure and titin oxidation and ubiquitination. (A) Experimental set-up, male Wistar rats were subjected to aortocaval fistula to develop volume overload-induced hypertrophy, (B) passive tension–sarcomere length relations of control (Ctrl) and volume overload (VO) cardiomyocytes in 4-month group with or without GSH treatment at sarcomere length (SL) 1.8–2.3 μm. (C) Passive tension–sarcomere length relations of control (Ctrl) and volume overload (VO) cardiomyocytes in 8-month group with or without GSH treatment at sarcomere length (SL) 1.8–2.3 μm. All force recordings were normalized for cardiomyocyte cross-sectional area. Curves represent third-order polynomial regressions. Data are shown as nonlinear regression fit of the means/medians; n = 5–6. * p < 0.05 Ctrl vs. VO 8-month, † p < 0.05; 8-month VO + GSH vs. 8-month VO, using one-way ANOVA Tukey multiple comparisons test. (D) S-glutathionylation of N2B-titin from left ventricular tissue. (E) GSSG/GSH ratio, (F) ubiquitination of titin, (G) ubiquitin level. Data are shown as mean ± SEM; n = 5–6. # p < 0.05 Ctrl vs. VO.; * p < 0.05 4-months vs. 8-months using one-way ANOVA Tukey multiple comparisons test.

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  Figure 2. Inflammatory pathways. (A) Passive tension–sarcomere length relations of control (Ctrl) and volume overload (VO) cardiomyocytes in 4-month group with or without IL-6 inhibitor (IL-6i) treatment at sarcomere length (SL) 1.8–2.3 μm. (B) Passive tension–sarcomere length relations of control (Ctrl) and volume overload (VO) cardiomyocytes in 8-month group with or without IL-6 inhibitor treatment at sarcomere length (SL) 1.8–2.3 μm. All force recordings were normalized to cardiomyocyte cross-sectional area. Curves represent third-order polynomial regressions. Data are shown as nonlinear regression fit the means/medians; n = 5–6. * p < 0.05 Ctrl vs. VO 8-month, † p < 0.05; 8-month VO + IL-6i vs. 8-month VO, using one-way ANOVA Tukey multiple comparisons test. (C) Interleukin-6 protein level, (D) interleukin-18 protein level, (E) tumor necrosis factor alpha protein level, (F) phosphorylated/total endothelial nitrogen monoxide synthase (eNOS) protein ratio, (G) phosphorylated/total nuclear factor kappa B protein ratio, (H) phosphorylated/total nuclear factor of activated T-cells (NFAT) protein ratio. Data are shown as mean ± SEM; n = 4–6. * p < 0.05 4-months vs. 8-months, # p < 0.05 Ctrl…

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  Figure 3. Downstream signaling pathway of cardiac hypertrophic growth and histone modifications. (A) Phosphorylated/total mTOR protein ratio, (B) sequestosome 1 (p62) protein level, (C) autophagy marker light chain 3 protein level, (D) phosphorylated/total histone 3 protein ratio, (E) acetylated/total histone 3 protein ratio, (F) monomethylated/total histone 3 protein ratio, (G) dimethylated/total histone 3 protein ratio. Data are shown as mean ± SEM; n = 4–6. * p < 0.05 4-months vs. 8-months, # p < 0.05 Ctrl vs. VO using one-way ANOVA Tukey multiple comparisons test.

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  Figure 4. Proteases of the protein quality control system. (A) Procaspase 3 expression level from left ventricle tissue. (B) Active caspase 3 level, (C) cathepsin levels, (D) procaspase 9 expression level, (E) active caspase 9 level, (F) calpain levels. Data are shown as mean ± SEM; n = 4–6. * p < 0.05 4-months vs. 8-months, # p < 0.05 Ctrl vs. VO using one-way ANOVA Tukey multiple comparisons test.

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  Figure 5. Chaperon proteins of the protein quality control system. (A) Phosphorylated/total heat shock protein 27 ratio, (B) phosphorylated/total heat shock protein 70 ratio, (C) phosphorylated/total αβ-crystallin ratio. Data are shown as mean ± SEM; n = 4–6. * p < 0.05 4-months vs. 8-months, # p < 0.05 Ctrl vs. VO using one-way ANOVA Tukey multiple comparisons test.