Physical inactivity by tail suspension alters markers of metabolism, structure, and autophagy of the mouse heart

Figures

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  FIGURE 1. Cross‐sectional area assessment of heart cardiomyocytes upon tail suspension. (a) Histological section of the heart from a control (CTR) condition. (b) Histological section of the heart from a tail suspension (TS) condition. (c) Assessment of the mean cardiomyocyte (CM) cross‐sectional area (CSA) between control (CTR) and tail suspension (TS) conditions. (d) Assessment of the mean cardiomyocyte cross‐sectional area related to body weight (BW) between control (CTR) and tail suspension (TS) conditions. Bar = 50 μm. 40× magnification. Eighty CM was counted by animal. All experiments were performed with CTR = 10 mice; TS = 11 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test.

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  FIGURE 2. Analysis of cardiac damage and pathological hypertrophy genes. (a) Atrial natriuretic peptide (Nppa); (b) Brain natriuretic peptide (Nppb); (c) α‐skeletal muscle Actin (Acta1); (d) Tumor necrotic factor α (Tnf). CTR = control condition; TS = tail suspension condition. All experiments were performed with CTR = 9 mice; TS = 10 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test.

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  FIGURE 3. Analysis of fibrosis‐related genes in cardiomyocytes. (a) Collagen 1 (Col1a1); (b) Fibronectin (Fn1); (c) Transforming growth factor beta 1 (Tgfb1). CTR, control condition; TS, tail suspension condition. (d) Picrosirius Red staining of CTR and TS conditions. (e) Quantification of fibrosis area (μm2). Bar = 200 μm. 40× magnification. All experiments were performed with CTR = 9 mice; TS = 10 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test. Levels of significance were **p < 0.01.

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  FIGURE 4. Assessment of cardiac cell regeneration markers. (a) Yes‐associated protein (Yap); (b) Serum response factor (Srf). CTR, control condition; TS, tail suspension condition. All experiments were performed with CTR = 9 mice; TS = 10 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test. Levels of significance were ***p < 0.001.

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  FIGURE 5. Investigation of protein degradation‐ and protein synthesis‐related genes. (a) Tripartite Motif Containing 63 (Trim63); (b) F‐Box Protein 32 (Fbxo32); (c) Ribosomal protein S6 kinase (P70s6kb1); (d) Mechanistic target of rapamycin (Mtor1). CTR, control condition; TS, tail suspension condition. All experiments were performed with CTR = 9 mice; TS = 10 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test. Levels of significance were **p < 0.01, and ***p < 0.001.

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  FIGURE 6. Evaluation of protein synthesis. (a) Phospho‐mTOR (Ser2448)/ total mTOR; (b) Bands quantified of p‐mTOR/ total mTOR normalized with Vinculin (1) (corresponds to loading control for total proteins) and Vinculin (2) (corresponds to loading control for p‐mTOR); (c) Phospho‐P70S6K1 (Thr421/Ser424)/ total P70S6K1; (d) Bands quantified of p‐P70S6K1/total P70S6K1 normalized with Vinculin (3) (corresponds to loading control for phosphorylated proteins). CTR, control condition; TS, tail suspension condition. All experiments were performed with CTR = 5 mice; TS = 5 mice. All gels were done simultaneously. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test. Level of significance is p < 0.05.

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  FIGURE 7. Evaluation of autophagy genes expression. (a) Microtubule Associated Protein 1 Light Chain 3 Beta (Map1lc3b); (b) Autophagy Related 7 (Atg7); (c) BCL2 Apoptosis Regulator (Bcl2); (d) Unc‐51 Like Autophagy Activating Kinase 1 (ULK); (e) Beclin‐1 (Becn‐1). CTR, control condition; TS, tail suspension condition. All experiments were performed with CTR = 10 mice; TS = 11 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test. Levels of significance were *p < 0.05, **p < 0.01.

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  FIGURE 8. Analysis of genes involved in cardiac electrophysiological processes. (a) Gap Junction Protein Alpha 1/connexin 43 (Gja1); (b) Aquaporin 1 (Aqp1); (c) Aquaporin 7 (Aqp7); (d) ATPase Sarcoplasmic/Endoplasmic Reticulum Ca 2+ Transporting 2 (Atp2a2); (e) Solute carrier family 8 sodium/calcium exchanger (Slc8a1). CTR, control condition; TS, tail suspension condition. All experiments were performed with CTR = 10 mice; TS = 11 mice. Normality was assessed with D'Agostino and Pearson and depending on the outcome, differences between CTR and TS were analyzed using either the unpaired Student's t test or Mann–Whitney U test. Levels of significance were *p < 0.05.