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. 2023 Jan;11(2):e15574.
doi: 10.14814/phy2.15574.

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

Affiliations

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

Ana Victoria Rojo-García et al. Physiol Rep. 2023 Jan.

Abstract

Sedentary behavior has become ingrained in our society and has been linked to cardiovascular diseases. Physical inactivity is the main characteristic of sedentary behavior. However, its impact on cardiovascular disease is not clear. Therefore, we investigated the effect of physical inactivity in an established mouse model on gene clusters associated with cardiac fibrosis, electrophysiology, cell regeneration, and tissue degradation/turnover. We investigated a sedentary group (CTR, n = 10) versus a tail suspension group (TS, n = 11) that caused hindlimb unloading and consequently physical inactivity. Through histological, protein content, and transcript analysis approaches, we found that cardiac fibrosis-related genes partly change, with significant TS-associated increases in Tgfb1, but without changes in Col1a1 and Fn1. These changes are not translated into fibrosis at tissue level. We further detected TS-mediated increases in protein degradation- (Trim63, p < 0.001; Fbxo32, p = 0.0947 as well as in biosynthesis-related [P70s6kb1, p < 0.01]). Corroborating these results, we found increased expression of autophagy markers such as Atg7 (p < 0.01) and ULK1 (p < 0.05). Two cardiomyocyte regeneration- and sarcomerogenesis-related genes, Yap (p = 0.0535) and Srf (p < 0.001), increased upon TS compared to CTR conditions. Finally, we found significant upregulation of Gja1 (p < 0.05) and a significant downregulation of Aqp1 (p < 0.05). Our data demonstrate that merely 2 weeks of reduced physical activity induce changes in genes associated with cardiac structure and electrophysiology. Hence, these data should find the basis for novel research directed to evaluate the interplay of cardiac functioning and physical inactivity.

Keywords: cardiomyocytes; heart; mouse; physical inactivity; tail suspension.

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Conflict of interest statement

The authors declare no conflicts of interests.

Figures

FIGURE 1
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.
FIGURE 2
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.
FIGURE 3
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.
FIGURE 4
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.
FIGURE 5
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.
FIGURE 6
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.
FIGURE 7
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.
FIGURE 8
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.

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