Skeletal muscle-derived musclin attenuates glycolysis, oxidative stress, and pulmonary hypertension through the NPR3/AKT/mTORC1 pathway

Figures

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  Figure 1 Skeletal muscle-derived musclin ameliorates pulmonary arterial remodeling and right ventricular hypertrophy in an experimental PH model (A) Serum musclin levels in mice in different groups were determined by ELISA (n = 8). (B,C) Representative western blots and corresponding quantification of musclin expression in right quadricep muscles from mice in different groups (n = 6). (D) Representative H&E staining images (scale bar: 20 μm), Masson staining images (scale bar: 20 μm), immunohistochemical staining images of α-SMA (scale bar: 20 μm), and echocardiography images of the mice in the different groups. (E–K) Quantification of (E) RVSP (n = 7), (F) RVHI (n = 8), (G) percentage of pulmonary wall thickness/total thickness (n = 7), (H) percentage of muscularization in PAs (n = 6), (I) PAT/PET (n = 8), (J) RVID (n = 8), and (K) RVFWT (n = 8) in the mice of different groups. *P < 0.05, **P < 0.01, and ***P < 0.001. Student’s t test for (A and C). Post hoc test for LSD in (E, F, G, H, I, J, and K).

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  Figure 2 Musclin represses hypoxia-induced proliferation and migration of PASMCs (A) PASMCs were treated with vehicle or musclin (50 nM) and simultaneously incubated with or without hypoxia for 24 h. The viability of PASMCs from different groups was assessed via CCK-8 assay (n = 8). (B,C) Representative western blots and corresponding quantification of PCNA in PASMCs from different groups (n = 7). (D) PASMCs from different groups were stained with Ki-67 (green) and DAPI (blue). Representative images of Ki-67 staining (scale bar: 50 μm). The migration of PASMCs from different groups was measured via transwell and wound healing assays (scale bar: 200 μm). (E) The percentages of Ki-67-positive PASMCs in the different groups (n = 6). (F) Quantification of migrating cells from different groups (n = 8). (G) Quantification of wound healing rates in PASMCs from different groups (n = 8). *P < 0.05, **P < 0.01, and ***P < 0.001. Post hoc test for LSD in (A, C, E, F, and G).

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  Figure 3 Musclin suppresses glycolysis and oxidative stress in hypoxia-challenged PASMCs (A,B) PASMCs from different groups were stained with DHE (red). Representative images and the corresponding intensity of DHE staining (n = 7). Scale bar: 50 μm. (C,D) Representative western blots and corresponding quantification of NOX4 and SOD1 in PASMCs from different groups (n = 7). (E,F) The MDA content and relative GSH level in the supernatant of PASMCs from different groups (n = 5). (G,H) Glycolysis and glycolytic capacity in PASMCs from different groups were determined by the ECAR (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. Post hoc test for LSD in (B, D, E, F, and H).

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  Figure 4 Musclin inhibits the proliferation and migration of hypoxia-stimulated PASMCs by binding to NPR3 (A) PASMCs were treated with musclin (50 nM) for 24 h and then subjected to co-IP analysis. (B,C) Representative western blots and corresponding quantification of NPR3 in PASMCs from different groups (n = 8). (D,E) Glycolysis and glycolytic capacity in PASMCs from different groups were determined by the ECAR (n = 3). (F) PASMCs from different groups were stained with DHE (red). Scale bar: 50 μm. PASMCs from different groups were stained with Ki-67 (green) and DAPI (blue). Scale bar: 50 μm. The migration of PASMCs from different groups was measured via transwell assay. Scale bar: 200 μm. (G) The relative intensity of DHE in PASMCs from different groups (n = 6). (H) The percentages of Ki-67-positive PASMCs in the different groups (n = 6). (I) Quantification of migrating cells from different groups (n = 6). *P < 0.05 and **P < 0.01. Post hoc test for LSD in (C, E, G, H, and I).

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  Figure 5 Musclin suppresses the proliferation and migration of hypoxia-stimulated PASMCs by inhibiting mTORC1 activity (A,B) PASMCs were treated with vehicle or musclin (50 nM) and simultaneously incubated with or without hypoxia for 24 h. Representative western blots and corresponding quantification of p-S6/S6 and p-4EBP1/4EBP1 in PASMCs from different groups (n = 7). (C,D) Representative western blots and corresponding quantification of TSC1, p-S6/S6, and p-4EBP1/4EBP1 in PASMCs from different groups (n = 6). (E,F) Glycolysis and glycolytic capacity in PASMCs from different groups were determined by the ECAR (n = 3). (G) PASMCs from different groups were stained with DHE (red). Scale bar: 50 μm. PASMCs from different groups were stained with Ki-67 (green) and DAPI (blue). Scale bar: 50 μm. The migration of PASMCs from different groups was measured via transwell assay. Scale bar: 200 μm. (H) The relative intensity of DHE in PASMCs from different groups (n = 7). (I) The percentages of Ki-67-positive PASMCs in the different groups (n = 6). (J) Quantification of migrating cells from different groups (n = 7). *P < 0.05, **P < 0.01, and ***P < 0.001. Post hoc test for LSD in (B, D,…

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  Figure 6 Skeletal muscle-derived musclin ameliorates MCT-induced PH by reducing mTORC1 activity in Pas (A,B) Representative western blots and corresponding quantification of p-S6/S6 and p-4EBP1/4EBP1 in PAs from mice in different groups (n = 7). (C,D) Representative western blots and corresponding quantification of TSC1, p-S6/S6, and p-4EBP1/4EBP1 in PAs from mice in different groups (n = 6). The activities of (E) HK and (F) LDH in the PAs of the mice in the different groups (n = 8). (G,H) Representative western blots and corresponding quantification of SOD1 and NOX4 in PAs from mice in different groups (n = 6). (I) Representative H&E staining images (scale bar: 20 μm) and echocardiography images of the mice in the different groups. Quantification of (J) RVSP (n = 8), (K) RVHI (n = 7), (L) the percentage of pulmonary wall thickness/total thickness (n = 8), and (M) PAT/PET (n = 8) in the mice in the different groups. *P < 0.05, **P < 0.01, and ***P < 0.001. Post hoc test for LSD in (B, D, E, F, H, J, L, and M). Post hoc test for Dunnett’s T3 test in (K).

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  Figure 7 Skeletal muscle-derived musclin suppresses the proliferation and migration of PASMCs and pulmonary hypertension partly by attenuating glycolysis and oxidative stress in an NPR3/AKT/mTORC1-dependent manner Overexpression of musclin in skeletal muscle via AAV6 transfection results in increased musclin release into the circulation from skeletal muscle. Musclin suppresses AKT phosphorylation and thus inhibits subsequent mTORC1 activation by binding to NPR3. Decreased AKT/mTORC1 activity subsequently attenuates glycolysis, oxidative stress, proliferation, and migration in PASMCs, thereby ameliorating PH progression.