IGF-1 potentiates sensory innervation signalling by modulating the mitochondrial fission/fusion balance

Abstract

Restoring the contractile function of long-term denervated skeletal muscle (SKM) cells is difficult due to the long period of denervation, which causes a loss of contractility. Although sensory innervation is considered a promising protective approach, its effect is still restricted. In this study, we introduced insulin-like growth factor-1 (IGF-1) as an efficient protective agent and observed that IGF-1 potentiated the effects of sensory protection by preventing denervated muscle atrophy and improving the condition of denervated muscle cells in vivo and in vitro. IGF-1-induced Akt phosphorylation suppressed the mitochondrial outer-membrane protein Mul1 expression, which is a key step on preserving contractile property of sensory innervated SKM cells. Mul1 overexpression interfered with the balance between mitochondrial fusion and fission and was a key node for blocking the effects of IGF-1 that preserved the contractility of sensory-innervated SKM cells. Activation of AMP-activated protein kinase α (AMPKα), a mitochondrial downstream target, could block the effects of IGF-1. These data provide novel evidence that might be applied when searching for new approaches to improve the functional condition of long-term denervated SKM cells by increasing sensory protection using the IGF-1 signalling system to modulate the balance between mitochondrial fusion and fission.

Figure 1. IGF-1 promoted the effects of sensory protection in vivo.

(A) Cryosections of GAS muscles were stained with dystrophin. Representative images are shown. Scale bar = 50 μm. Mean CSA of GAS muscles in the sham operation, denervation, IGF-1, sensory protection and sensory protection + IGF-1 groups at the 8th week after surgery (n = 5). (B) Mean GAS muscle wet weight for the different treatments. (C) The total protein extracted from GAS muscle samples was immunoblotted with antibodies against MyHC1 and GAPDH. GAPDH was used as a loading control. (D) Quantification of MyHC1 protein levels (n = 5). (E) Quantification of the number of mitochondria in GAS muscle cells (n = 5). Bar graphs with error bars showing the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. IGF-1 potentiated the effects of sensory innervation in vitro.

(A) Double immunofluorescence labelling of α-actin (Green) and NF-200 (Red) indicated the SKM cell status in the SKM, SKM + DRG, SKM + IGF-1, and SKM + DRG + IGF-1 groups. Scale bar = 200 μm. (B) Quantification of the average length of each SKM cell (n = 5). (C) Quantification of the average area of each SKM cell (n = 5). (D) Double immunofluorescence labelling of α-actin (green) and NF-200 (red) showed dense networks of axons on the surface of SKM cells. Scale bar = 50 μm. (E) The total protein extracted from SKM cells was immunoblotted with antibodies against MyHC1 and GAPDH. GAPDH was used as a loading control. (F) Quantification of MyHC1 protein levels (n = 5). (G) Mitochondrial copy number in SKM cells (n = 5). (H) Mitochondrial morphology observed using MitoTracker Red staining in living SKM cells. The right column is an enlarged view of the box in the left column. The solid arrows in the zoomed area highlight the filamentous mitochondria, and the hollow arrows indicate the particulate mitochondria. Scale bar = 20 μm. (I) Cytosolic Cyt C protein immunoreactive bands. (J) Quantification of cytosolic Cyt C protein levels (n = 5). Bar graphs with error bars showing the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. IGF-1 improved sensory-innervated SKM cell status by Akt phosphorylation.

(A) Immunoreactive bands of phosphorylated Akt and GAPDH. (B) The pAkt protein expression was normalized to that of the SKM group and is shown as the fold change (n = 5). (C) Immunoreactive bands of phosphorylated Akt. (D) Quantification of pAkt protein levels at different time points (n = 5). (E) Immunoreactive bands of phosphorylated Akt and GAPDH in the SKM + DRG, SKM + DRG + IGF-1 and SKM + DRG + LY294002 + IGF-1 groups. (F) Quantification of pAkt protein levels with LY294002 administration (n = 5). (G) Quantitative analysis of mtDNA copy number per nuclear genome in SKM cells (n = 5). (H) MitoTracker Red staining for mitochondrial fission/fusion. The solid arrows and the hollow arrows indicate filamentous and particulate mitochondria, respectively. Scale bar = 20 μm. (I) Cytosolic Cyt C protein immunoreactive bands. (J) Quantification of cytosolic Cyt C protein levels (n = 5). (K) MyHC1 protein immunoreactive bands. (L) The expression of MyHC1 protein was normalized to that of the SKM + DRG group and is shown as the fold change (n = 5). (M) MyHC1 protein immunoreactive bands for exploring the effect of LY294002 to innervated SKM cells. (N) Quantification of MyHC1 protein levels (n = 5). (O) Levels of atrogin-1 and MuRF1 mRNA (n = 5). Bar graphs with error bars showing the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. IGF-1 promoted the effects of sensory innervation by suppressing Mul1 expression.

(A) Quantification of Mul1 mRNA for LY294002 administration (n = 5). (B) Immunoreactive bands of Mul1 and GAPDH after LY294002 treatment. (C) Quantification of Mul1 protein levels (n = 5). (D) Mul1 mRNA levels in the SKM + DRG (non-coding lentivirus) and SKM + DRG (Mul1 lentivirus) groups (n = 5). (E) Mul1 protein immunoreactive bands after lentivirus transfection in the above two groups. (F) Quantification of Mul1 protein levels (n = 5). (G) Mul1 mRNA levels in the SKM + DRG (non-coding lentivirus), SKM + DRG (non-coding lentivirus) + IGF-1 and SKM + DRG (Mul1 lentivirus) +IGF-1 groups (n = 5). (H) Mul1 protein immunoreactive bands after lentivirus transfection. (I) Quantification of Mul1 protein levels after lentivirus transfection (n = 5). (J) The immunofluorescence staining to confirm the validity of Mul1 lentivirus transfection. GFP (green) shows the successful infection of the lentivirus, and the Mul1 (red) labelling indicates the expression of Mul1 protein. (K) MitoTracker Red staining for mitochondrial fission/fusion of SKM cells infected by the lentivirus. Scale bar = 20 μm. (L) Quantitative analysis of mtDNA copy number per nuclear genome in SKM cells (n = 5). (M) Cytosolic Cyt C protein immunoreactive bands after lentivirus transfection. (N) Quantification of cytosolic Cyt C protein levels (n = 5). (O) Levels of atrogin-1 and MuRF1 mRNA (n = 5). (P) Immunoreactive bands of MyHC1 and GAPDH. (Q) Quantitated MyHC1 protein abundance (n = 5). Bar graphs with error bars showing the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. AMPKα activation of sensory-innervated SKM cells was modulating by IGF-1.

(A) Immunoreactive bands of pACC, pAMPKα, AMPKα and GAPDH for the 6-hour AICAR treatment. (B) Quantification of the pACC levels (n = 5). (C) Quantification of the pAMPKα levels (n = 5). (D) Levels of atrogin-1 and MuRF1 mRNA for the 2-day AICAR treatment (n = 5). (E) MyHC1 immunoreactive bands for the 2-day AICAR treatment. (F) Quantification of the MyHC1 protein levels (n = 5). Bar graphs with error bars showing the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Schema of the mechanism of IGF-1 on enhancing sensory innervation efficacy.

IGF-1-induced Akt phosphorylation suppressed Mul1 expression, restored the balance between mitochondrial fusion and fission, and inhibited activation of AMPKα to preserve the intrinsic contractility of sensory-innervated SKM cells.