Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of mitochondrial dysregulation

Abstract

Background: Nitric oxide (NO), generated in skeletal muscle mostly by the neuronal NO synthases (nNOSμ), has profound effects on both mitochondrial bioenergetics and muscle development and function. The importance of NO for muscle repair emerges from the observation that nNOS signalling is defective in many genetically diverse skeletal muscle diseases in which muscle repair is dysregulated. How the effects of NO/nNOSμ on mitochondria impact on muscle function, however, has not been investigated yet.

Methods: In this study we have examined the relationship between the NO system, mitochondrial structure/activity and skeletal muscle phenotype/growth/functions using a mouse model in which nNOSμ is absent. Also, NO-induced effects and the NO pathway were dissected in myogenic precursor cells.

Results: We show that nNOSμ deficiency in mouse skeletal muscle leads to altered mitochondrial bioenergetics and network remodelling, and increased mitochondrial unfolded protein response (UPR(mt)) and autophagy. The absence of nNOSμ is also accompanied by an altered mitochondrial homeostasis in myogenic precursor cells with a decrease in the number of myonuclei per fibre and impaired muscle development at early stages of perinatal growth. No alterations were observed, however, in the overall resting muscle structure, apart from a reduced specific muscle mass and cross sectional areas of the myofibres. Investigating the molecular mechanisms we found that nNOSμ deficiency was associated with an inhibition of the Akt-mammalian target of rapamycin pathway. Concomitantly, the Akt-FoxO3-mitochondrial E3 ubiquitin protein ligase 1 (Mul-1) axis was also dysregulated. In particular, inhibition of nNOS/NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent-protein kinases induced the transcriptional activity of FoxO3 and increased Mul-1 expression. nNOSμ deficiency was also accompanied by functional changes in muscle with reduced muscle force, decreased resistance to fatigue and increased degeneration/damage post-exercise.

Conclusions: Our results indicate that nNOSμ/NO is required to regulate key homeostatic mechanisms in skeletal muscle, namely mitochondrial bioenergetics and network remodelling, UPR(mt) and autophagy. These events are likely associated with nNOSμ-dependent impairments of muscle fibre growth resulting in a deficit of muscle performance.

Keywords: Akt-FoxO3-Mul-1 axis; Akt-mTOR pathway; Autophagy; Fibre growth; Mitochondrial bioenergetics; Mitochondrial network; Muscle exercise; Muscle structure; Nitric oxide synthase and signalling; Unfolded protein response.

Figures

Figure 1

Mitochondrial metabolism is impaired in skeletal muscles of NOS1-/- mice. Fibres were isolated from different muscles of wild-type and NOS1-/- mice at P120. (A) Mitochondrial membrane potential measured in fibres isolated from flexor digitorum brevis muscles, loaded with TMRM and treated with 5 μM oligomycin (Olm) or 4 μM FCCP. TMRM staining was monitored in six to ten fibres obtained from at least three different animals per experimental group. Data are expressed by setting the initial value as 1. (B) ATP production on mitochondria isolated from tibialis anterior and diaphragm muscles, at 10 minutes after substrate addition. Data are expressed by setting the initial value as 1. (C-D) Oxygen consumption on fibres isolated from tibialis anterior and diaphragm muscles, supplied with specific CI, CII and CIV mitochondrial complex substrates, as indicated in the Methods. (E) Quantitative analysis of the mtDNA copy number. Data are expressed by normalizing mtDNA values versus nuclear DNA. Each histogram represents the data obtained from at least five different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control.

Figure 2

Mitochondrial morphology, UPRmtand autophagy in skeletal muscles of NOS1-/- mice.Tibialis anterior muscles were isolated from wild-type and NOS1-/- mice at P120. (A)In vivo imaging of the mitochondrial network by two-photon confocal microscopy. Muscles were transfected with the mitochondrially targeted red fluorescent protein pDsRed2-Mito. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (B) TEM images detecting the presence of abnormal, enlarged subsarcolemmal mitochondria (asterisks) or autophagic vacuoles (arrowheads) in NOS1-/- muscles. The inset depicts a multivesicular body in NOS1-/- fibres taken at higher magnification. The images are representative of results obtained from at least three different animals per experimental group. (C-D) Subsarcolemmal mitochondrial ultrastructure analysis by TEM. Data represent the quantification of the mitochondrial area and morphometric analysis of mitochondrial cristae complexity. Each histogram represents the data obtained from at least three different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control. (E) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. The image is representative of results obtained from at least five to seven different animals per experimental group. (F)In vivo imaging of autophagosome formation by two-photon confocal microscopy. Muscles were transfected with YFP-LC3. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (G) Western blot analysis of LC3 lipidation. Actin was used as the internal standard. The image is representative of results obtained from at least 10 different animals per experimental group.

Figure 3

NO signalling, UPRmt, and autophagy on myogenic precursor cells. Cells were differentiated for six hours in the absence (control) or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM), and ODQ +8 Br-cGMP (2.5 mM). (A) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. (B) Confocal microscopy imaging of cells transfected with YFP-LC3. Mitochondrial morphology was detected by mitochondrial matrix-specific protein cyclophillin D (CypD) staining. Scale Bar: 10 μm. Images are representative of at least three to five independent experiments..

Figure 4

NO signalling and autophagic pathway on myogenic precursor cells. (A) Western blot analysis of LC3 lipidation in cells differentiated for six hours in the absence or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM), and ODQ +8 Br-cGMP (2.5 mM). Actin was used as the internal standard. Image is representative of at least five independent experiments. (B) qPCR analysis of mRNA levels for p62, Bnip3 and Atg4 in cells differentiated for six hours in the absence (control) or in the presence of L-NAME ODQ, and KT5823. Values are expressed as the fold change over control. Each histogram represents the data obtained from at least five independent experiments. * P <0.05 versus respective control.

Figure 5

NO signalling, FoxO3, and ubiquitin ligases. Western blot analysis of phosphorylated FoxO3 levels (pFoxO3) or mitochondrial ubiquitin ligase Mul-1 expression in tibialis anterior(A-B) and diaphragm (C-D) of wild-type and NOS1-/- mice at P120. FoxO3 or actin were used as the internal standard. The images are representative of results obtained from at least four to ten different animals per experimental group. (E) Western blot analysis of Mul-1 expression in myogenic precursor cells differentiated in the absence or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM) and ODQ +8 Br-cGMP (2.5 mM). Actin was used as the internal standard. The image is representative of at least five independent experiments. qPCR analysis of mRNA levels for atrogin-1, muRF1 and MUSA1 in tibialis anterior(F) and diaphragm (G) muscles of wild-type and NOS1-/- mice at P120. Values are expressed as the fold change over wild-type. Each histogram represents the data obtained from at least five to eight different animals per experimental group. * P <0.05 versus the respective wild-type control.

Figure 6

Skeletal muscle phenotype of wild-type and NOS1-/- mice at P120. (A) Weight of tibialis anterior, gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. The muscle size is relative to body weight. Each histogram represents the data obtained from at least 10 different animals per experimental group. (B) The number of myofibres in tibialis anterior. Each histogram represents the data obtained from at least four to five different animals per experimental group. Laminin staining of tibialis anterior(C-E) and diaphragm (F-H) muscles. (C, F) Immunohistochemical images. Scale bar: 100 μm. (D, G) Representative distribution of CSA values. (E, H) Quantification of CSA. Images and quantifications represent the data obtained from at least four to seven different animals per experimental group. *P <0.05, **P <0.01, and ***P <0.001 versus the respective wild-type control.

Figure 7

Skeletal muscle phenotype of wild-type and NOS1-/- mice at P10. (A-C) Laminin staining of hind limb muscles. (A) Immunohistochemical images. Scale bar: 100 μm. (B) Representative distribution of CSA values. (C) Quantification of CSA. Images and quantifications represent the data obtained from at least five different animals per experimental group. (D) Number of myonuclei per fibre in hind limb muscles. Each histogram represents the data obtained from at least three different animals per experimental group. (E) Western blot analysis of myosin (MF20) and MyoD expression in myogenic precursor cells isolated from wild-type and NOS1-/- mice and differentiated for increasing times. Calnexin was used as the internal standard. Images are representative of at least three independent experiments. (F) Confocal microscopy imaging of myogenic precursor cells isolated from wild-type and NOS1-/- mice and differentiated for 48 hours. Mitochondrial morphology was detected by mitochondrial matrix-specific protein cyclophillin D staining. Scale Bar: 10 μm. Images are representative of at least three independent experiments. * P <0.05 versus the respective wild-type control.

Figure 8

Skeletal muscle phenotype of wild-type and NOS1-/- mice at P30. (A-C) Laminin staining of tibialis anterior muscles. (A) Immunohistochemical images. Scale bar: 100 μm. (B) Representative distribution of CSA values. (C) Quantification of CSA. Images and quantifications represent the data obtained from at least five different animals per experimental group. Western blot analysis in tibialis anterior: (D) phosphorylated S6, 4E-BP1 and Akt levels, (E) phosphorylated FoxO3 levels or mitochondrial ubiquitin ligase Mul-1 expression. S6, 4E-BP1, Akt, FoxO3 or actin were used as the internal standard. The images are representative of results obtained from at least four different animals per experimental group. *P <0.05 versus the respective wild-type control.

Figure 9

Skeletal muscle function in wild-type and NOS1-/- mice. (A) WBT measurements determined by dividing the average of the top ten or top five forward pulling tensions, respectively, by the body weight. (B) Running distance calculated during one bout of exhaustive treadmill running (day 1) and after repeated challenges (days 2 and 3). (C) Treadmill runtime to exhaustion calculated as the averages obtained at day 1 to 3. Each histogram represents the data obtained from at least four to five different animals per experimental group. (D) TEM analysis performed in tibialis anterior muscles of both unchallenged (no run) and challenged (exhaustive running) mice. The images are representative of results obtained from at least three different animals per experimental group. (E) EBD uptake in tibialis anterior muscles after the treadmill running. Scale Bar: 100 μm. The images are representative of results obtained from at least four different animals per experimental group. (F) CK serum levels (units per litre) of mice after treadmill running. Each histogram represents the data obtained from at least four different animals per experimental group. *P <0.05, **P <0.01 and ***P <0.001 versus the respective wild-type control. WBT and treadmill running were performed on animals at P120.