Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice

Abstract

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7 days) and consumed drinking water with or without 1 mM sodium nitrate. Compared with the contralateral control limb, 3 days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P < 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3 days decreased FSR in both SS (P = 0.009) and IMF (P = 0.031) mitochondria. Additionally, 3 days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7 days of immobilization. However, in contrast to 3 days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7 days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3 day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Keywords: immobilization; intermyofibrillar mitochondria; mitochondrial reactive oxygen species; mitochondrial respiration; nitrate; protein synthesis; subsarcolemmal mitochondria.

Figure 1. Dietary nitrate supplementation preserves mitochondrial protein synthesis rates after 3 days of immobilization, without preventing immobilization‐induced muscle atrophy

A–D, 3 days of single‐limb immobilization in mice (A) decreased muscle mass of the soleus (B and C) and gastrocnemius + plantaris (D) muscles in comparison to the control limb, an effect that occurred (in the soleus) regardless of nitrate consumption. E, 3 days of immobilization decreased myofibrillar protein synthesis rates. F–H, in isolated mitochondria (F), nitrate supplementation preserved mitochondrial protein synthesis rates in both SS (G) and IMF (H) subfractions. Individual values are shown as black circles (n = 6–9). Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. Data are shown as the mean ± SD. Abbreviations: Cav1, caveolin 1; COX IV, cytochrome c oxidase subunit 4; FSR, fraction synthesis rate; GLUT4, glucose transporter type 4; H₂O, standard water; Homog; whole‐muscle homogenate; IMF, intermyofibrillar; NO₃, nitrate; SERCA2, sarco/endoplasmic reticulum calcium ATPase 2; SS, subsarcolemmal. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. Dietary nitrate supplementation prevents immobilization‐induced impairments in mitochondrial protein content, respiration, and reactive oxygen species after 3 days of immobilization

A–D, 3 days of immobilization decreased mitochondrial protein content (A), reduced maximal mitochondrial respiration (B) and increased maximal (but not submaximal) mitochondrial ROS (C and D) in standard water‐consuming mice. E–H, dietary nitrate completely attenuated these impairments. I and J, 4‐HNE protein content was decreased after 3 days of immobilization in standard water‐consuming mice (I), but not in nitrate‐consuming mice (J). K–M, immobilization with or without nitrate did not alter the ability of SERCA‐derived ADP from Ca²⁺ (K) to support mitochondrial respiration (L and M). Individual values are shown as black circles (n = 7–9). Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. Data are shown as the mean ± SD. Abbreviations: 4‐HNE, 4‐hydroxynonenal; ANT1, adenine nucleotide translocase 1; C, control limb; CPA, cyclopiazonic acid; CI, OXPHOS complex I; CII, OXPHOS complex II; CIII, OXPHOS complex III; CV, OXPHOS complex V; H₂O, standard water; I, immobilized limb; JO₂, oxygen consumption rate; K _m, Michaelis–Menten constant; mH₂O₂, mitochondrial hydrogen peroxide emission; NADH, nicotinamide adenine dinucleotide; NO₃, nitrate; OD, optical density; OXPHOS; oxidative phosphorylation; P_i, inorganic phosphate; PM, pyruvate + malate; Ponc., Ponceau stain; ROS, reactive oxygen species; SERCA, sarco/endoplasmic reticulum calcium ATPase; SR, sarcoplasmic reticulum. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Three days of immobilization alters signalling proteins in skeletal muscle, regardless of nitrate consumption

A–C, regardless of nitrate consumption, CaMKII and AMPK phosphorylation was increased in the immobilized limb. D–F 3 days of immobilization increased Parkin protein content in nitrate‐consuming mice, decreased LC3‐I protein content in both groups, and decreased LC3‐II protein content in nitrate‐consuming mice. Individual values are shown as black circles (n = 5–8). Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. Data are shown as the mean ± SD. Abbreviations: AMPK, 5‐AMP‐activated protein kinase; C, control limb; CaMKII, Ca²⁺/calmodulin‐dependent protein kinase II; DRP1, dynamin‐related protein 1; H₂O, standard water; I, immobilized limb; LC3, microtubule‐associated proteins 1A/1B light chain 3; MFN1, mitofusin 1; MFN2, mitofusin 2; mTOR, mammalian target of rapamycin; NO₃, nitrate; OD, optical density; OPA1, dynamin‐like 120 kDa protein; p, phosphorylated protein; t, total protein. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. Content of mitochondrial and mitophagy‐related proteins in isolated mitochondria are not altered by 3 days of immobilization, whereas SS and IMF mitochondria display inherent differences

A–E, content of mitochondrial and mitophagy–related proteins in SS mitochondria (A–C) and IMF mitochondria (A, D and E) were not influenced by 3 days of immobilization regardless of nitrate consumption. A and F, at baseline in the control limb of standard water‐consuming mice, content of MFN1, MFN2, DRP1, Parkin, and LC3‐II was lower in IMF compared with SS mitochondria. G, protein FSR in IMF mitochondria was lower than in SS mitochondria (3 and 7 day control limb of standard water‐consuming mice). In B–E, individual values are shown as black circles (n = 5–8). MFN1, DRP1 and Parkin protein content in IMF mitochondria could not be quantified accurately for the effect of immobilization with or without nitrate owing to the low content of these proteins; therefore, MFN1, DRP1, and Parkin were only analysed in IMF mitochondria for SS vs IMF differences. Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. In F and G, individual values are shown as grey squares (n = 5–7 in F, n = 16 in G). Data were analysed using Student's paired two‐tailed t tests within each mouse (SS vs. IMF). ^* P < 0.05 vs SS mitochondria. Data in F are regraphed from B and D for comparative purposes between SS and IMF mitochondria. Data in G are regraphed from Figs 1G , 1H, 5F ,G for comparative purposes between SS and IMF mitochondria. Data are expressed as the mean ± SD. Abbreviations: 4‐HNE, 4‐hydroxynonenal; CI, OXPHOS complex I; CII, OXPHOS complex II; CIV, OXPHOS complex IV; COX IV, cytochrome c oxidase subunit 4; DRP1, dynamin‐related protein 1; FSR, fractional synthesis rate; H₂O, standard water; IMF, intermyofibrillar; LC3, microtubule‐associated proteins 1A/1B light chain 3; MFN1, mitofusin 1; MFN2, mitofusin 2; NO₃, nitrate; OD, optical density; PINK1, PTEN‐induced kinase 1; SS, subsarcolemmal. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5. Seven days of immobilization induces muscle atrophy and reduces protein synthesis rates, regardless of nitrate consumption

A–C, 7 days of single‐limb immobilization decreased muscle mass of the soleus (A and B) and gastrocnemius + plantaris (A and C) muscles compared with the control limb, an effect that was not prevented by dietary nitrate. D–G, immobilization decreased myofibrillar protein synthesis rates (D) and mitochondrial protein synthesis rates (E–G), regardless of nitrate consumption. Individual values are shown as black circles (n = 7–13). Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. Data are expressed as the mean ± SD. Abbreviations: Cav1, caveolin 1; COX IV, cytochrome c oxidase subunit 4; EDL, extensor digitorum longus; FSR, fractional synthesis rate; Gast, gastrocnemius; GLUT4, glucose transporter type 4; H₂O, standard water; Homog, whole‐muscle homogenate; IMF, intermyofibrillar; NO₃, nitrate; SERCA2, sarco/endoplasmic reticulum calcium ATPase 2; Sol, soleus; SS, subsarcolemmal; TA, tibialis anterior. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Dietary nitrate supplementation prevents immobilization‐induced reductions in mitochondrial respiratory capacity and mitochondrial protein content after 7 days of immobilization

A and B, 7 days of immobilization decreased maximal mitochondrial respiration in standard water‐consuming mice (A), and dietary nitrate attenuated this impairment (B). C–E, mitochondrial protein content was decreased after 7 days of immobilization in standard water‐consuming mice (C and D), but not nitrate‐consuming mice (D and E), while 4‐HNE was not altered in either group. Individual values are shown as black circles (n = 8 or 9). Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. Data are expressed as the mean ± SD. Abbreviations: 4‐HNE, 4‐hydroxynonenal; ANT1, adenine nucleotide translocase 1; C, control limb; CI, OXPHOS complex I; CII, OXPHOS complex II; CIII, OXPHOS complex III; CIV, OXPHOS complex IV; CV, OXPHOS complex V; COX IV, cytochrome c oxidase subunit 4; Cyt C, cytochrome c; G, glutamate; H₂O, standard water; I, immobilized limb; JO₂, oxygen consumption rate; NO₃, nitrate; OD, optical density; OXPHOS; oxidative phosphorylation; PM, pyruvate + malate; RCR, respiratory control ratio; S, succinate. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7. Mitochondrial bioenergetics are impaired after 7 days of immobilization, while dietary nitrate prevents this response

A and B, mitochondrial respiration after 7 days of immobilization was decreased across a range of ADP concentrations (A), despite a decrease in the apparent K _m for ADP (B). C and D, maximal (succinate‐supported; C) and submaximal (+100 μM ADP; D) mitochondrial ROS emission rates were increased in the immobilized limb of standard water‐consuming mice. E–H, dietary nitrate prevented all of these responses. Individual values are shown as black circles (n = 8–11). Data were analysed using Student's paired two‐tailed t tests within each group (control vs. immobilized limb). ^* P < 0.05 vs control limb. Data are expressed as the mean ± SD. Abbreviations: H₂O, standard water; JO₂, oxygen consumption rate; K _m, Michaelis–Menten constant; mH₂O₂, mitochondrial hydrogen peroxide emission; NO₃, nitrate. [Colour figure can be viewed at wileyonlinelibrary.com]