Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3

Abstract

Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD(+)/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD(+) precursor, was previously shown to boost NAD(+) levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD(+) levels are a promising treatment strategy for mitochondrial myopathy.

Keywords: NAD+; mitochondrial myopathy; nicotinamide riboside; treatment; unfolded protein response.

© 2014 The Authors. Published under the terms of the CC BY license.

Figures

Figure 1. Nicotinamide riboside (NR) induces mitochondrial biogenesis and effectively ameliorates morphological and genetic landmarks of mitochondrial myopathy

1. A Skeletal muscle NAD+ content in 23- to 27-month-old Deletors and WT mice (n = 5 in each group).

2. B Quantification of muscle fibers showing decreased COX activity in histochemical analysis of frozen sections of quadriceps femoris muscle (total fibers n = 2000 counted per mouse); diet started before disease manifestation (pre-manifestation, “pre”; Deletor NR n = 5; Deletor CD n = 4); diet started after disease manifestation (post-manifestation, “post”; Deletor NR n = 12; CD n = 11).

3. C Quantification of the overall histochemical COX activity in muscle. Total intensity of activity in the quadriceps femoris muscle measured (n = 6 in each group).

4. D Mitochondrial respiratory complex protein and ATPase protein amounts in quadriceps femoris muscle. Western blot analysis of post-manifestation NR- and CD-fed mouse groups. Representative results shown. CI, respiratory chain complex I, 39-kDa subunit; CII, complex II, 70-kDa subunit; CIV, complex IV, cytochrome c oxidase, COI subunit, 40 kDa; CV, complex V, ATPase, alpha subunit, 55 kDa. Tubulin indicates the loading control.

5. E–H Quantification of the respiratory chain complex amounts from Western blots (Fig 1D). (Deletor NR n = 8; Deletor CD n = 8, WT NR n = 6, WT CD n = 6).

6. I Multiple mtDNA deletions in quadriceps femoris muscle. Long-range PCR amplification. 16.6 kb: amplification product from full-length mtDNA; bracket: smear representing deleted mtDNA species; 549 bp, product from 12S rRNA gene in mtDNA, not carrying deletions and representing total mtDNA. Representative image (pre-treatment Deletor NR n = 5; Deletor CD n = 4; post-manifestation Deletor NR n = 12; Deletor CD n = 11).

7. J Quantification of mtDNA deletion load (H). Pre- and post-manifestation NR- and CD (pre-manifestation; Deletor NR n = 5; Deletor CD n = 4), (post-manifestation; Deletor NR n = 12; CD n = 11).

8. K mtDNA copy number in quadriceps femoris muscle. Quantitative PCR analysis, using beta-actin gene as a nuclear two-copy control gene (post-manifestation Deletor NR n = 12; Deletor CD n = 11, WT NR n = 8, WT CD n = 7).

9. L Citrate synthase (CS) activity in quadriceps femoris muscle (n = 5 in each group). Numbers above columns indicate P-values. All values are presented as mean ± s.e.m. (Student's t-test).

NR, nicotinamide riboside; CD, chow diet; Del, Deletor; WT, wild-type mice; pre, pre-manifestation age group; post, post-manifestation age group; COX, cytochrome c oxidase; CI-CIV respiratory chain complexes I-IV; CV, ATP synthase; AU, arbitrary units. Source data are available for this figure.

Figure 2. NR induces skeletal muscle mitochondrial mass and prevents the development of mitochondrial ultrastructural abnormalities in mitochondrial myopathy

1. A–C Deletor quadriceps femoris muscle ultrastructure on chow diet shows increased amount of swollen mitochondria with concentric crista abnormalities (arrows) especially in subsarcolemmal regions.

2. D–F Deletors on NR diet. Mitochondria with normal ultrastructure and dense matrices are prominent subsarcolemmally (D–F), especially around the vessels (E, arrows), and also intramyofibrillar mitochondria show increased volume and matrix density (D, arrows). NR-fed Deletors show high crista density (F).

3. G WT mice on CD, showing subsarcolemmal mitochondria (arrow).

4. H WT mouse on NR diet with remarkable accumulation of subsarcolemmal mitochondria with dense matrices, and high crista density.

5. I Quantification of mitochondrial crista density in Deletors and WT mice on CD or NR diets. In image analysis, a “measuring stick” of 1 μm, placed perpendicular to the crista lamellae, was used to count crista content. A total of 20 μm of mitochondrial length was calculated per each sample electron micrograph. n = 3 in each group.

Data information: Numbers above columns indicate P-values. All values are presented as mean ± s.e.m. (Student's t-test). Bars in electron micrographs indicate magnification. Del, Deletor; WT, wild-type mice; CD, chow diet; NR, nicotinamide riboside; V, blood vessel; E, erythrocyte.

Figure 3. Decreased brown adipose tissue (BAT) function in Deletors and the effects of nicotinamide riboside treatment for whole-body metabolism, BAT, and liver

1. A, B Oxygen consumption of post-manifestation NR- and CD-treated Deletor and WT mice. CLAMS® metabolic cage analysis, the final 24-h recording from a total of 72 h. “Light” indicates the hours of lights on, mice inactive. “Dark” indicates the dark hours, mice active. “Cold” indicates cold exposure at 4°C for 6 h. The P-values are from left to right: ** = 0.0092, *** = 0.001, ** = 0.0095.

2. C, D Carbon dioxide production, as in A–B. **P = 0.013.

3. E–H Lipid content of BAT in post-manifestation NR- or CD-fed Deletors and WT mice. Oil Red O lipid staining on frozen sections with hematoxylin–eosin counterstaining. Red indicates lipid pools in BAT. (E) Deletors on CD, (F) Deletors on NR, (G) WT on CD, (H) WT mice on NR diet.

4. I–L BAT ultrastructure in NR- or CD-fed Deletors and WT mice. Electron micrographs of Deletor (I, on CD; J on NR) or WT (K on CD, L on NR).

5. M BAT, quantification of mitochondrial crista density, as in Fig 2I.

6. N BAT, mtDNA copy number, post-manifestation NR- or CD-fed Deletors and WT mice. Analysis by quantitative PCR. (post, n = 12 Deletor NR, n = 11 Deletor CD, n = 8 WT NR, n = 7 WT CD).

7. O Liver, mtDNA copy number, post-manifestation study groups (n = 12 Deletor NR, n = 11 Deletor CD, n = 8 WT NR, n = 7 WT CD).

8. P–S Liver ultrastructure in NR- or CD-fed post-manifestation mice. Deletors (P, on CD; Q on NR) and WT mice (R on CD, S on NR). Arrows indicate ribosomes, accumulating in NR-fed mice to close contact with mitochondria. Representative electron micrographs.

Data information: The bars indicate magnification; data shown in A–D (two-way ANOVA); all values are presented as mean ± s.e.m. (Student's t-test).

Figure 4. Skeletal muscle of Deletor mice have enhanced mitochondrial unfolded protein response (UPRmt), expression of fatty acid oxidation enzymes, and deacetylation of FOXO1, a downstream target of Sirt1

1. A Skeletal muscle, total FOXO1 (total-F), and acetylated FOXO1 (ac-F) protein levels. Western blot analysis, Deletor and WT mice, on CD or NR (n = 4 for each group); tubulin as a loading control.

2. B Quantification of acetylated FOXO1, from Western blot results (n = 4 in each group).

3. C Quantification of total FOXO1 (B), from Western blot results (n = 4 in each group).

4. D–F Muscle mRNA expression of fatty acid transport and oxidation proteins CD36, ACOX1, MCAD (post-manifestation Deletor NR n = 12; Deletor CD n = 11; WT NR n = 8; WT CD n = 7; arbitrary units).

5. G Muscle UPRmt proteins HSP70, HSP60, ClpP in post-manifestation Deletors, and WT mice on NR- or CD-fed diet. Western blot analysis, beta-actin as a loading control.

6. H Serum FGF21 in Deletors and WT mice on CD or NR diet, post-manifestation (Deletor NR n = 12; Deletor CD n = 11; WT NR n = 8; WT CD n = 7). ELISA analysis.

Data information: All values are presented as mean ± s.e.m. (Student's t-test). Source data are available for this figure.