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. Author manuscript; available in PMC: 2017 Oct 19.
Published in final edited form as: Sci Transl Med. 2016 Oct 19;8(361):361ra139. doi: 10.1126/scitranslmed.aaf5504

Fig. 4. In vivo monitoring of NAD+ provides an efficacy marker for improvements in the phenotype of NR-treated mdx mice.

Fig. 4

Sixteen-week-old mdx mice received a dietary supplement with NR (400 mg/kg per day) for 12 weeks. (A) 31P MRS measurements of increasing NAD+ levels upon NR treatment in mdx mice (mdx, n = 5; mdx NR, n = 10). (B) The recovery of mdx muscle NAD+ levels was verified by the analysis of gastrocnemius extracts by mass spectrometry (n = 5). (C) PARP activity (n = 4) and total protein PARylation was reduced (CD, 1.00 ± 0.31; NR, 0.23 ± 0.07; P = 0.035), whereas (D) NAMPT (CD, 1.00 ± 0.06; NR, 1.20 ± 0.23) and NMNAT3 (CD, 1.00 ± 0.23; NR, 2.64 ± 0.30; P = 0.012) content increased, after NR treatment of mdx mice. (E) Unchanged ATP levels were measured by HPLC in gastrocnemius (mdx, n = 7; mdx NR, n = 10). (F) In vivo 31P MRS measurements of mitochondrial energetics revealed an improvement in the PCr/ATP ratio (mdx, n = 5; mdx NR, n = 10), unchanged resting ATPase activity (mdx, n = 5; mdx NR, n = 10), and a recovery of ATPmax (mdx, n = 5; mdx NR, n = 10) in NR-treated mdx mice. NR-treated mdx animals hence function at a reduced fraction of their capacity (ATPmax) to meet ATP demand. The enhancement of NAD+ metabolism with NR prevented improved (G) ATP5A (CD, 1.00 ± 0.15; NR, 1.18 ± 0.15), UQCRC2 (CD, 1.00 ± 0.24; NR, 2.21 ± 0.57), MTCO1 (CD, 1.00 ± 0.53; NR, 1.62 ± 0.16), SDHB (CD, 1.00 ± 0.38; NR, 2.96 ± 0.57; P = 0.023), and TOM20 (CD, 1.00 ± 0.15; NR, 2.63 ± 0.22; P = 0.002) protein levels, using HSP90 (D) or Ponceau loading control, (H) mitochondrial complexes as evidenced by blue native PAGE of isolated gastrocnemius mitochondria, using a Coomassie loading control, (I) CS activity in frozen gastrocnemius extracts (mdx, n = 9; mdx NR, n = 6), and (J) cytochrome c oxidase staining of soleus (arrow) and gastrocnemius (asterisks) muscle fibers. (K) NR increased the running distance and time during an endurance treadmill test in mdx mice (mdx, n = 9; mdx NR, n = 12). (L and M) Hematoxylin and eosin (H&E) and Masson’s trichrome staining of hearts from control C57BL/10 and mdx male mice at 16 months of age with and without NR treatment. Representative images of the (L) whole heart and (M) magnified sections showing necrosis and fibrosis (n = 4).