NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation
Authors: Xu Wang
Anthony A Sauve
Philip L Marshall
Dongryeol Ryu
Shama R Iyer
Richard M Lovering
Johan Auwerx
Kevin E Conley
David J Marcinek
Matthew D Campbell
Carles Cantó
Laurent Mouchiroud
Keir J Menzies
Amir Safi Ali
Eduardo R Ropelle
Karim Gariani
Gary M Knowels
Vincenzo Sorrentino
Bernard J Jasmin
Davi A G Mázala
Eva R Chin
Hongbo Zhang
Stéphanie Bellemin
Ludivine Walter
Journal: Science Translational Medicine (2016)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Neuromuscular diseases; inherited mutations; skeletal muscle weakness; degeneration; mRNA transcripts
Affiliations: Laboratory of Integrative and Systems Physiology, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland., Laboratory of Molecular Biology of Exercise, School of Applied Science, University of Campinas, CEP 13484-350 Limeira, São Paulo, Brazil., Department of Kinesiology, School of Public Health, University of Maryland, College Park, MD 20742, USA., Interdisciplinary School of Health Sciences, University of Ottawa Brain and Mind Research Institute and Centre for Neuromuscular Disease, Ottawa, Ontario K1H 8M5, Canada., Department of Radiology, University of Washington, Seattle, WA 98195, USA., Centre de Génétique et de Physiologie Moléculaires et Cellulaires, Université Claude Bernard Lyon 1, CNRS UMR 5534, 69622 Villeurbanne, France., Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD 21201, USA., Department of Pharmacology, Weill Cornell Medical School, New York, NY 10065, USA., Nestlé Institute of Health Sciences, 1015 Lausanne, Switzerland., Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada., Laboratory of Integrative and Systems Physiology, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.  .

Abstract:  Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr-/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. 

Retracted: No

PubPeer comments:
 Readers should become aware of a recent article that couldn t reproduce part of the results of Ryu et al., 2016  Complementary NAD+ replacement strategies fail to functionally protect dystrophin-deficient muscle https: skeletalmusclejournal.biomedcentral.com articles 10.1186 s13395-020-00249-y From Discussion:  However, both NAD-modulating strategies employed in our study failed to improve muscle function. **We were largely unable to reproduce the results of the Auwerx group, who observed a significant reduction of plasma creatine kinase and a nearly 50% protection from eccentric challenge in the same strain of MDX mice treated with NR for only 12u2009weeks [10].** The discrepancies may derive from the fact that Ryu et al. assessed hindlimb torque around the knee joint instead of the ankle, and administered an NR-triflate salt to mice, instead of the NR chloride salt used in all neutraceutical formulations. Nonetheless, our finding that neither GSK978A nor NR had any effect on the performance of MDX muscle over time raises the question of whether biochemical imbalance is pathologically relevant in the absence of a central structural component like dystrophin. The favorable pharmacokinetics and brain penetrance of GSK978A suggest that the compound may have better efficacy in certain neurodegenerative disorders, which feature NAD depletion, such as Cockayne syndrome or xeroderma pigmentosa [39]. These and other indications for small molecule NAD-modulators warrant further investigation. , 
 #1 In response to this comment, the publication by Frederick et al., 2020, confirmed our observations of NAD dysfunction in mouse models of muscular dystrophy. Our groups and others have determined that NAD dysfunction in muscle and other tissues can be deleterious during aging and disease and that nicotinamide riboside (NR) can be used as a treatment compound to boost NAD levels in tissues to improve functional endpoints, especially when related to tissue energy metabolism. Specifically related to muscular dystrophy, we have shown that when a high dose of NR is given to mdx mice, a mouse model of muscular dystrophy, we can replete muscle NAD levels over the course of a 12 week treatment . We demonstrated that this NR treatment regimen can protect from an acute bout of eccentric muscle damage, while also increasing molecular markers of mitochondrial metabolism, and improving the state of inflammation and fibrosis in different muscles including the heart of mdx mice. Importantly, Frederick et al. were not able to replete muscle NAD levels in mdx mice for either their acute study or for their chronic study when using NR or their other compound of interest namedu00a0GSK978Au00a0(Figures 3d, 3e and 6e). This suggests that muscle NAD stores were not affected by their treatments and thereby confounding their conclusions. In contrast to our treatments, that performed by Frederick et al. provided a low dose NR treatment to mdx mice for either 7 days or 20 weeks in drinking water that was refreshed weekly. It should be noted that once dissolved in water, NR degrades by about 50% in u00b1 48hrs, which is well known in the field and corresponds with our own observations. Beyond this issue, Frederick et al. dissolved NR chloride water at doses of 200mg kg, which would provide 176mg kg of NR to mdx mice. In our study we provided a diet formulation of NR triflate at 400mg kg to provide 252mg kg of NR; the food was refreshed every 2 days to avoid issues related to NR degradation. Despite no measurable changes in muscle NAD for any mdx treatments, the Federick et al. study assessed the phenotype of mdx mice by performing a test that appeared to be more of a test of muscle regeneration rather than a test of susceptibility to injury and therefore did not address the same muscle phenotype as our work. Specifically,u00a0they performed eccentric contractions in vivo then waited either days or weeks before doing tetanic contractions of muscleu00a0in vivo. This is contrasted to our study where we examined muscle susceptibility to injury during repeated eccentric contractions by simultaneously measuring the percent loss in muscle torque, which is a gold-standard test for mdx mice. Further to this, for eccentric contractions the Frederick et al. study subjected u201chindlimbs to a series of 40 lengthening contractile stimuli, consisting of a sub-tetanic stimulation of 100u2009Hz at 200u2009u03bcs pulse for 0.4u2009s, while the motorized footplate applied an eccentric rotational torque,u201d which was followed isometric titanic force measurements on different days. In our study, we obtained the maximal isometric torque for each animal where the pulse amplitude was adjusted to optimize twitch tension, and the optimal position of the leg was determined by measuring twitches at different lengths of the quadriceps. A maximal tetanic contraction was then obtained to determine maximal contractile tension (P0). Maximal tetanic contractions were then performed repeatedly over time, with the final tension expressed as percentage of P0.u00a0 With regards to their use of serum creatine kinase (CK) to measure muscle damage, the starting level (time 0) of serum CK for each of the mdx mouse cohorts exhibited dramatic variability, as the averages between mdx groups varied by about 25% from the lowest group to the highest group despite not yet starting treatments (Figure 4c). This may be due to the fact that animals included in the study had a rather wide age range of 7 to 9 weeks, which, as they correctly state, is near the most dynamic period of muscle necrosis and growth in mdx mice. Combined with this, there is a dramatic reduction in creatine kinase levels over the course of the study, despite one or more bouts of eccentric challenge, that may have contributed to the masking of any potential differences between cohort treatments. u00a0 In summary, the statement by Frederick et al. that u201cneither GSK978A nor NR had any effect on the performance of MDX muscle over time raises the question of whether [NAD] biochemical imbalance is pathologically relevantu201d, is in our opinion, compromised for the reasons explained above; the most important confounder being that their treatments were not able to recover the NAD imbalance in mdx muscle, in either acute or chronic experiments, making a functional improvement also improbable. This is a response formulated by Profs. Keir Menzies, Johan Auwerx, Dongryeol Ryu and Hongbo Zhang. ,