REVIEW Open Access

A need for NAD+ in muscle development,homeostasis, and agingMichelle F. Goody1 and Clarissa A. Henry1,2*

Abstract

Skeletal muscle enables posture, breathing, and locomotion. Skeletal muscle also impacts systemic processes suchas metabolism, thermoregulation, and immunity. Skeletal muscle is energetically expensive and is a major consumerof glucose and fatty acids. Metabolism of fatty acids and glucose requires NAD+ function as a hydrogen/electrontransfer molecule. Therefore, NAD+ plays a vital role in energy production. In addition, NAD+ also functions as acosubstrate for post-translational modifications such as deacetylation and ADP-ribosylation. Therefore, NAD+ levelsinfluence a myriad of cellular processes including mitochondrial biogenesis, transcription, and organization of theextracellular matrix. Clearly, NAD+ is a major player in skeletal muscle development, regeneration, aging, anddisease. The vast majority of studies indicate that lower NAD+ levels are deleterious for muscle health and higherNAD+ levels augment muscle health. However, the downstream mechanisms of NAD+ function throughoutdifferent cellular compartments are not well understood. The purpose of this review is to highlight recent studiesinvestigating NAD+ function in muscle development, homeostasis, disease, and regeneration. Emerging researchareas include elucidating roles for NAD+ in muscle lysosome function and calcium mobilization, mechanismscontrolling fluctuations in NAD+ levels during muscle development and regeneration, and interactions betweentargets of NAD+ signaling (especially mitochondria and the extracellular matrix). This knowledge should facilitateidentification of more precise pharmacological and activity-based interventions to raise NAD+ levels in skeletalmuscle, thereby promoting human health and function in normal and disease states.

BackgroundThe laws of thermodynamics state that energy remainsconstant within a closed system. Energy is neither creatednor destroyed; it just changes form. In biology, the flow ofenergy through organisms, tissues, and cells is a universalrequirement to sustain life. Energy in the form of dietarynutrients is metabolized into adenosine triphosphate(ATP) and other molecules that power necessary cellularprocesses. Therefore, energy is potentially the mostimportant information that cells interpret. As such, itwould logically follow that multiple, highly nuanced cellu-lar mechanisms for nutrient and energy sensing wouldevolve due to intense selective pressures. This exquisitetailoring of form to function is perhaps best exemplified inthe mitochondrial reticulum that facilitates quick energytransfer throughout the muscle cell [1]. Oxidized

nicotinamide adenine dinucleotide (NAD+) and itsreduced form, reduced nicotinamide adenine dinucleotide(NADH), are critical molecules involved in energy gener-ation because NAD+/NADH participate in oxidation-reduction (redox) reactions in the tricarboxylic acid(TCA) cycle. In this context, NAD+ is not destroyed, it isreversibly reduced. However, NAD+ is also a cosubstratein multiple post-translational modifications such as deace-tylation and ADP-ribosylation. These reactions result inconsumption of NAD+, necessitating replenishment ofcellular NAD+ stores. Thus, maintaining the balancebetween NAD+ degradation and biosynthesis is extremelyimportant for cellular homeostasis. In this review, we willdiscuss evidence and mechanisms of NAD+ function inskeletal muscle development, homeostasis, and disease.In addition to mediating posture and locomotion, skel-

etal muscle is a metabolically important organ. Skeletalmuscles sense, produce, store, and utilize nutrients andenergy transfer molecules that enable muscle to partici-pate in systemic, energy-expensive processes such astemperature regulation and immunity. Skeletal muscle is

* Correspondence: clarissa.henry@maine.edu1School of Biology and Ecology, University of Maine, Orono, ME 04469, USA2Graduate School of Biomedical Sciences and Engineering, University ofMaine, Orono, ME 04469, USA

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Goody and Henry Skeletal Muscle (2018) 8:9 https://doi.org/10.1186/s13395-018-0154-1

plastic and readily adapts to change. Examples of skeletalmuscle plasticity include hypertrophy in response toweight bearing exercise, atrophy in response to disuse,and fiber type switching due to changes in gene expres-sion, cellular metabolism, or innervation. It seems likelythat there is coordination between skeletal muscle’senergy use/sensing and the signaling pathways that regu-late its remarkable phenotypic plasticity.Muscle diseases have a negative effect on health, life-

span, and/or quality of life. Genetic muscle diseasescaused by mutations result in a variety of ultrastructuraldefects in muscle cells and progressive loss of musclemass and function via multiple different mechanisms.Inflammatory or metabolic diseases (such as diabetes,obesity, autoimmune diseases, cancer, and infections)can result in loss of skeletal muscle as well. Given theintegration and interdependence of the nervous andmuscular systems, neural disorders or injuries can alsoimpair muscle tissue structure and function. Addition-ally, skeletal muscle is lost as a natural part of the agingprocess, and this loss is exacerbated in a condition calledsarcopenia. Adequate skeletal muscle mass prior toillness, injury, or aging is predictive of recovery andhealthy aging (reviewed in [2]). Skeletal muscle performsa diverse set of crucial functions in organisms and pro-moting muscle health and exploiting its plasticity couldimprove locomotion, metabolism, and quality of life inmany disorders.In this review, we will discuss the recent data that

document conserved roles for NAD+ in skeletal muscledevelopment, regeneration, aging, and disease as well asinterventions targeting skeletal muscle and affectingNAD+ that suggest promising therapeutic benefits. Wewill also highlight gaps in our knowledge and proposeavenues of future investigation to better understand whyand how NAD+ regulates skeletal muscle biology.

Introduction to NAD+A role for NAD+ in cellular ATP production hasbeen known for approximately a century. NAD+ wasoriginally referred to as “cozymase” when first discov-ered as a hydrogen and electron acceptor and donorin redox reactions (reviewed in [3]). The metabolismof glucose in cells that can occur in either the pres-ence or absence of oxygen (aerobic/oxidative vs.anaerobic/glycolytic, respectively) requires NAD+.NAD+ plays roles in multiple, diverse cellular pro-cesses in addition to redox reactions. NAD+ is acosubstrate in deacetylation reactions as well as inmono- and poly-ADP-ribosylation. While NAD+ cancycle between its oxidized and reduced forms inredox reactions, it is cleaved when it functions as acosubstrate. Thus, NAD+ must be constantly re-synthesized from dietary precursors in order to

maintain cellular pools of NAD+ as well asappropriate NAD+/NADH ratios (Fig. 1). It is notentirely clear whether the critical aspect of NAD+biology in skeletal muscle is levels of NAD+ or theNAD+/NADH ratio [4]. For simplicity, we will gener-ally just refer to NAD+ levels.Given the large demand for NAD+ as a cosubstrate in

many cellular reactions, it has been estimated that gramsof NAD+ precursors would need to be ingested daily inorder to maintain physiological NAD+ concentrations[5]. As the recommended daily allowance of dietaryNAD+ precursors is only in the milligram range, thissuggests that there are multiple biosynthetic pathwaysthat generate NAD+ from different dietary precursorsand that the nicotinamide byproduct formed upon NAD+cleavage is efficiently recycled for NAD+ biosynthesis [6].NAD+ can be formed from dietary ingestion of the

amino acid tryptophan or forms of vitamin B3/niacin(nicotinic acid, nicotinamide, and nicotinamide riboside).Quinolinic acid, a metabolite of tryptophan, can also bea starting point for NAD+ biosynthesis (Fig. 1). The denovo pathway of NAD+ biosynthesis from tryptophaninvolves seven enzymatic steps. In contrast, the salvagepathways of NAD+ biosynthesis from niacin involve onlytwo or three enzymatic steps. Important enzymesinvolved in NAD+ biosynthesis are nicotinamide phos-phoribosyltransferase (NAMPT), which acts on nicotina-mide; NRK1 and/or NRK2, which act on nicotinamideriboside; and the NMNAT family of enzymes, which acton multiple substrates in de novo and salvage NAD+biosynthesis (Fig. 1).

Conserved roles for NAD+ as a mediator of healthNAD+ is linked to healthspan and lifespan in organismsseparated by millions of years of evolution. There is aconserved decline in the NAD+/NADH ratio as worms,mice, and humans age [7–9]. Lower levels of NAD+ andlower NAD+/NADH ratios are correlated with func-tional decline and diseases of aging and mitochondrialdysfunction, such as metabolic and neurodegenerativediseases. Enhancing NAD+ levels is sufficient to increaselifespan in worms [7, 10], and in worm and mousemodels of ataxia telangiectasia [11]. NAD+ levels arefurther implicated in human health in that dietary defi-ciencies in NAD+ precursors can cause pellagra. Pellagraresults in skin, gastrointestinal, psychological, and neu-rodegenerative symptoms and is fatal without B vitaminor other appropriate NAD+ precursor supplementation.Given the conserved decline of NAD+ with age and theability of NAD+ to increase lifespan in worms, it hasbeen hypothesized that the loss of skeletal muscle massand function due to aging or disease could be caused, atleast in part, by lower availability of NAD+. We will re-view roles for NAD+ in muscle development,

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homeostasis, disease, and aging and highlight interven-tions that restore NAD+ levels and ameliorate muscledysfunction.

NAD+ compartmentalization in skeletal muscleSkeletal muscle is energetically expensive. Skeletalmuscle cells take in glucose and store it as glycogen.When there is a need for more energy within musclefibers, glycogen is converted to glucose that is used togenerate ATP. Skeletal muscle cells can containthousands of mitochondria, which are where theaerobic/oxidative metabolism of glucose occurs. AsNAD+ is required as a hydrogen and electron acceptorand donor for aerobic cellular metabolism, NAD+localization to mitochondria is important for musclefunction. Mitochondria are the subcellular compart-ments that have the highest level of NAD+ in skeletalmuscle cells. However, 95% of the NADH in skeletalmuscle is also localized to mitochondria. Therefore, theNAD+/NADH ratio is relatively low in skeletal musclemitochondria despite there being high levels of NAD+[12–14]. The observation that mitochondria contain thegreatest amount of NAD+ in skeletal muscle cellsreflects the prioritization of redox and ATP-generatingreactions for muscle biology; however, NAD+ is involvedin many cellular reactions as discussed below.NAD+ localization to other organelles facilitates the

additional NAD+-consuming reactions that occur inmuscle homeostasis (Fig. 2). A cytosolic pool of NAD+participates in the anaerobic/glycolytic metabolism ofglucose into ATP. This type of cellular metabolism ismore rapid but less efficient in ATP generation than theaerobic/oxidative metabolism that occurs in mitochon-dria. Metabolites of cytosolic NAD+, such as nicotinic

acid adenine dinucleotide phosphate (NAADP), cyclicADP-ribose (cADPR), O-acetyl-ADPR (OAADPR), andADPR, regulate calcium ion channels which canpromote calcium influx from the extracellular environ-ment or calcium release from internal stores (reviewedin [6, 15, 16]), (Fig. 2). Like ATP, calcium ions arerequired for functionality of the contractile apparatus instriated muscle and calcium ions are also importantsecondary messengers in signal transduction events.Considering that skeletal muscle evolved a subcellularorganelle specifically designated for storage and rapidrelease of calcium ions (the sarcoplasmic reticulum), therole that NAADP, cADPR, OAADPR, and ADPR play inregulating calcium mobilization in skeletal muscle is aburgeoning area of research.In the nucleus, NAD+ is used as a cosubstrate for

sirtuin-mediated deacetylation and mono-ADP-ribosylation reactions as well as poly-ADP-ribosylationreactions carried out by PARP enzymes (Fig. 2). Sirtuinsregulate gene expression and protein function via deace-tylation of histones or other proteins, respectively.Sirtuin activity is generally beneficial for cellular andorganismal health [7, 17]. PARP activity is stimulated byoxidative stress [18, 19]. PARPs are involved in repair ofDNA damage. PARP-mediated repair of DNA damage iscertainly a beneficial process in times of nutrient excessand oxidative stress; however, PARP activity drasticallyreduces the pool of nuclear NAD+ and essentially out-competes sirtuins for their shared cosubstrate. There-fore, the reduction of oxidative stress and the genetic orpharmacological inhibition of PARPs can increase theNAD+ available for sirtuin-mediated deacetylation reac-tions. This has shown to be a promising therapeuticapproach for some muscle conditions [20–23].

Fig. 1 NAD+ biosynthetic pathways. NAD+ can be synthesized via de novo, Preiss-Handler, and multiple salvage pathways. NAD+ precursors andmetabolites are represented in gray boxes. The enzymes involved in each conversion are listed above the arrows. The cellular outputs regulatedby NAD+ or metabolites are listed in white boxes with black outlines. For explanation of the acronyms, please see the abbreviations list in themain body of the text

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Adhesion of muscle cells to their extracellular matrix(ECM) microenvironment is critical for muscle develop-ment and homeostasis (reviewed in [24]). Muscle fibers,muscle fiber bundles, and whole muscle are surroundedby ECM. One substructure within the ECM is the base-ment membrane. Basement membranes are specializedparts of the ECM that serve a myriad of functions, play-ing both structural and signaling/organizational roles.Laminin, a major component of basement membranes,is necessary for basement membrane assembly [25].NAD+ is found in the extracellular environment sur-rounding cells [6, 16], where it is a cosubstrate for ecto-ADP-ribosyltransferases. NAD+ is important for normalstructure of the extracellular microenvironment (Fig. 3).There is a membrane tethered, extracellular mono-ADP-ribosyltransferase, ART1, that ADP-ribosylates Integrinalpha7 (ITGA7) at multiple sites in a NAD+concentration-dependent manner [26–30]. This ADP-ribosylation by ART1 is not readily reversible and leadsto an increased laminin-binding affinity of Integrinalpha7beta1 in differentiated myotubes. Thus, this ADPribosylation event might be a protective mechanism thatincreases adhesion to laminin when the sarcolemma hasbeen compromised and NAD+ leaks into the extracellu-lar space [27]. Another example of NAD+ function inthe plasma membrane proximal compartment of musclecells involves NRK enzymes. Yeast and human NRK en-zymes participate in an alternative salvage pathway thatgenerates NAD+ [31]. NRK2 (aka MIBP2, ITGB1BP3)was found to bind to the cytosolic tail of Integrin beta1and regulate adhesion of cultured mouse muscle cells to

laminin-111 but not fibronectin [32]. We found thatzebrafish Nrk2b localizes to the myotendinous junctionand is required for proper organization of laminin-111in the developing myotendinous junction. ExogenousNAD+ rescues laminin integrity at the myotendinousjunction in Nrk2b-deficient zebrafish [33], indicatingthat zebrafish Nrk2b also functions to generate NAD+.Laminin organization at the myotendinous junction is anovel role for NAD+ and the mechanism is stillunknown. Taken together, the above data suggest thepotential of an additional pool of NAD+, the membraneproximal pool, that mediates skeletal muscle physiology(Fig. 3).NAD+ is also localized to vesicular compartments

within cells, such as the Golgi, endoplasmic reticulum,peroxisomes, and lysosomes. It is tempting to speculatethat compartmentalization of NAD+ allows for finerspatial and temporal control of NAD+-dependent signal-ing. To our knowledge, roles for NAD+ in function ofthese organelles in skeletal muscle have not beenelucidated and this represents a major area for futureresearch. We will focus on new data implicating themembrane proximal, mitochondrial, and nuclear/cyto-plasmic pools of NAD+ in skeletal muscle developmentand health.

NAD+ in muscle development and regenerationPeriods of muscle development and/or growth may becoordinated with nutrient availability. As NAD+ pro-vides cells with information about nutrient levels, it islikely that NAD+ is involved in regulating these cellular

Fig. 2 Compartmentalization of NAD+ pools in skeletal muscle. Diagram of a striated skeletal muscle fiber. NAD+ is localized to mitochondrial,nuclear, cytosolic, and membrane proximal pools in muscle cells. Additional NAD+ compartments not diagrammed here include vesicularcompartments. The NAD+/NADH ratio is higher in the nuclear and cytosolic compartments compared to the mitochondrial compartment. Theratio is unknown in the membrane proximal compartment in muscle. Enzymes that consume NAD+ and their relative subcellular localizations arefound within black or white boxes. Integrin receptors and membrane channels that transport NAD+ and calcium across the sarcolemma can beseen in the diagram

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transitions. A decrease in NAD+ levels is observed inmyotubes compared to myoblasts (both in C2C12s andprimary muscle cells) [34]. Thus, the current thinking isthat decreased NAD+ levels are permissive for muscledifferentiation. The regulation of muscle differentiationby NAD+ in cultured mouse myoblasts seems to bemediated by SIRT1 activity. SIRT1 represses muscledifferentiation: SIRT1 complexes with myoD and theacetyltransferase p300/CBP-associated factor (PCAF) toinhibit muscle gene transcription in vitro [34]. Fulco andcolleagues hypothesized that nutrient sensing of glucosewould be integrated with NAD+ levels because this

would provide a mechanism where nutrition and myo-genesis could be coordinated [4]. They found that energysensing feeds into NAD+ levels by modulating the levelsof NAMPT. NAMPT activity is promoted by AMPK,which is increased with glucose restriction. Thus, glu-cose restriction activates NAMPT activity, resulting inincreased NAD+, increased SIRT1 activity, and repres-sion of muscle differentiation [4] (Fig. 4). There is noevidence that primary (embryonic) myogenesis requiresmodulation of NAD+ levels in vivo. There is, however,data indicating that secondary (fetal) myogenesis mayintegrate nutrient sensing with NAD+ levels and SIRT1

c

a b

e e1d d1

Fig. 3 Hypothesized mechanism of membrane proximal NAD+ action in muscle-ECM adhesion. (A–B) Model of membrane proximal NAD+regulation of subcellular protein localization, post-translational modification, laminin-binding affinity, and ECM organization in muscle. (A)Damage scenario. NRK enzymes localize to cytoplasmic tails of Integrin receptors for laminin and generate an intracellular membraneproximal pool of NAD+. In response to damage, intracellular membrane proximal NAD+ generated by NRK enzymes is actively transportedor leaks across the sarcolemma into the extracellular environment. Extracellular membrane proximal NAD+ is consumed by ART enzymesas ADP-ribose moieties are added to Integrin receptors for laminin in mono-ADP-ribosylation reactions. Intracellular localization of Paxillinto cell-matrix adhesion complexes is disrupted. Extracellular organization of laminin is disrupted. (B) NAD+-mediated damage responsescenario. Due to the movement of intracellular membrane proximal NAD+ to the extracellular environment, ADP-ribosylation of Integrinreceptors for laminin change to a high-affinity binding conformation. Laminin organization is increased, Paxillin localization to cell-matrixadhesion complexes is restored, and Integrin-laminin binding is augmented. (C–E) Reprinted with permission from [33]. c Model ofgenetic mosaic experiment to determine if wild-type Nrk2b is sufficient in a cell autonomous manner for subcellular localization of beta-Dystroglycan and/or Paxillin to cell-matrix adhesions. Fluorescent dextran labeled wild-type cells were transplanted into a Nrk2b-deficientbackground and subcellular localization of beta-Dystroglycan and Paxillin were determined by immunohistochemistry. (D–E) Side mount,anterior left, 26 hpf nrk2b morphant hosts with transplanted wild-type cells (red) and beta-Dystroglycan (blue) or Paxillin antibody staining(green). (D-D1) Beta-Dystroglycan robustly localizes to MTJs in nrk2b morphants in the presence and the absence of transplanted wild-type cells. (E-E1) Robust Paxillin localization to cell-matrix adhesion complexes in nrk2b morphants is rescued in the transplanted wild-type cells, but not in the Nrk2b-deficient cells surrounding them. Therefore, Nrk2b is cell autonomously sufficient for subcellularlocalization of Paxillin, likely through generation of membrane proximal NAD+

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activity to mediate muscle growth in vivo. Rats born tocalorie-restricted mothers are smaller due to deficits insecondary myogenesis [35]. These data support thatmuscle differentiation and growth are linked to nutrientavailability via NAD+. However, the mechanisms regu-lating which signal transduction cascades and whichprotein-protein interactions are invoked downstream ofNAD+ inputs are unclear. There are additionalunanswered questions regarding roles for NAD+ inmodulating SIRT1 activity during muscle developmentand growth, such as how are NAD+ levels decreased toalleviate SIRT1-mediated repression of differentiation,and how are NAD+ levels restored after differentiation?One factor that may contribute to changes in NAD+

levels in the transition from myoblasts to myotubes isPARP-1 (Fig. 4). PARP-1 covalently attaches branchedADP-ribose chains to proteins using NAD+ as a sub-strate. Thus, PARP-1 is a major NAD+ consumer andNAD+ levels tend to be inversely related to PARP-1levels in multiple cell types. This phenomenon has beenobserved in C2C12 myotubes exposed to oxidativestress. H2O2 activated PARP-1 and a subsequentdecrease in NAD+ levels were observed [23]. DecreasedNAD+ did not reduce SIRT1 protein expression;however, SIRT1 activity, as measured by PGC1-alphaacetylation, was reduced [23]. PARP-1 levels are high inmyoblasts and lower in myotubes (C2C12 and a ratskeletal muscle line L6) [22]. Thus, changing levels ofPARP-1 likely play a role in SIRT1 activity during muscledifferentiation via modulation of NAD+ levels.An additional effect of PARP-1 levels is sensitivity to

oxidative stress. PARP-1 is induced by oxidative stressand plays a role in DNA repair. However, if leftunchecked, PARP-1 activity depletes NAD+ levels. Thisadversely affects electron transport and ATP synthesisand can lead to necrosis. It is also possible that the

downregulation of PARP-1 levels in developed myotubesprovides a mechanism for increased resistance to oxida-tive stress [22]. Inhibition of PARP-1 catalytic activityincreased protection against oxidative stress in C2C12myotubes and overexpression of PARP-1 sensitized thesecells to oxidant-induced injury. Thus, in the future it willbe interesting to determine mechanisms regulatingPARP-1 expression during muscle differentiation, to askwhether PARP-1 plays a significant role in differentiationby modulating NAD+ levels, and, if so, whether PARP-1interacts with nutrient sensing by AMPK.One question is whether muscle development and

muscle repair show similar responses to NAD+ levelsduring differentiation. Muscle repair is mediated bymuscle satellite cells that reside next to muscle fibersunder the basement membrane [36, 37]. These self-renewing cells are usually quiescent, but they proliferateand differentiate in response to muscle injury. Recentdata suggest SIRT1 plays a role in actively maintainingquiescence in satellite cells by repressing transcription ofmyogenic genes. Quiescent satellite cells use oxidative-phosphorylation (OXPHOS) to generate energy [38].Metabolic reprogramming is a hallmark of satellite cells,and Ryall and colleagues asked whether these cellsshifted their metabolic state when activated. Theyobserved that satellite cells switch from primarilyOXPHOS to increased glycolysis and glutaminolysiswhen activated and proliferating [38]. One consequenceof this metabolic shift is that, similar to muscle develop-ment, NAD+ levels decrease, SIRT1 activity decreases,and this change is permissive for myogenic gene tran-scription [38]. Inactivation of SIRT1 in satellite cellsresults in initially smaller mice with smaller musclefibers at postnatal day 9, although the mice recover bypostnatal day 14 [38]. In the future, it would be quiteinteresting to determine the mechanism of size recovery.

Fig. 4 Signaling pathways by which NAD+ regulates mitochondrial biogenesis and homeostasis in muscle. Arrows denote promotion and “T”ssignify inhibition of mitochondrial biogenesis or homeostasis. Interventions in green boxes promote NAD+ levels and interventions in red boxesdeplete NAD+ levels, thereby regulating mitochondria number and function. For explanation of the acronyms, please see the abbreviations list inthe main body of the text

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Taken together, these data indicate that muscle repairand muscle development both rely on changing NAD+levels and that this process mechanistically linksmetabolism/energy availability with differentiation inboth contexts.

Membrane proximal NAD+ in muscle developmentThe ECM protein laminin is necessary for primary myo-tome development. In mice, laminin is thought to act asa barrier that keeps myogenic cells inside the myotome.When laminin polymerization is compromised, myocytesdisperse into the sclerotome region [39]. Similarly, inlamb1 or lamc1 mutant zebrafish, some fast-twitchmuscle cells extend into adjacent myotomes rather thanstopping at myotendinous junctions [40]. Surprisingly,NAD+ biosynthesis plays a role in potentiating lamininorganization in the basement membrane at the myoten-dinous junction. Zebrafish deficient for Nrk2b exhibitdefects in laminin organization that are rescued withexogenous NAD+ supplementation. Although the mech-anism is not known, NAD+ appears to act via the lam-inin receptor Integrin alpha6 and promotes subcellularlocalization of Paxillin to cell adhesion complexes [33](Fig. 3). This suggests that, at least in zebrafish musclecells, the NAD+ synthesized in the membrane proximalcompartment during muscle development modulates theECM. Interestingly, there were no observed muscledefects in NRK2 knockout mice [41]. Analysis of NRK1and NRK2 single and double knock out cells in aprimary muscle culture system demonstrated someredundancy between NRK1 and NRK2 in salvage NAD+biosynthesis from NR [41]. It is possible that the func-tional redundancy between NRK1 and NRK2 observedin mouse muscle cells does not occur in zebrafish andcould explain the discrepancy in muscle developmentphenotypes between the two species. Regardless of thisdifference, NAD+ was subsequently implicated in C.elegans muscle development and function [42] and inmouse skeletal muscle health [43–46]. Therefore, a rolefor NAD+ biosynthesis in muscle development andfunction appears to be well conserved.

NAD+ and aging muscleAging is considered by some to be the tail end of devel-opment. If changes that occur throughout the lifespanare thought of as part of a developmental continuum,then it logically follows that developmental robustnessand plasticity likely impact the aging process. The loss ofskeletal muscle mass and function with aging is calledsarcopenia. Sarcopenia decreases quality of life andincreases the risk of metabolic diseases. Although thereis some controversy regarding whether age decreasesmitochondrial content and ATP production (reviewed in[47]), the data in general seem to suggest that

mitochondrial dysfunction is a major driver of muscleaging [48–52] (Fig. 5). As described above, mitochon-drial homeostasis is strongly influenced by NAD+ viaSIRT1. One key target of SIRT1 is PGC-1alpha, whichpromotes mitochondrial biogenesis [53]. SIRT1 alsopromotes mitochondrial biogenesis by potentiating mito-chondrial transcription of oxidative phosphorylationgenes in a PGC-1alpha-independent manner [54]. Thispathway involves stabilization of HIF-1alpha, and AMPKappears to mediate the switch between these two SIRT1-dependent pathways of mitochondrial biogenesis [54].NAD+ levels are reduced in aged muscle in rats, mice,and humans [9, 54, 55], leading to reduced SIRT1activity, and reduced mitochondrial homeostasis. Themechanisms by which NAD+ levels are modulated areof extreme interest because understanding these mecha-nisms could lead to approaches to slow or even halt theloss of muscle mass and function during aging. Here, wewill discuss recent insights into the mechanisms under-lying decreased NAD+ with aging and highlight inter-ventions that increase NAD+ and skeletal musclefunction.

NAMPT levels decrease during aging, increasing NAMPTimproves aged muscleIn aged rats, NAD+ levels have been shown to decreasedue to lower levels of the rate limiting enzyme in NAD+biosynthesis, NAMPT [56]. Knock out of NAMPT in skel-etal muscle of mice (mNKO mice) demonstrated thatNAMPT is required for muscle function [45]. NAD+ andATP levels were decreased by 85 and 60%, respectively, inmNKO mice. At 3 months, functional defects were notimmediately apparent on a treadmill test: mNKO micetired at similar rates to controls. However, when extensordigitorum longus (EDL) muscle was repeatedly electricallystimulated ex vivo, force generation was significantlyreduced [45]. This deficiency, apparent in response torepeated electrical stimulation, could be because mito-chondria have been found to recover more slowly in agedvs young mice, at least in whole lumbrical muscle [49].mNKO mice were only slightly smaller than wild-typesiblings; however, histological changes (smaller fiber diam-eter, increased Evans blue infiltration, and increasedcentral nuclei) were apparent at 3 months. Thesephenotypes worsened through time and were classified as“NAD+-deficient myopathy” [45]. Thus, mNKO miceappear to be a good model for investigating the effects ofreduced NAD+ on skeletal muscle biology in the contextof aging on a developmental continuum.NAMPT is not only necessary for muscle function, it

is sufficient to combat the decline in muscle perform-ance with aging. Overexpression of NAMPT in cardiacand skeletal muscle throughout life increased healthspan[45]. Overexpression of NAMPT in skeletal muscle

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resulted in improved exercise capacity in aged mice.NAMPT overexpression increased NAD+ levels and,because of this higher baseline, NAD+ levels in agedmice overexpressing NAMPT in skeletal muscle weresimilar to those of young control mice [45]. This wasdespite NAMPT overexpressing mice still experiencing adecline in NAD+ levels with aging. Therefore,interventions that bolster NAD+ levels prior to andduring aging, such as NAMPT overexpression, maintainthe functional capacity and promote the healthy aging ofskeletal muscle.So how can NAMPT levels be increased during aging?

Multiple studies in three different mammals indicatethat exercise increases NAMPT. Exercise trainingincreased NAMPT levels, NAD+, and SIRT1 activity inaged rats [56]. NAMPT protein was increased 16% inhumans in the leg that conducted one-legged knee-extensor exercises compared to the untrained leg [57].This result is quite compelling, but it suggests thatresistance training with the goal of increasing NAMPTexpression would need to target all muscle groups. Asimilar increase in NAMPT expression was observed inthe quadriceps of endurance-trained mice (this was theonly muscle examined) [57]. Electrostimulation of pri-mary skeletal muscle cells in vitro also increasedNAMPT expression [58]. One study found that exercisetraining increased SIRT3 expression but not NAMPTlevels in male rats [59]. The reason for this discrepancyis not clear. However, these studies mostly suggest thatexercise training or electrical stimulation could be atractable approach to increase NAMPT levels untilpharmacological interventions are identified/improved.Experiments elucidating the mechanisms regulating

the exercise-mediated increase in NAMPT expressionthus far implicate AMPK. Stimulation of AMPK is suffi-cient to increase NAMPT protein in skeletal muscle:

4 weeks of activating AMPK with the adenosinemonophosphate (AMP) analog AICAR (5-aminoimida-zole-4-carboxamide ribonucleotide) increased NAMPTexpression. AICAR treatment in AMPK knockout micedid not increase NAMPT expression [57]. These experi-ments showed that AMPK activation is sufficient toincrease NAMPT expression. However, a critical experi-ment showed that AMPK is not necessary for theexercise-induced increase in NAMPT expression in vivo:NAMPT expression was increased in endurance-trainedAMPK knockout mice [57]. In the future, it will be inter-esting to determine the AMPK-independent mechanismof increased NAMPT expression in an effort tomaximize NAD+ levels in muscle via exercise and/orpharmacological interventions.

Other approaches to increase NAD+ and SIRT1 activityduring agingA likely benefit of increased NAD+ availability in muscleduring aging is potentiation of SIRT1 activity. Manymethods of enhancing SIRT1 activity have been discov-ered and shown to have positive effects on musclehealthspan. Activation of SIRT1 by a small moleculeagonist, SRT2104, resulted in increased muscle mito-chondrial size and better endurance in aged mice [60].SRT2104 also reduced loss of muscle mass in the 2-week hindlimb suspension model of muscle atrophy[60]. Another method involves activating NADH-quinone oxidoreductase 1 (NQ01) with beta-lapachone[61]. NQ01 uses NADH as an electron donor and thusdepletes NADH and increases the NAD+/NADH ratio[62, 63]. Aged mice fed with beta-lapachone showedincreased oxygen consumption, increased heat gener-ation, and improved skeletal muscle mitochondrialbiogenesis [61]. Another approach to enhance NAD+levels is to provide intermediates in NAD+ biosynthesis.

a b

Fig. 5 Model of the pathological observations in aged muscle and their reversal by augmentation of nuclear and mitochondrial NAD+ levels.a Fewer mitochondria, fewer muscle stem cells, reduced levels of NAD+ in nuclei and mitochondria, and reduced nuclear SIRT-1 activity havebeen observed in aged muscle. b Repletion of NAD+ levels in nuclei and mitochondria via various interventions increases mitochondria, increasesmuscle stem cells, and increases nuclear and mitochondrial NAD+ levels thereby increasing SIRT-1 activity. One mechanism involves nuclearSIRT-1-mediated regulation of TFAM and PGC1alpha gene expression and their effect on mitochondrial metabolism

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Mice fed NMN for 12 months were more active and hadimproved oxidative metabolism without overt sideeffects [43]. The particularly interesting aspect of thisstudy is that microarray analysis did not detect acommon set of “NMN response genes” between differenttissues. This suggests that the response to enhancedNAD+ metabolism is tissue specific. Similar to the aboveexperiments, supplementation with NAM for 5 weeksenhanced SIRT1 activity in young and aged rats [64].However, combining exercise training with NAMsupplementation decreased SIRT1 levels [64]. NAM caninhibit SIRT1 and PARP-1 under stress conditions topreserve NAD+ levels. Thus, NAM supplementationmay not be as effective as other methods of enhancingNAD+ levels and SIRT1 activity in aging muscle.Modulating levels of the major NAD+ consumer

PARP-1 have also been shown to be effective in main-taining muscle function during aging. In young mice,isometric exercise increases activity of both SIRT1 andPARP-1, but SIRT1 then binds PARP-1 and inhibits itsactivity [20]. Thus, young mice are protected fromexcess activation of PARP-1 and the potential for necro-sis that NAD+ depletion can incur. In contrast, agedmuscle has higher PARP-1 activity, lower NAD+content, and lower SIRT1 activity, resulting in decreasedperformance. The higher PARP-1 activity is partially dueto increased acetylation of PARP-1 by GCN5 [20]. NAD+levels may also indirectly regulate PARP-1 activity. DBC1(deleted in breast cancer 1) binds and inhibits PARP-1 ina NAD+-dependent manner in vitro [65]. Whether thisoccurs in skeletal muscle is unknown, but this could be anadditional mechanism that regulates PARP-1 activityduring aging. Inhibition of PARP-1 by injecting the inhibi-tor PJ34 in aged mice increased NAD+ content, SIRT1activity, muscle mitochondrial biogenesis, and perform-ance [20]. PGC-1alpha was not required, which raises thequestion of whether the increased mitochondrial biogen-esis is due to stabilization of HIF-1alpha. Taken together,these results suggest that regulation of NAD+-dependentenzymes, such as decreasing PARP-1 activity, decreasingGCN5 activity, or activating SIRT1, could prevent or slowthe age-associated decline in skeletal muscle mass andfunction.

NR supplementation rejuvenates aged muscle stem cells(MuSCs)It is easy to imagine that there could be crosstalkbetween the cellular pathways involved in lifespan exten-sion. The mitochondrial unfolded protein response(UPRmt) is an adaptive pathway that mediates proteosta-sis in response to stress. Activation of UPRmt correlateswith longevity in worms and mice [66]. Thus, it isperhaps not surprising that gene expression analysis ofaged MuSCs showed reduced UPRmt, decreased cell

cycle, and increased proinflammatory secretome geneexpression [46]. NAD+ biosynthesis was found to feedinto the UPRmt response. Nicotinamide riboside (NR)treatment increased levels of UPRmt proteins, increasedMuSC number in young and aged mice, and quickenedmuscle regeneration after cardiotoxin-induced injury inboth young and old mice [44]. Importantly, infiltrationof fibro-adipogenic progenitors (FAPs) was decreasedafter injury in NR-treated mice. Knockout of SIRT1 inMuSCs abrogated the beneficial effects of NR, indicatingthat, in this context, NR acts through increased NAD+and the ensuing increase in SIRT1 activity [46]. TheECM microenvironment provides a niche that regulatesMuSC behavior [67]. As mentioned above, NAD+ playsa role in modulating organization of the ECM duringmuscle development [33]. Thus, one future directionwould be to determine if NR supplementation affectedMuSC responses by acting on the ECM in the MuSCniche. It would also be interesting to investigate whetherNAD+ bioavailability feeds into other pathways thatpromote longevity.

NAD+ in myopathies and dystrophiesNormal aging and sarcopenia result in decreased muscleperformance; however, some individuals lose musclemass and function due to genetic muscle diseases.Muscular dystrophies and myopathies are stereotypicallycharacterized by muscle wasting, weakness, andimpaired locomotion (Fig. 6). Although significant pro-gress has been made with regard to identifying diseasecausing genes and pathological mechanisms, these con-ditions are still incurable. One major challenge thatremains is to understand if therapeutic crossover existsbetween the treatments for Duchenne/Becker musculardystrophy (DMD) and the other types of musculardystrophies. This is an important question becausetherapeutic strides are being made for the most commontype of muscular dystrophy, DMD, but treatment formost other types of muscle diseases lags behind. There-fore, in the future it is critical to determine whetherdifferences in cellular pathology affect treatment options.Given the critical role of NAD+ in muscle development,homeostasis, and aging, it is tempting to speculate thatpotentiating NAD+ biosynthesis could be a beneficialadjuvant therapy for a broad spectrum of musculardystrophies and myopathies.Resveratrol is a grape polyphenol that has received

attention for its potential to extend lifespan. Resveratrolcan directly activate SIRT1 [68] as well as indirectlyactivate SIRT3 through increasing the mitochondrialNAD+/NADH ratio [69]. Thus, resveratrol may have thepotential to improve muscle function in aging and/ordisease. Resveratrol was tested for possible efficacy inthe mdx mouse model of DMD [70]. Thirty-two weeks

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of resveratrol supplementation resulted in reductions inboth muscle mass lost and fibrosis. The decreasedfibrosis presumably resulted from reduced transcriptionof collagen 1a1, 1a2, and fibronectin [70]. Resveratrolwas provided in the diet; thus, it is not known in whichtissues/cell types resveratrol functions to reduce fibrosis.Angiotensin-II is known to promote fibrosis viaNAD(P)H oxidase-generated reactive oxygen species(ROS) in myoblasts [71]. These results suggest thatmyoblasts, in addition to fibroblasts and macrophages,can promote fibrosis. Given that CD45+ inflammatorycells were still observed in mdx muscle despiteresveratrol treatment [70], it would be interesting todetermine whether resveratrol reduced transcription

of ECM genes in immune cells, myofibroblasts, orboth.Another method of increasing NAD+ biosynthesis was

shown to be beneficial in the mouse model of DMD.NAD+ repletion with NR supplementation improvedmuscle function in mdx mice [44]. At least some of thisimprovement is likely due to the NR-mediated increasein number and function of MuSCs in mdx mice [46].Taken together, these data suggest that NAD+ repletion,via multiple mechanisms, is a worthwhile avenue toexplore in humans with DMD.Could NAD+ repletion benefit other types of muscular

dystrophies? As mentioned above, we showed thatNrk2b is necessary for normal muscle ECM

c e f

d

a b

Fig. 6 Cellular and molecular mechanisms of amelioration of muscle disease due to NAD+ repletion. (A, B) Model of pathological changesobserved in animal models of diseased muscle (A) and the improvement in these disease phenotypes due to provision of dietary NAD+precursors or NAD+ (B). (C, D) Anterior left, dorsal top, side-mounted, 3 dpf embryos stained with phalloidin to visualize actin. Fiber detachmentis readily observed in dag1 morphants (C, white arrowheads), whereas dag1 morphants supplemented with NAD+ display less fiber detachment(D). (E, F) Transmission electron micrographs showing normal muscle ECM (white arrows), disrupted muscle ECM (red arrows), normal sarcomerestructure (white arrowheads), and disrupted sarcomere structure (red arrowheads) in a zebrafish model of muscular dystrophy with and withoutNAD+ supplementation. (E) Dag1-deficient zebrafish. (F) Dag1-deficient zebrafish supplemented with NAD+

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development [33]. Laminin in the basement membranewas disorganized in Nrk2b-deficient zebrafish andrescued with exogenous NAD+. We found that muscleatrophy in a zebrafish model of primary dystroglycano-pathy due to Dag1 deficiency is preceded by disorga-nized laminin in the muscle ECM. We asked whetherexogenous NAD+ would improve laminin organizationin Dag1-deficient zebrafish. We found that either sup-plementation with NAD+ or EmergenC, which containsvitamin B precursors to NAD+, improved lamininorganization, reduced muscle degeneration, andimproved swimming in Dag1-deficient zebrafish [72].We identified the Integrin alpha6 and alpha7 receptorsas well as intracellular localization of the focal adhesionprotein Paxillin as downstream mechanisms. Usinggenetic mosaic analysis, we showed that the ECM micro-environment plays a critical role in maintenance ofmuscle cell-ECM adhesion and fiber viability and thustissue homeostasis [72]. These results and others suggestthat understanding the mechanisms downstream ofNAD+-mediated ECM organization could lead to newtherapeutic approaches (Fig. 6).Increasing NAD+ bioavailability also appears to be

beneficial for a myopathy. NR supplementation delaysthe progression of mitochondrial myopathy by stimulat-ing the UPRmt as well as inducing mitochondrialbiogenesis [73]. Given the dynamics of mitochondrialremodeling in muscle homeostasis and disease, it seemscritical to understand mitochondrial dynamics in liveorganisms. Generation of tools that allow observation ofmitoflashes in living zebrafish provided insight into howmitoflashes change in development and disease [74]. Amitoflash is a burst of ROS that mediates mitochondrial-cytosol/nuclear signaling [75]. It is not currently knownwhether or how mitoflashes affect the progression ofmuscle disease. Transgenic zebrafish expressing thebiosensor mt-cpYFP [76] showed that mitoflashes areshorter early in muscle development but become longeras development proceeds [74]. Transgenic zebrafishmodeling DMD showed the expected decrease inmitochondria numbers, although whether this reductionwas due to decreased mitochondrial biogenesis was notinvestigated. Interestingly, the mitoflashes that wereobserved in zebrafish with muscle disease were longerthan those of wild-type zebrafish [74]. The signalingevents downstream of longer bursts of ROS are notknown, but do provide an interesting avenue of investi-gation to pursue in terms of muscle disease pathologyand progression.The above data show that increasing NAD+ levels,

whether by supplementing with resveratrol, NR, orEmergenC, is beneficial for multiple types of muscledisease. The mechanisms hypothesized to be responsiblefor ameliorating disease include increased: mitochondrial

biogenesis, UPRmt, MuSC numbers, and lamininorganization in the muscle ECM (Fig. 6). IncreasedNAD+ levels also correlated with reductions in bothfibrosis and FAPs. However, none of these studiesintegrated or looked for connections between all ofthe different facets of muscle phenotypic improve-ment. The ECM microenvironment is involved infibrosis as well as the health of muscle fibers andMuSCs. Thus, it would be interesting in the future toexamine whether there is coordination between mito-chondrial health and muscle ECM structure orwhether these are two entirely separate mechanismsof NAD+ action. It also seems important to investi-gate whether NAD+ supplementation is beneficialacross the spectrum of muscular dystrophies/myop-athies to determine if there are muscle diseases thatare resistant to NAD+ supplementation. There is onestudy suggesting that genotype may impact theresponse to exogenous NAD+. Dystrophic myoblasts(from mdx mice) showed an increase in ERK1/2phosphorylation in response to exogenous NAD+,whereas control myoblasts did not [77]. The physio-logical significance of these results is not known, butthey do highlight that NAD+ may have varying effectsin different genetic environments. Thus, addressingwhether activating NAD+ biosynthesis exhibits thera-peutic crossover for multiple dystrophies and myop-athies should be a priority. Understanding howmechanisms downstream of increased NAD+ causecertain muscle outputs should also be prioritized asthis could provide a way to elicit desired muscleresponses to NAD+ in different genetic backgrounds.

NAD+ in muscle metabolic diseasesSkeletal muscle mass and function are not only criticalfor daily life but also whole-body protein metabolismand thus play a critical role in metabolic diseases such asobesity and type 2 diabetes (reviewed in [2]). Metabolicdiseases are common and debilitating conditions. Giventhe roles of skeletal muscle and NAD+ in metabolicregulation, it is possible that NAD+ supplementationcould improve metabolic health.PARP-2 is a major NAD+ consumer and thus indir-

ectly affects SIRT1 activity and metabolic health bymodulating NAD+ levels [21]. High fat diet-fed micehave elevated PARP-2 expression in skeletal muscle,which would be predicted to act as a NAD+ sink andlower SIRT1 activity [21]. Therefore, PARP-2 inhibitionis a potential strategy to augment NAD+ levels inskeletal muscle in the context of obesity. Mohamed et al.conducted a microRNA (miR) screen to identify poten-tial miRNAs that modulate PARP-2 expression. Theyidentified miR-149 as necessary and sufficient todecrease PARP-2 expression, increase SIRT1 activity,

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and increase mitochondrial biogenesis in C2C12 cells[21]. Taken together, these results suggest that targetingeither PARP-2 expression or enhancing miR-149 expres-sion could be potential approaches to promoting meta-bolic health via NAD+ repletion in skeletal muscle.Interestingly, boosting NAD+ levels by 50% in mouse

skeletal muscle via tissue-specific overexpression ofNAMPT did not induce mitochondrial biogenesis orprotect these mice from the negative metabolic conse-quences of a high fat diet [78]. Therefore, augmentingNAD+ in tissues other than striated muscle is necessaryfor promoting mitochondrial function and oxidativemetabolism [78]. The tissues in which NAD+ repletionis required for whole body metabolic improvement areunknown and are important targets to elucidate formetabolic therapies.Human trials that analyze the consequences of

increasing NAD+ on muscle are limited. However, onerecent multicenter trial provided patients with type 2diabetes either placebo or the NAD+ precursor acipimoxfor 2 weeks [79]. Muscle biopsies allowed for examin-ation of muscle respiration and gene expression after thetreatment. Although mitochondrial DNA content didnot differ between the treatments, respiration was higherin acipimox-treated muscle. Upregulation of gene setsthat have been previously positively correlated withNAD+ supplementation were observed in this case [79].While there were side effects to this particular type ofNAD+ augmentation in these type 2 diabetics, these datasuggest that roles for NAD+ in skeletal muscle andmetabolic health are conserved in humans. These dataalso support the promise of pharmacologically or enzy-matically modulating NAD+ biosynthesis and/orutilization as a therapy for many human genetic andmetabolic disorders.

Future perspectivesWhy is it important to elucidate downstream mecha-nisms of NAD+ action if, in general, the studiespresented here all show benefits to NAD+ supplementa-tion? There is correlative evidence that increased NAD+can be deleterious in the context of multiple musclecancers. Higher NAMPT expression levels correlate withincreased tumor grade for leiomyosarcomas, as well asembryonal, alveolar, and pleomorphic rhabdomyosarco-mas [80]. Thus, it is critically important to learn down-stream signaling pathways affected by NAD+ signalingsuch that the benefits of NAD+ can be leveragedwithout potentially deleterious side effects. This is espe-cially true because NAMPT expression not onlycorrelates with tumor grade for muscle cancers but alsocorrelates with invasion, metastasis, and chemotherapy re-sistance for gastric, thyroid, and prostate cancers [81–83].Although current studies are focused on developing

NAMPT inhibitors that are administered along withnicotinic acid to help alleviate toxic side effects ofNAMPT inhibitors, it is likely that identifying downstreamtargets in normal and cancerous cells would lead toimproved efficacy with fewer side effects.

ConclusionsIt is clear that NAD+ plays a beneficial role in musclehealth. The mechanisms underlying promotion ofmuscle development and homeostasis by NAD+ are bestunderstood in the context of sirtuin regulation in thenucleus, mitochondria, and cytosol. The role of NAD+in other cellular compartments—particularly the vesicu-lar and membrane proximal compartments—in musclehealth is currently understudied. Future research willlikely delve into not only the mechanisms of NAD+action in these different compartments but also theinterplay and signaling between NAD+ pools within andbetween cells.

AbbreviationsADP: Adenosine diphosphate; ADPR: Adenosine diphosphate-ribose; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMP: Adenosinemonophosphate; AMPK: Adenosine monophosphate kinase; ART: ADP-ribosyltransferase; ATP: Adenosine triphosphate; C. elegans: Caenorhabditiselegans; C2C12: Immortalized mouse myoblast cell line; cADPR: Cyclic ADP-ribose; CD45: Cluster of differentiation 45; Dag1: Dystroglycan;DMD: Duchenne/Becker muscular dystrophy; DNA: Deoxyribonucleic acid;ECM: Extracellular matrix; EDL: Extensor digitorum longus; ERK1/2: Extracellular signal-regulated protein kinase 1/2; FAP: Fibro-adipogenicprogenitors; GCN5: Histone acetyltransferase GCN5; H2O2: Hydrogenperoxide; HIF-1alpha: Hypoxia inducible factor-1alpha; IL-6: Interleukin-6;ITGA7: Integrin alpha7; ITGB1BP3: Integrin beta1 binding protein3;L6: Immortalized rat skeletal myoblast cell line; lamb1: Laminin beta1;lamc1: Laminin gamma1; mdx: Mouse DMD model; MIBP2: Muscle integrinbinding protein2; miR: microRNA; mNKO: Muscle NAMPT knock out;MuSC: Muscle stem cell; NA: Nicotinic acid; NAAD: Nicotinate adeninedinucleotide; NAADP: Nicotinic acid adenine dinucleotide phosphate;NAD+: Oxidized nicotinamide adenine dinucleotide; NADH: Reducednicotinamide adenine dinucleotide; NADPH: Reduced nicotinamide adeninedinucleotide phosphate; NADS: NAD+ synthase; NAM: Nicotinamide;NAMN: Nicotinate mononucleotide; NAMPT: Nicotinamidephosphoribosyltransferase; NAPRT: Nicotinate phosphoribosyltransferase;NAR: Nicotinic acid riboside; NEFA: Nonesterified fatty acid; NMN: Nicotinamidemononucleotide; NMNAT: Nicotinamide nucleotide adenylyltransferase;NQO1: NADH-quinone oxidoreductase 1; NR: Nicotinamide riboside; NRK1/2: Nicotinamide riboside kinase 1 and 2; Nrk2b: Nicotinamide riboside kinase 2b;OAADPR: O-acetyl-ADPR; OXPHOS: Oxidative phosphorylation; PARP: Poly (ADP-ribose) polymerase; PCAF: p300/CBP-associated factor; PGC1-alpha: Peroxisomeproliferator-activated receptor gamma coactivator 1-alpha; PJ34: PARP inhibitorVIII; QA: Quinolinic acid; QAPRT: Quinolinate phosphoribosyltransferase;Redox: Oxidation-reduction; ROS: Reactive oxygen species; SIRT: Sirtuin;SR: Sarcoplasmic reticulum; SRT2104: Selective activator of SIRT1;TFAM: Mitochondrial transcription factor A; UPRmt: Mitochondrial uncoupledprotein response; VHL: Von Hippel-Lindau tumor suppressor

AcknowledgementsThe authors would like to thank the members of the Henry lab for reviewingthe manuscript. Thank you to Kell Fremouw for assisting with artisticrenderings.

FundingMarch of Dimes Foundation Award Number: #1-FY14-284, NIH R15HD088217.

Goody and Henry Skeletal Muscle (2018) 8:9 Page 12 of 14

Availability of data and materialsNot applicable.

Authors’ contributionsMFG and CAH both wrote, edited, and approved the final manuscript.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Received: 28 November 2017 Accepted: 22 February 2018

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