The Effects of Nicotinamide Adenine Dinucleotide (NAD) on Muscle Degeneration

Introduction

Nicotinamide adenine dinucleotide (NAD) is one of the most important coenzymes in the

eukaryotic cells. NAD and its dependent precursors are major components participating in many

crucial biochemical processes of cellular respiration and energy production in order for animals

to survive and thrive. Human muscular dystrophy and neuromuscular diseases, such as

Duchenne, Becker, Merosin-deficient muscular dystrophies, and mitochondrial myopathies, are

considered extremely dangerous, and are associated with premature death in nearly all patients

affected. Treatments for these diseases and disorders in general have rather been disappointing as

the efficacy of long-term therapy is yet to be established. Abnormal muscle degeneration or

myasthenia does not only appear in muscle dystrophy and neuromuscular diseases, but also

obesity, aging, and other livers and heart diseases, etc. The purpose of this literature review is to

examine studies regarding the correlation between the levels of nicotinamide adenine

dinucleotide (NAD) and the progress of abnormal muscle degeneration. Despite overall

inconclusiveness, combined current studies indicate that higher level of NAD helps slow down

the progress of abnormal muscle degeneration through improving the structures and activities of

muscles.

NAD and Muscle Cells (Myocytes)

Research has long reported that NAD deficiency in myocytes have association with

abnormal muscle degeneration, and that NAD-boosting strategies are capable of enhancing

mitochondrial biogenesis and metabolic functions in muscle tissues, especially skeletal muscles.

Sankhyan et al. conducted a diagnostic case study on a 12-year-old boy with calpainopathy, a

1
disease characterized by symmetric and progressive weakness of proximal (limb-girdle) muscles

caused by a deficiency of the enzyme calpain 3. Their patients obtained muscle biopsy showed

features of muscular dystrophy and lobulated fibers on reduced nicotinamide adenine

dinucleotide-tetrazolium (NADH-TR) staining (Sankhyan et al. 151). Similarly, Stllberger et al.

also conducted a diagnostic case study on a 32-year-old female patient with cardiac failure, who

recurrent creatine-kinase elevation and reduced NAD staining on muscle biopsy suggested

metabolic myopathy (Stllberger et al. 231). Both case studies observations support previous

scholars reports on the positive correlation of NAD and abnormal muscle degeneration. In

correspondence with previous reports, Goody et al.s zebrafish model of muscular dystrophy

experimentation indicates that nicotinamide riboside kinase 2b (Nrk2b)-deficient embryos, at

myotendinous junction (MTJ) loci where Laminin is not properly polymerized, muscle fibers

elongate into adjacent myotomes and are abnormally long (Goody et al. 820). According to

Goody et al., Nrk2b, a NAD-dependent precursor, plays a specific role in modulating subcellular

localization of discrete cell-matrix adhesion complexes (CMACs) components that in turn plays

roles in musculoskeletal development (Goody et al. 809).

Dynamic modulation of Activation Pathways

Research certifies that NAD in its oxidized (NADH) and reduced (NAD+) forms play

fundamental roles in transport activity and glycogen store on myocytes, specifically skeletal

muscle energy metabolism. M. Goody et al. proposed research on molecular mechanisms

underlying a novel pathway called Nrk2b that restores muscle structure and function in

dystrophic zebrafish. They found that abnormal muscle degeneration related diseases result from

mutations that disrupt adhesion of muscle fibers to their surrounding basement membrane (BM),

a substructure within the extracellular matrix (ECM) (Goody et al. 1). NAD+ and Emergen-C

2
treatment with the activation of the Nrk2b-mediated NAD+ biosynthesis pathway regulates

subcellular localization of Paxillin and focal adhesion kinase (FAK); resulting in downstream

signaling events that increase organization and structure of the BM microenvironment, which not

only significantly reduces muscle degeneration but also improves the swimming ability of

dystrophic zebrafish (Goody et al. 2). FAK and Paxillin are two well-known cytoplasmic

proteins that modulate dynamic adhesion. Diverse in test subjects to Kubwabo et al., H. Zhang et

al. studied the life span of laboratory mice. The study concluded that the reduction of the NAD+

pool reduces the cells' ability to activate the mitochondrial unfolded protein response (mtUPR)

and stress response prohibitins; while restoration of the NAD+ pool, through nicotinamide

riboside (NR), a NAD+ precursor, is able to delay senescence of not only muscle stem cells, but

also neural and melanocyte stem cells (Zhang et al. 1437). Both research findings suggest that

NAD, as reductant or oxidant, has significant contribution in stabilizing and improving muscle

cells.

In further study of human muscular dystrophy and neuromuscular diseases for more

effective treatments, scholars have conducted multitude researches and experimentations on

varies biochemical drugs and muscles cells types. Different in approach from M. Goody et al.,

Tokin et al.s research introduces another pathway of activation: placing SIRT1 at the crossroad

between energy homeostasis. The SIRT1 gene is a nicotinamide adenine dinucleotide (NAD+)-

dependent deacetylase that removes acetyl groups from various proteins and coordinate many

important cellular functions such as cell cycle, metabolism, apoptosis and autophagy, sensing

and responding to DNA damage. Current evidences illustrate that this pathway helps maintain

fiber strength and regeneration from damage in the skeletal muscle (Tokin et al. 74). J. Tonkin et

al. examined muscles strength and their adaptive capacity in heavily impair patients with skeletal

3
muscle diseases, especially those during aging. Although, issues concerning the role of SIRT1 in

pathogenesis remain unanswered, the researchers concluded that SIRT1 has influence on muscle

insulin resistance induced by acute glucose oversupply, providing evidences of increasing fiber

strength, and regeneration from damage in skeletal muscle (Tonkin et al. 375). Scientists have

long reported that aging associates with a reduced muscle strength and functionality that are the

consequences of mitochondrial dysfunction. In further confounding the role of SIRT1 in

potential longevity, Ho et al.s experimental data disputes J. Tonkin et al.s affirmation,

justifying that there is not enough evidence to reject the null hypothesis, which states that there is

no significant difference in the means of potential longevity between population receiving

SIRT1-abundant treatment and population receiving placebo. If SIRT1 genuinely has positive

influence on muscle strength and regeneration from damage, then abundant level of SIRT1

should improve muscle cells productivity and vigorous condition, therefore increasing their

potential longevity. Ho et al. performed experiment to determine whether the replicative lifespan

of human vascular smooth muscle cells (SMCs) is impacted by enhanced expression of SIRT1.

They infected proliferating human SMCs with retrovirus containing SIRT1 cDNA. The cultures

of adult SMCs were generally short-lived, and there was a modest (1.30 0.93-fold, P < 0.01)

increase in cumulative population doublings in SIRT1-overexpressing cells (SIRT1SMCs)

compared to vector-infected SMCs (Ho et al. 3083). Their p-value is less than the significance

level () of 0.05, suggesting that an increasing expression of SIRT1 in aging SMCs has little

effect on SMC lifespan, despite an otherwise aging-related decline in SIRT1 expression (Ho et

al. 3084).

The Effects of NAD

4
 NAD is a ubiquitous pyridine nucleotide that functions as an essential reduction-

oxidation (redox) cofactor in cellular metabolism. Recent preclinical studies present the potential

of NAD+ on increasing oxidative phosphorylation and improve metabolic health. Weijer et al.,

who experimented on mice with type II diabetes (T2D), stated that NR or NMN

supplementation in mice resulted in elevated NAD+ levels, sirtuin 1 (SIRT1) activation, and

beneficial adaptation in mitochondrial gene expression profiles. Longer-term NR or NMN

supplementation in mice fed a high-fat diet additionally improved mitochondrial function and

metabolic health (Weijer et al. 1195). Nicotinamide riboside (NR) and nicotinamide

mononucleotide (NMN) functions as precursors to NAD. In assent to the effectiveness of NAD

and its precursors with Weijer et al., Tokin et al. notes that SIRT1 has been involved in the

protection against metabolic disorders and cancers, and overall enhancement of life span (Tokin

et al. 372). As previously mentioned, this enhancement of life span theory contradicts with Ho et

al.s credence on potential longevity, where SIRT1-deficient mouse embryonic fibroblasts have

extended, rather than shortened, replicative lifespan (Ho et al. 3081). Yet, in consensus to Tokin

et al. on the potency of NAD+ in mitochondrial biogenesis, Goody et al. found that Nrk2b cell

autonomously modulates the molecular composition of CMACs in vivo, and that Nrk2b-

mediated NAD+ is required for normal Laminin polymerization at the MTJ. In yeast and human

cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative

salvage pathway (Goody et al. 820).

In contrast to Tokin et al. and Goody et al., Frederick et al.s investigation in the degree

which increasing NAD concentration exerts tissue-autonomous effects on oxidative metabolism

on Nampt+/WT mice, disagrees with previous affirmations on the positive correlation between

the levels of NAD and effective muscle functions. Frederick et al.s investigation results provide

5
evidence that a modest increase in the steady-state level of NAD is insufficient to improve

muscle oxidative function or ameliorate consequences of high fat diet feeding and does not alter

the NAD/NADH ratio (Frederick et al. 46). Mice between the ages of 3 and 9 months, was

induced to obesity with high fat diet (HFD) containing 60% calories from fat, and later received

NAM and NR as supplementation treatments. These treatments are homogeneous to those of

Weijer et al.s experimentation. The mices muscle performance was tested on treadmill,

voluntary wheel running, and grip strength, and their quadriceps muscle concentrations of redox-

sensitive metabolites were measured. Their one-way analysis of variance with Bonferroni's

multiple comparisons post hoc test (two-tailed unpaired Student's t-test) extracted p < 0.05,

indicating that theres not much improvement from the treatments. Expression of the transcript

for nicotinamide N-methyltransferase, which competes with NAMPT to metabolize NAM, was

low in skeletal muscle and unaffected by genotype (Frederick et al. 1555). Interestingly, they

did find that overexpression of the NAD-dependent deacetylase SIRT1 in the brain has recently

been shown to improve muscle physiology, whereas overexpression of the enzyme in the muscle

itself was ineffective (Frederick et al. 1557).

Conclusion

Despite overall controversies and different approaches from current studies, scholars

generally agree that higher level of nicotinamide adenine dinucleotide (NAD) has adequate effect

in decelerating the progress of abnormal muscle degeneration or myasthenia. The dynamic

modulation of activation for any particular pathways, and specific tissues or cell types that are

capable of responding to the change in levels of NAD and its precursors, are uncertain and

require further research. The combination of these findings suggests that evolution of different

classes of NAD-dependent enzymes are presumably to be accompanied by additional layers of

6
regulations, which are responsible for modifying the enzyme activities in different contexts. A

deeper understanding of the tissues and signals that mediate specific processes and effects will

greatly facilitate the development of NAD-boosting therapeutics to combat comorbidities of

muscular dystrophy and neuromuscular diseases, obesity and aging, etc. Muscle-specific

transgenic approach contributes progressive breakthroughs and understandings toward dissecting

the mechanisms, which NAD availability influences mammalian physiology in battling human

muscle-related diseases and disorders.

Works Cited

Frederick, David W, et al. Increasing NAD Synthesis in Muscle via Nicotinamide

Phosphoribosyltransferase is Not Sufficient to Promote Oxidative Metabolism. The Journal

of Biological Chemistry, vol. 290, no. 3, Jan. 2015, pp.1546-58. MEDLINE,

doi:10.1074/jbc.M114.579565. Web. 25 Sep. 2016.

Goody, Michelle F., et al. NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular

Dystrophy. NAD+ and Paxillin Rescue Muscular Dystrophy, vol. 10, no. 10, Oct. 2012, pp.

11-7. ScienceDirect. Web. 20 Sep. 2016.

Goody, Michelle F., et al. Nrk2b-mediated NAD+ Production Regulates Cell Adhesion and is

Required for Muscle Morphogenesis in Vivo: Nrk2b and NAD+ in Muscle Morphogenesis.

Developmental Biology, vol. 344, no. 2, 2010, pp. 809-26. ScienceDirect,

doi:10.1016/j.ydbio.2010.05.513. Web. 20 Sep. 2016.

7
Ho, Cynthia, et al. SIRT1 Markedly Extends Replicative Lifespan if the NAD + Salvage

Pathway is Enhanced. FEBS Letters, vol. 583, no. 18, 2009, pp. 3081-5. John Wiley & Sons,

Inc., doi:10.1016/j.febslet.2009.08.031. Web. 29 Sep. 2016.

Sankhyan, Naveen, et al. Progressive Weakness in a 12-Year-Old Boy. Journal of Clinical

Neuroscience, vol. 18, no. 12, Dec. 2011, pp. 1751-52. Churchill Livingston,

doi:10.1016/j.jocn.2010.07.134. Web. 21 Sep. 2016.

Stllberger, Claudia, et al. Complete Atrioventricular Block and Reversible Systolic

Dysfunction in left Ventricular Hypertrabeculation/Non-compaction with Metabolic

Myopathy. Cardiology in the Young, vol. 21, no. 2, Apr. 2011, pp. 229-32. MEDLINE

Complete, doi:10.1017/S1047951110001666. Web. 21 Sep. 2016.

Tonkin, Joanne, et al. SIRT1 Signaling as Potential Modulator of Skeletal Muscle Diseases.

Current Opinion in Pharmacology, vol. 12, no. 3, Jun. 2012, pp. 372-6. ScienceDirect, doi:

10.1016/j.coph.2012.02.010. Web. 20 Sep. 2016.

Weijer, Tineke Van De, et al. Evidence for a Direct Effect of the NAD+ Precursor Acipimox on

Muscle Mitochondrial Function in humans. Diabetes, vol. 64, no. 4, Apr. 2015, pp.1193-

1201. Cengage Learning, Inc., doi.org/10.2337/db14-0667. Web. 29 Sep. 2016.

Zhang, Hongbo, et al. NAD Repletion Improves Mitochondrial and Stem Cell Function and

Enhances Life Span in Mice. Science, vol. 352, no. 6292, Jun. 2016, pp. 1436-43. MEDLINE

Complete. Web. 24 Sep. 2016.