Accepted Manuscript

Small molecule nicotinamide N-methyltransferase inhibitor activates senescent

muscle stem cells and improves regenerative capacity of aged skeletal muscle

Harshini Neelakantan, Camille R. Brightwell, Ted Gfoch. Graber, Rosario

Maroto, Hua-Yu Leo Wang, Stanton F. McHardy, John Papaconstantinou,
Christopher S. Fry, Stanley J. Watowich

PII: S0006-2952(19)30046-2
DOI: https://doi.org/10.1016/j.bcp.2019.02.008
Reference: BCP 13414

To appear in: Biochemical Pharmacology

Received Date: 8 January 2019

Accepted Date: 7 February 2019

Please cite this article as: H. Neelakantan, C.R. Brightwell, T.G. Graber, R. Maroto, H.L. Wang, S.F. McHardy, J.
Papaconstantinou, C.S. Fry, S.J. Watowich, Small molecule nicotinamide N-methyltransferase inhibitor activates
senescent muscle stem cells and improves regenerative capacity of aged skeletal muscle, Biochemical
Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.02.008

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Small molecule nicotinamide N-methyltransferase inhibitor activates senescent

muscle stem cells and improves regenerative capacity of aged skeletal muscle

Harshini Neelakantan,1# Camille R. Brightwell,2,3# Ted G. Graber,4 Rosario Maroto,3

Hua-Yu Leo Wang,5 Stanton F. McHardy, 5 John Papaconstantinou,1 Christopher S.

Fry,3,6* Stanley J. Watowich1*

1Department of Biochemistry and Molecular Biology, University of Texas Medical

Branch, Galveston, Texas 77550 USA

2Department of Cell Biology, Neuroscience and Anatomy, University of Texas Medical

Branch, Galveston Texas USA

3Department of Nutrition and Metabolism, University of Texas Medical Branch,

Galveston, Texas USA

4Division of Rehabilitation Sciences, University of Texas Medical Branch, Galveston,

Texas USA
5Department of Chemistry and Center for Innovative Drug Discovery, University of

Texas at San Antonio, San Antonio, Texas USA

6Shriners Hospitals for Children, Galveston, Texas USA

#Co-first authors
*Corresponding authors
ABSTRACT

Aging is accompanied by progressive declines in skeletal muscle mass and strength

and impaired regenerative capacity, predisposing older adults to debilitating age-related

muscle deteriorations and severe morbidity. Muscle stem cells (muSCs) that proliferate,

differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are

increasingly dysfunctional upon aging, impairing muscle recovery after injury. While

regulators of muSC activity can offer novel therapeutics to improve recovery and reduce

morbidity among aged adults, there are no known muSC regenerative small molecule

therapeutics. We recently developed small molecule inhibitors of nicotinamide N-

methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles

and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity,

and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi)

treatment will rescue age-related deficits in muSC activity to promote superior

regeneration post-injury in aging muscle. 24-month old mice were treated with saline

(control), and low and high dose NNMTi (5 and 10 mg/kg) for 1-week post-injury, or

control and high dose NNMTi for 3-weeks post-injury. All mice underwent an acute

muscle injury (barium chloride injection) locally to the tibialis anterior (TA) muscle, and

received 5-ethynyl-2’-deoxyuridine systemically to analyze muSC activity. In vivo

contractile function measurements were conducted on the injured TA muscle and

tissues collected for ex-vivo analyses, including myofiber cross-sectional area (CSA)

measurements to assess muscle recovery. Results revealed that muscle stem cell

proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting

nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of
larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared

to controls. Prolonged NNMTi treatment post-injury further augmented myofiber

regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC

activity translated not only to larger myofibers after injury but also to greater contractile

function, with the peak torque of the TA increased by ~70% in NNMTi-treated mice

compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts,

where NNMTi treatment promoted and enhanced myoblast differentiation with

supporting changes in the cellular NAD+/NADH redox states. Taken together, these

results provide the first clear evidence that NNMT inhibitors constitute a viable

pharmacological approach to enhance aged muscle regeneration by rescuing muSC

function, supporting the development of NNMTi as novel mechanism-of-action

therapeutic to improve skeletal muscle regenerative capacity and functional recovery

after musculoskeletal injury in older adults.

Keywords: muscle regeneration, aged muscle, therapeutics, nicotinamide N-

methyltransferase, inhibitor, muscle stem cells, satellite cells

1. INTRODUCTION

The population of older (60+ years of age) adults is rapidly expanding in the United

States and throughout the world, placing ever-increasing strains on health care

resources and an urgent need for improved approaches to elder care [1]. One of the

most significant impacts of aging is the progressive decline in skeletal muscle mass and

strength [2, 3], with concomitant deteriorations in physical function and mobility that are

strongly associated with numerous chronic diseases and increased mortality [4, 5].

While all older individuals experience muscle degeneration, approximately 30% of

adults over 60 years of age and 50% of adults over 80 years of age develop sarcopenia,

a geriatric disease characterized by significant and objective defects in muscle mass,

strength, and function [6-8]. Sarcopenic elderly individuals are at a 2- to 5-fold increased

risk for permanent disability and greatly diminished quality of life arising from

progressive muscle degeneration, decreased muscle function, and poor muscle quality

that predispose them to debilitating falls and substantial disease burden [4].

Furthermore, as muscle regenerative capacity of older adults becomes increasingly

compromised, it leads to delayed and impaired recovery following muscle injury [9-11],

decreased mobility and independence, increased hospitalization costs [12], and higher

mortality rates [13].

Muscle stem cells (muSCs; also termed satellite cells) are responsible for

mediating skeletal muscle repair by proliferating and differentiating into fusion-

competent myoblasts and facilitating myofiber regeneration following injury [14-16]. The

major driving factor for delayed and impaired recovery of aged muscle following injury

appears to be a significant decrease in muSC regenerative capacity and function [10,

11, 17]. These events occur independent of sarcopenia [18], resulting in the formation of

dysfunctional senescent muSCs that are no longer activated by muscle injury or

turnover stimuli and present compromised ability to proliferate, differentiate, fuse, and

promote the repair and replacement of myofibers [19, 20]. Additionally, muSC

abundance has been observed to decline in some aged skeletal muscle tissues [21] as

muSCs develop diminished intrinsic capacity for reversible quiescence, which further

reduces the population of muSCs supporting the post-injury repair cascade [19].

Applications of muSCs in promoting muscle repair have been preclinically investigated

[16]; however, there are no approved pharmacological therapeutics that can safely and

effectively enhance muSC activation and regenerative capacity to promote recovery

from injury among aging adults.

Nicotinamide adenine dinucleotide (NAD+) is a fundamental cellular regulator of

energy metabolism, mitochondrial function, and bioenergetics [22, 23] that declines with

age [24-26]. Age-related diminished NAD+ contributes to altered skeletal muscle

metabolism and mitochondrial function [25, 27], progressive muscle degeneration and

loss of function [28], which further link to increased muSC senescence, reduced muSC

function, dysfunctional muscle regeneration, and impaired muscle repair [29, 30].

Recently, several studies have observed that nutraceutical supplements such as

nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide

analogues (e.g., acipimox), which increase intracellular NAD+ levels and stimulate

muscle NAD+ biosynthetic pathways (e.g., NAD+ salvage pathway), can improve muscle

metabolic, mitochondrial, and muSC function, and accelerate muscle regeneration in

aged mice [29, 31, 32]. Thus, pharmacologically enhancing NAD+ levels in aging
skeletal muscle tissues may provide a viable approach to improve the regenerative

function of aged muSCs.

The cytosolic enzyme nicotinamide N-methyltransferase (NNMT) has been newly

discovered to modulate the levels of nicotinamide precursors required for NAD+

biosynthesis, and thus plays a crucial role in regulating the NAD+ salvage pathway and

cellular metabolism [33]. NNMT is expressed in skeletal muscle, with progressively

greater expression associated with aging in muscle tissues [34, 35]. Importantly, NNMT

is a dominant component of the gene expression signature for sarcopenia [36],

overexpression of which could lead to significantly reduced levels of NAD+ and

associated declines in muscle mass, strength, and function that accompany aging [37,

38]. We recently discovered and developed first-in-class small molecule inhibitors of

NNMT that have amenable drug-like properties and selectively inhibit NNMT activity in

vivo [38, 39]. In the present study, we generated proof-of-concept data demonstrating

the efficacy of a novel NNMT small molecule inhibitor to accelerate muscle regeneration

in an aged mouse muscle injury model. Our results indicated that treatment of aged

mice with a small molecule NNMT inhibitor (NNMTi) enhanced proliferation, fusion, and

regenerative capacity of muSCs, subsequently increasing functional performance of

skeletal muscle. Similar results were demonstrated in vitro with C2C12 cells that closely

mimic muSCs, where NNMTi treatment promoted myoblast differentiation and

supporting metabolic changes in NAD+ salvage pathway-related cellular metabolites. To

the best of our knowledge, this is the first study to convincingly demonstrate that NNMT

inhibition rescues age-related muSC deficits and serves as a suitable therapeutic

approach to reactivate skeletal muSCs in aging skeletal muscle, thus mitigating age-
associated impaired muscle regeneration and improving skeletal muscle remodeling

and function following injury.

2. MATERIALS AND METHODS

2.1 Chemicals. NNMT inhibitor (NNMTi), 5-amino-1-methylquinolium was synthesized

by a previously established synthetic method [39]. Internal standard (IS) for LC/MS/MS

studies was a deuterated 5-amino-1-methylquinolinium analogue that was produced

using a modified synthetic scheme [39]. Other standards for LC/MS/MS studies were

purchased from established commercial suppliers; S-(5′-Adenosyl)-L-methionine (SAM),

NAD+, nicotinamide (NA), and NADH (reduced form of NAD+) were obtained from

Sigma-Aldrich (St. Louis, MO, USA). 1-methylnicotinamide (1-MNA) and S-Adenosyl-L-

homocysteine (SAH) were obtained from Cayman Chemical Company (Ann Arbor, MI,

USA).

2.2 Quantitative measurement of NNMT expression in young and aged muscle

tissue. 20-40 mg of tibialis anterior (TA) muscle tissue were collected from young (4-

month-old) and aged (24-month-old) C57Bl/6 mice, pulverized on ice, added to RIPA

buffer (Cell Signaling Technology, Danvers, MA, USA; product #9806) containing

protein phosphatase inhibitor cocktail (P-5726, Sigma Aldrich, St. Louis, MO) (1:10 w/v),

and homogenized using a hand-held tissue homogenizer (Tissue-TearorTM, Model

985370, BioSpec Products, Inc., Bartlesville, OK, USA). Homogenized samples were

centrifuged at 10,000 g for 10 min at 4°C. Supernatants were collected and stored at -

20°C until further processing. Protein concentration in samples were determined using
the bicinchoninic acid (BCA) protein assay (PierceTM BCA Protein Assay Kit, Thomas

Scientific, Swedesboro, NJ, USA). Briefly, 40 μg of tissue homogenates from young and

aged TA muscle samples were separated using sodium dodecyl sulfate polyacrylamide

gel electerophoresis (SDS-PAGE). Separated proteins were transferred onto a

polyvinylidene fluoride (PVDF) membrane and probed for NNMT protein expression

using anti-NNMT primary antibody (ab58743, rabbit polyclonal; Abcam, Cambridge, MA,

USA) and HRP-conjugated polyclonal goat anti-rabbit secondary antibody (ab205718;

Abcam) via western blotting. Membrane was striped and re-probed for β-actin using

anti-β-actin primary antibody (ab101173, monoclonal mouse; Abcam) as the loading

control. Western blots were analyzed for NNMT and β-actin expression using ImageJ

software (NIH). NNMT protein expression (bands detected at ~27 kDa) was normalized

to β-actin expression (bands detected at ~42 kDa) and compared between young and

aged TA muscle tissues. Samples were run and analyzed in technical duplicates.

2.3 Efficacy of NNMTi to activate and improve regenerative capacity of muSC in

an aged mouse muscle injury model. Aged, 24-month-old (N=48), male C57Bl/6 mice

were obtained from the National Institute of Aging (NIA). Upon arrival, mice were single-

housed and allowed to acclimate to the controlled environment vivarium maintained at a

constant temperature (21-23°C) and humidity (40-50%) on a 12-hour light-dark cycle

(lights on 0600-1800 h) for at least 7 days before initiation of experiments. Food and

water were available ad libitum. All experiments were carried out in accordance with the

Guide for the Care and Use of Laboratory Animals and with approval from the

Institutional Animal Care and Use Committee at the University of Texas Medical Branch .
2.3.1. Short-term treatment effects of NNMTi on muscle regeneration after injury.

Following acclimation, mice were randomized into control (saline; n=13), low dose

NNMTi (5 mg/kg; n=10), and high dose NNMTi (10 mg/kg; n=13) treatment groups,

ensuring similar mean average body weights across the three cohorts. Mice received

twice daily (BID) subcutaneous (SC) injections of saline or NNMTi for 2 weeks (1 week

pre-injury and 1 week post-injury). Mice were weighed and the body weights were

recorded every other day. On day 8 (following 1 week of treatment), mice were briefly

anesthetized using isoflurane and 30 L of barium chloride (BaCl2) toxin (1.2%

concentration) was injected locally into the TA muscle of one hindlimb to induce muscle

injury, followed by carprofen for analgesia. Starting on the day of the injury (day 8) and

on days 10, 12, and 14, mice received a single intraperitoneal (IP) injection of 5-ethynyl-

2´-deoxyuridine (EdU) (150 ug in saline) to track muSC proliferation and fusion into

regenerating TA muscle fibers. All groups of mice continued to receive BID, SC

injections of saline or NNMTi on days 8 through 14. At the terminal end of the study, a

sub-cohort of mice from the 3 treatment groups (n=6/group) were deeply anesthetized

using ketamine/xylazine cocktail anesthetic (IP injection) and the blood was drawn by

cardiac puncture procedure, following which muscle tissues were harvested for

additional processing. Remaining mice (n=7/group in control and high dose groups)

were subject to TA contractile function measurements (measures described in detail

below) under isoflurane-induced anesthesia to assess muscle strength. Terminal bleeds

were performed on all remaining mice and the tissues harvested and flash frozen for ex-

vivo analyses as described below.

2.3.2. Long-term treatment effect of NNMTi on complete muscle recovery after

injury. A separate cohort of 24-month-old, aged mice were randomized into control

(saline; n=6) and high dose NNMTi (10 mg/kg; n=6) treatment groups, ensuring similar

mean average body weights across the two cohorts. In contrast to the short-term

treatment regimen (1-week post-injury treatment) described above, mice received twice

daily (BID) subcutaneous (SC) injections of saline or NNMTi for 4 weeks; pretreatment

for 7 days prior to BaCl2 injury to one TA muscle, followed by 3 weeks of post-injury

treatment to examine a more complete muscle recovery. Muscle tissues were harvested

and flash frozen for ex-vivo analyses as described below.

2.4 Effect of NNMTi on in vivo contractile physiology determined by dorsiflexor

torque measurement. This protocol was adapted and modified from previously

published literature [40, 41]. Briefly, mice were anesthetized with isoflurane (3-4% for

induction and ~2.5% for maintenance of anesthesia via a nose cone; oxygen maintained

at ~1.5 l/min with a VetEquip vaporizer), the BaCl2 injured leg trimmed (Wahl Bravmini;

Sterling, IL, USA), and set on a platform (Aurora Scientific 809c in situ testing

apparatus; Aurora, ON, Canada) heated to 37°C (with an Anova Industries Model 10

water circulator; Stafford, TX, USA). The leg was then mounted into a clamp to hold it

static at the knee, with the tibia perpendicular to the femur and the foot perpendicular to

the tibia. The foot was secured in a mount connected to the force transducer, with the

ankle wedged in place and with a piece of tape holding the foot to the mount. Platinum

needle electrodes were set percutaneously, approximately near the top of the knee and
halfway down TA, between the TA and the gastrocnemius (needles were adjusted to

find the optimal placing for maximum force production with minimum current and

minimal antagonistic muscle response). With an Aurora Physiology system (Model

6650LR Force Transducer, Dual Mode lever System, Hi power Bi-Phase Stimulator,

Signal Interface, and software: Dynamic Muscle Control v5.500 and Dynamic muscle

Analysis version 5.300), the proper maximal current used to stimulate muscle

contraction was determined by eliciting a twitch (pulse duration 0.2 s) with the stimulus

starting at 5 mA and increased incrementally to ~50 mA until the maximum twitch torque

at the minimum amperage needed to stimulate this torque was recorded. This minimum

current was then maintained throughout a torque-frequency curve to find the maximum

torque output (twitch1, 10 Hz, 40 Hz, 80 Hz, 100 Hz, 120 Hz, 150 Hz, 180 Hz, 200 Hz,

250 Hz and a final twitch2). A twitch1/twitch2 minimum ratio of 90% determined muscle

integrity post-procedure (<90% may indicate extreme muscle fatigue or damage). No

mice failed the twitch1/twitch2 test. Peak torque per gram of body mass and peak torque

normalized to CSA (average cross-sectional area of fibers) per animal were recorded as

outcomes, group averaged, and compared between control and high dose NNMTi (10

mg/kg)-treated mice. Data from one treated mouse was not included in analysis since

torque could not be reliably measured above background levels.

2.5 Immunohistochemistry. For all immunohistochemical analyses, dissected muscles

from mice were mounted in Tissue Tek (O.C.T. Compound, Sakura Finetek, Torrance,

CA, USA) at resting length and frozen in liquid nitrogen-cooled 2-methylbutane and

stored at −80°C until analysis. 7 m-thick sections were cut with a cryostat (HM525-NX,
Thermo Fisher Scientific, Waltham, MA, USA) and allowed to air dry for 1 h prior to

storage at -20 °C.

2.5.1 NNMT expression in 4-month old young and 24-month old aged tibialis

anterior (TA) muscle. For NNMT immunohistochemical staining, sections were fixed in

4% paraformaldehyde (PFA). Slides were blocked for 1 h in 1% TSA blocking reagent

and incubated overnight at 4 °C in primary antibody against NNMT (ab58743, Abcam,

Cambridge, MA; 1:100). The following day, slides were incubated for 1 h at room

temperature with IgG anti-rabbit secondary conjugated to AF555 (no. A21428, 1:500;

Life Technologies/Thermo Fisher Scientific, Waltham, MA USA) and AF488-cojugated

wheat germ agglutinin (WGA) (no. W11261, Life Technologies/Thermo Fisher Scientific)

to denote myofiber borders. Slides were incubated in 4′,6-diamidino-2-phenylindole

(DAPI; 10 nM, LifeTechnologies/Thermo Fisher Scientific) for 10 min and washed and

mounted with Vectashield fluorescence mounting media (Vector Laboratories,

Burlingame, CA, USA).

2.5.2 muSC immunohistochemical analyses. Pax7 is a transcription factor unique to

muSCs and a routinely used marker to identify muSCs; laminin is routinely used to

denote muscle fiber borders. For Pax7-EdU-laminin staining, sections were fixed in 4%

PFA followed by antigen retrieval using sodium citrate (10mM, pH 6.5) at 92°C for 20

min. Slides were then placed in 3% hydrogen peroxide in PBS for 7 min to block

endogenous peroxidase activity followed by a second blocking step in Mouse-on-Mouse

Blocking Reagent (Vector Laboratories, Burlingame, CA, USA). Sections were

incubated overnight at 4 °C in primary antibodies against Pax7 (DSHB, Iowa City, IA,

USA; 1:100) and laminin (L9393, Sigma-Aldrich, St Louis, MO, USA; 1:100). Next,

slides were incubated with biotinylated anti-mouse IgG secondary antibody for Pax7

(no. 115-065-205, Jackson ImmunoResearch, West Grove, PA, USA; 1:500) and anti-

rabbit IgG secondary antibody for laminin (Alexa Fluor 647, no. A11034, Life

Technologies/Thermo Fisher Scientific; 1:500) followed by incubation with streptavidin-

horseradish peroxidase (HRP) included with a commercially available tyramide signal

amplification kit (TSA, Life Technologies/Thermo Fisher Scientific). TSA-Alexa Fluor

488 was used to visualize Pax7 antibody binding. Following all Pax7-laminin steps,

slides were permeabilized in 0.1% Triton-X100 in phosphate-buffered saline and

incubated with the Life Technologies Click-iT Kit per manufacturer’s instructions, with

EdU visualized with Alexa Fluor 555. Slides were incubated in 4′,6-diamidino-2-

phenylindole (DAPI; 10 nM, LifeTechnologies/Thermo Fisher Scientific) for 10 min,

washed, and mounted using Vectashield fluorescence mounting media (Vector

Laboratories).

2.5.3 Image acquisition and quantification. Immunohistochemical images were

captured at 200X total magnification room temperature with a Zeiss upright microscope

(AxioImager M1; Zeiss, Göttingen, Germany). muSC abundance was evaluated by

Pax7 staining along with laminin and DAPI; only cells that were Pax7+ /DAPI+ within the

laminin border were counted. Proliferating muSC included only those that were

Pax7+/EdU+/DAPI+ within the laminin border. muSC fusion to myofibers was determined

by newly acquired myonuclei classified as Pax7 -/EdU+/DAPI+. Mean myofiber cross-

sectional area (CSA) was measured by manually tracing myofiber area using the

laminin border. Only injured myofibers (fibers with central myonucleation) were included

in CSA analysis.

Image analysis was performed in a blinded manner using AxioVision Rel

software (v4.9). To assess the reliability, accuracy, and reproducibility of the image

quantification procedures, three blinded raters assessed images from six mice; two

mice were randomly selected from each treatment group for validation of counts of

muSC abundance, proliferating muSCs, and muSC fusion (saline: n=2, low dose

NNMTi: n=2, high dose NNMTi: n=2). Intraclass correlation coefficients (ICCs) were

calculated for each variable to determine reliability of the data. ICC estimates and 95%

confidence intervals were calculated using SPSS statistical software (IBM Corp. IBM

SPSS Statistics for Windows, Version 25. Armonk, NY, USA) based on a mean-rating,

consistency, 2-way mixed-effects model with 3 raters across 6 mice. ICC values below

0.5 indicate poor reliability, 0.5 to 0.75 indicate moderate reliability, 0.75 to 0.9 indicate

good reliability, and values greater than 0.90 are indicative of excellent reliability and

reproducibility [42, 43].

2.6. Plasma chemistry panel. Blood samples were collected from each animal

immediately upon sacrifice, processed to isolate plasma, frozen in liquid nitrogen, and

shipped on dry ice to Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL;

College Station, TX, USA) for small animal serum chemistry analysis via standard

protocols. TVMDL is accredited by the American Association of Veterinary Laboratory

Diagnosticians and all results were confirmed with positive and negative controls.
Complete plasma measures included hepatic panel (AST, ALT, ALP, GGT, GLDH, total

bilirubin, albumin, globulin, AG ratio, total protein), renal panel (amylase, BUN, creatine

kinase, calcium, phosphorus), amylase, electrolytes (sodium, potassium, and chloride),

glucose, and cholesterol to validate systemic effects of repeated NNMTi treatment on

major organ functions.

2.7 Differentiation of C2C12 myoblasts. C2C12 myoblast cells (CRL-1772, American

Type Culture Collection; Manassas, VA, USA) were cultured with standard growth

media (DMEM, 4.5 g/L glucose, L-glutamine, sodium pyruvate, 10% FBS, 1%

penicillin/streptomycin) and grown to 80% confluency before initiating differentiation. To

begin differentiation of myoblasts, growth media was replaced by differentiation media

(DMEM, 4.5 g/L glucose, L-glutamine, sodium pyruvate, 2% Horse serum, 1%

penicillin/streptomycin); low nutrient condition and increased cell-to-cell contact

stimulated differentiation of myoblasts by inhibiting proliferation and promoting the

formation of multi-nucleated myotubes. Differentiation media was replaced every other

day to maintain myotubes in culture for 3-4 days, during which time experiments as

described below were performed.

2.8.1 Immunocytochemical analysis of NNMT expression in C2C12 myoblasts and

differentiated myotubes. C2C12 myoblasts and myotubes grown on glass cover slips

were fixed in 4% PFA warmed to 37°C for 15 min, and then washed 3 times with PBS.

Myoblasts and myotubes were permeabilized with a 10 min wash in PBS + 0.2% Triton-

X100, and then blocked in 1% BSA made in PBS + 0.2% Triton-X100 for 1 hr. For
myoblasts, cells were incubated in primary antibody (1:100, NMMT cat #ab119758,

mouse IgG2b, Abcam) for 2 hr at room temp in 1% BSA, followed by PBS washing, and

then secondary antibody (Gt anti-Ms IgG2b AF488, cat # A-21141, ThermoFisher) for 1

hr followed by a co-stain with DAPI and mounting. For myotubes, cells were incubated

in primary antibody (NMMT, 1:100, cat #ab119758, mouse IgG2b, Abcam and myosin

heavy chain, 1:200, cat #M4276, mouse IgG1, Sigma) for 2 hr at room temp in 1% BSA,

followed by PBS washing, and then secondary antibody (Gt anti-Ms IgG2b AF488, cat #

A-21141 and Gt anti-Ms IgG1 AF555, cat # A-21127, ThermoFisher) for 1 hr followed by

a co-stain with DAPI and mounting. Images were captured at 200X total magnification.

2.8.2 Effect of NNMTi on C2C12 myoblast differentiation. To determine the effect of

NNMTi on C2C12 myoblast differentiation, myoblasts were cultured to confluency as

described above and stimulated to differentiate in the absence or presence of NNMTi

(10 and 30 µM concentrations) in differentiation media for 96 h. Concentrations of

NNMTi were chosen based on our previous published work in adipocytes, where

significant phenotypic and metabolic changes were observed in the presence of the

inhibitor relative to untreated conditions [38]. After 96 h of differentiation, media was

removed and cells were fixed in 4% PFA pre-warmed to 37°C for 15 min. PFA was then

removed and the cells were washed three times (3 min each wash) with PBS containing

0.2% Triton X-100. Cells were blocked for 1 h in 1% TSA (fTSA kit – compound D,

Invitrogen ThermoFisher Scientific #T-30955) in PBS/Triton. Following blocking, cells

were incubated for 2 h at room temperature with anti-myosin heavy chain (MHC)

primary antibody (Sigma #M4276 – from MY-32 clone) at 1:200 concentration made in
TSA/PBS/Triton diluent. Cells were washed in PBS/Triton and incubated for 1 h at room

temperature with goat anti-mouse secondary antibody (IgG1 AF488 conjugated;

Invitrogen #A21121) at 1:500 concentration made in PBS/Triton. Cells were washed,

incubated with DAPI (1:10,000 concentration; Invitrogen #D35471 in PBS) for 10 min to

stain nuclei, and washed finally with PBS/Trition before imaging. Four random field of

views were captured per well; total DAPI stained nuclei and MHC positive nuclei were

counted to calculate %MHC positive nuclei. Control and NNMTi-treated conditions were

run in duplicates with results averaged for each condition. %MHC positive nuclei

analyzed were averaged and compared between control and NNMTi-treated myotubes

with experiments repeated three times.

2.8.3. Effect of NNMTi treatment on NAD+ and NADH levels in C2C12 myoblasts

and myotubes. C212 myoblasts were differentiated to myotubes using the protocol

described above (see section 2.7) and maintained in culture for 4 days before NNMTi

treatment. On day 3 of differentiation, media was removed and replaced with fresh

differentiation media (control) or NNMTi (10 and 30 µM concentrations) made in

differentiation media for 24 h. Similarly, C2C12 myoblasts were cultured to ~40%

confluency and treated for 24 h with growth media alone (control) or in the presence of

NNMTi (10 and 30 µM concentrations) in growth media for 24 h. Following 24 h of

treatment with NNMTi, cells were treated with trypsin, centrifuged, and washed once in

ice cold PBS. Cells were re-suspended in 300 µL of 75% ethanol / 25% 10 mM HEPES

solution pre-heated to 80°C and extracted using a previously described protocol to

obtain soluble NAD+ and NADH metabolites [44]. Levels of NAD+ and NADH in the
extracted samples were assessed using a spectrophotometric enzymatic assay. Briefly,

samples were re-suspended in distilled water and treated with acid (0.1 M hydrochloric

acid) or base (0.1 M sodium hydroxide) and heated at 75°C for 30 min to remove all the

NADH and NAD+ in samples, respectively. Samples were then neutralized by

appropriately adding acid (0.1 M HCl/0.5 M bicene) or base (0.1 M NaOH/0.5 M bicene)

and treated with alcohol dehydrogenase (1x final; A3263-7, Sigma-Aldrich, St. Louis,

MO, USA) for 15 min. Following incubation, 10 µL of WST-1 reagent (Roche Applied

Science, Mannheim, Germany) to initiate the enzymatic reaction and the pink colored

product formed over time was spectrophotometrically measured via absorbance

detected at 450 nm wavelength and compared between control and treated samples.

2.9. Statistical analysis. Effects of NNMTi on muSC regeneration were expressed as

incidence of activated muSCs and fibers with myonuclei and analyzed using R statistical

software (R Core Team, 2018, version 3.5.1) by mixed effect logistic regression as a

function of dose group, adjusting for cohort (given that the experiments were conducted

across separate cohorts), and controlling for repeated measures effect. Differences

between dose groups were assessed by Tukey adjusted contrasts posthoc test. Fiber

CSA data were log-transformed (for 1-week post-injury treatment data to better

approximate normal distribution) or analyzed without transformation (3-week post-injury

treatment data) by mixed effect analysis of variance (ANOVA) followed by Tukey

multiple contrasts posthoc as a function of dose, adjusting for cohort (given that the

experiments were conducted across separate cohorts), and controlling for repeated

measures effect. Where applicable, statistical analysis for two-group comparisons was
conducted using unpaired Student’s t-test (e.g., serum chemistry results) or an

appropriate non-parametric analysis (where datasets did not pass normality test) using

Graphpad Prism (version 7.0). Two-way ANOVA with Sidak’s multiple comparison

posthoc test was used to compare frequency of fibers as a function of binned CSA/fiber

size in controls versus NNMTi treated groups using Graphpad Prism (version 7.0). One-

way ANOVA with Dunnett’s posthoc test was used to compare %MHC positive nuclei in

NNMTi-treated myotubes versus control using Graphpad Prism (version 7.0).

Absorbance data obtained at various timepoints for NAD + and NADH extracted control

and NNMTi-treated C2C12 cell samples were transferred to Graphpad Prism (version

7.0) to perform linear regression analysis and obtain slopes from the linear reaction

progress curves. Slopes for NAD+ and NADH extracted samples and the ratio of

NAD+/NADH slopes were compared using one-way ANOVA or Kruskal-Wallis test (for

datasets not passing normality) followed by Dunnett’s posthoc to compare NNMTi

treatments to controls. All statistical analyses were performed with an experiment-wise

error rate of α = 0.05, for a 95% level of confidence.

3. RESULTS

3.1 NNMT expression increases in aged TA muscle. Immunolabeling of NNMT

revealed NNMT-specific staining both in the TA muscle obtained from 24-month-old and

4-month-old mice (Fig. 1A, 1B). Expression of NNMT was qualitatively demonstrated to

be relatively greater in aged muscle based on the immunohistochemical evaluations of

the aged TA compared to young TA muscle tissues (Fig. 1A, 1B). To validate the

qualitative findings using a semi-quantitative approach, NNMT immunoblots were

established using aged and young mouse TA tissues (Fig. 1C). Consistent with the

differential immunohistochemical NNMT results in aged versus young TA tissue, the

expression level of NNMT protein in aged TA muscle was ~3-fold higher compared to

the level of NNMT protein in young TA tissue (Fig. 1D; p < 0.05 vs. NNMT expression in

young TA tissue). Taken together, these results support increased NNMT protein

expression in the skeletal muscle as a function of age, suggesting likely enhanced

NNMT activity in aged muscle tissue.

3.2 In vivo NNMT inhibition activates muSCs and promotes aged muscle

regeneration. Fig. 2A and 2B present representative images that highlight muSC

proliferation and fusion into damaged myofibers of aged TA muscle in control and

NNMTi (10 mg/kg, bid)-treated mice. As indicated by white arrowheads (rightmost

panels, Fig. 2A, 2B), NNMT inhibition increased the number of proliferating muSCs

(measured by counting Pax7+/EdU+/DAPI+ cells per unit area); however, the total

number of muSCs (i.e., Pax7+/DAPI+ cell counts per unit area) were unchanged in the

control, low NNMTi dose, and high NNMTi dose groups, and averaged 209 ± 23, 213 ±

44, and 205 ± 9 muSC/mm2, respectively (Fig. 2A, 2B, representative panels showing

Pax7+ positive cells indicated in green). Similarly, as indicated by yellow arrowheads

(rightmost panel, Fig. 2A and 2B), NNMT inhibition promoted greater fusion of muSCs

into damaged myofibers (measured by newly acquired myonuclei; Pax7-/EdU+/DAPI+

within laminin border), suggesting improved muscle regeneration following injury.

Consistent with these results, extensive quantification of numerous images

revealed a dose-related increase in the relative abundance of proliferating muSCs (Fig.

2C) and fibers with integrated myonuclei via muSC fusion (Fig. 2D) following 1-week

post-injury NNMTi treatment. The 5 mg/kg and 10 mg/kg doses of NNMTi tested

resulted in 60% and 75% higher incidence of proliferating/active muSCs, respectively,

relative to control (Tukey adjusted P values: p = 0.013, 5 mg/kg dose vs. control; p =

0.0007, 10 mg/kg vs. control). The odds ratio of active muSCs for control was 0.54 and

0.46 times the odds at the low and high doses, respectively. Although the higher

treatment dose produced ~11% higher incidence of activated muSC compared to the

lower treatment dose, this observed difference did not rise to the level of being

statistically significant (p > 0.05, n.s.) (Fig. 2C). ICC for muSC abundance was 0.944

[95% CI (0.765,0.992)] and for proliferating muSCs 0.754 [95% CI (-0.43,0.963)]

indicating excellent reliability of the data recorded by independent experimenters in a

blinded fashion.

Similarly, dose-dependent trends were observed with NNMTi treatment in the

incidence of newly acquired myonuclei via muSC fusion into damaged myofibers (Fig.

2D). The relative numbers of fibers with an EdU+ myonucleus increased 40% and 48%

with NNMTi treatment at 5 mg/kg and 10 mg/kg, respectively, relative to control. The

odds ratio of fused myonuclei for control were 0.58 and 0.53 times the odds at the low

and high NNMTi dose, respectively, and the Tukey adjusted P values bordered on the

cutoff generally considered statistically significant (p = 0.0686, 10 mg/kg dose vs.

control). ICC for muSC fusion was 0.971 [95% CI (0.878,0.996)] indicating excellent

reliability of data scored independently by blinded experimenters.

3.3 In vivo NNMT inhibition promotes muscle fiber growth after aged muscle

injury. Damaged TA muscle tissues were immunolabeled with laminin to trace fibers,

and the cross-sectional area (CSA) of the fibers was analyzed and compared between

control and NNMTi-treated tissues. Representative laminin labeled, muscle fiber-traced

images obtained from control (left panel) and NNMTi 10 mg/kg dose (right panel)

treatment groups are shown in Fig. 3A. One-week post-injury treatment with NNMTi at

the high dose (10 mg/kg) produced a significant 1.8-fold increase in the mean CSA of

damaged TA muscle fibers relative to control (Tukey adjusted P values: p = 0.0052, 10

mg/kg dose vs. control) (Fig. 3B). A similar (1.6-fold) dose-dependent increase was

observed at the high dose of NNMTi treatment compared to the low dose tested (Tukey

adjusted P values: p = 0.023, 10 mg/kg dose vs. 5 mg/kg), while the low dose of NNMTi

treatment did not significantly alter the muscle fiber mean CSA compared to control

(Tukey adjusted p > 0.05) (Fig. 3B). These results demonstrate that small molecule-

mediated in vivo NNMT inhibition improves muscle regeneration and growth, consistent

with the enhanced muSC activation and fusion observed following injury of the aged

muscle (Fig. 2).

The dramatic increase in muscle fiber size following a brief treatment with NNMTi

supported the testing of high dose NNMTi in a separate cohort of aged mice to assess

the impact of longer-term treatment on complete muscle recovery following injury. As

shown in Fig. 3C, the mean CSA of damaged muscle fibers from aged mice was

significantly (2- to 3-fold) larger at 3-weeks post-injury compared to 1-week post-injury

(Fig. 3C vs. 3B), indicating progressive longitudinal recovery of damaged muscle in

aged mice. Consistent with CSA results observed with 1-week post-injury treatment,
three-week post-injury treatment with 10 mg/kg NNMTi produced a statistically

significant and physiologically relevant 1.5-fold increase in the mean CSA of damaged

TA muscle fibers relative to control (Tukey adjusted p = 0.039). As noted in the Methods

section, a single aged mouse that showed unusually low CSA (included for

completeness and indicated by filled diamond scatter point in Fig. 3C) due to

unaccountable factors (i.e., lower CSA than control CSA in even the 1-week post-injury

data) was excluded from analysis. Taken together, these results suggest that in vivo

NNMT inhibition not only increases the rate of recovery by early activation of muSCs,

but also increases the overall magnitude of recovery following injury to aged muscle

(Tukey adjusted p > 0.05).

Further, binned analysis of the muscle fiber CSA size distribution demonstrated a

significant interaction between fiber size and NNMTi treatment (F(17, 396) = 3.315; p <

0.0001) as analyzed by a two-way ANOVA (Fig. 3D). Particularly, a significant

downward shift in the frequency of smaller sized myofibers (Fig. 3D; p = 0.03, NNMTi

treatment vs. control in the frequency of fibers up to 100 µm 2 CSA; p = 0.0002, NNMTi

treatment vs. control for 200 µm2 CSA bin) and prominent rightward and upward shifts

toward larger sized fibers (p = 0.07, NNMTi treatment vs. control in the frequency of

fibers at sizes greater than 1600 µm2 CSA) were noted in the damaged myofibers of

aged mice treated with the high dose NNMTi (Fig. 3D).

3.4 In vivo NNMT inhibition improves aged TA muscle contractile force. In vivo

dorsiflexor torque measurements were conducted 1-week post-injury on the damaged

TA muscle of control and NNMTi treated aged mice to assess muscle contractile
function properties at maximum applied force conditions. Peak torque output of the

damaged TA muscle, when normalized to body weight of each animal, was 67% higher

in NNMTi-treated group compared to control group. This increase was functionally and

statistically significant (p = 0.033 vs. control; Fig. 4A), and consistent with the increased

fiber size observed in the treated cohort. In contrast, peak torque output when

normalized to the muscle fiber size (i.e., normalized to regenerating TA myofiber CSA

and presented as torque per unit area, Fig. 4B) showed no difference between the

treated and control groups (p > 0.05; Fig. 4B); this lack of difference was largely driven

by the significantly larger muscle fiber mean CSA in the NNMTi-treated mice relative to

controls. These data clearly provide support for greatly improved functional strength in

aged muscle with in vivo NNMTi treatment, in addition to the improved muscle

regeneration response observed following injury.

3.5 Chronic administration of NNMTi produces no systemic toxicity. As shown in

Table 1, a complete plasma chemistry panel including major organ enzymes (liver,

pancreas, gastrointestinal, renal, endocrine), total lipid, plasma proteins, and major

electrolytes were profiled and compared between control and high dose (10 mg/kg)

NNMTi-treated plasma samples. No significant differences were noted in the levels of

the tested enzymes, glucose, cholesterol, plasma proteins, and electrolytes between

control and NNMTi-treated samples (Table 1). Amylase levels were noted to be higher

in all aged animals (regardless of control/treatment conditions) relative to young

animals, likely indicative of general age-related dysfunctions (Table 1). Moreover,

control and treated cohorts had similar body weight changes in all studies, suggesting
similar feeding patterns (data not shown). Taken together, 1-week post-injury daily

repeat-dosing of NNMTi was well tolerated with no untoward systemic toxicity or

behavioral implications in aged mice.

3.6 NNMTi treatment promotes differentiation of C2C12 myoblasts and alters

NAD+/NADH redox cofactors in differentiated C2C12 myotubes. In vitro experiments

were conducted to probe phenotypic and metabolic implications of NNMT inhibition in

C2C12 cells that capture many characteristics of muSCs. Prior to determining the

effects of NNMTi treatment in C2C12 cells, presence of NNMT target protein was

qualitatively confirmed both in C2C12 myoblasts and differentiated myotubes using

immunocytochemistry (Fig. 5A, 5B). NNMT expression was observed in C2C12

myoblast cells (Fig. 5A), differentiating myoblasts that were myogenic committed

precursor cells (Fig. 5B, elongated, MHC-expressing cells indicated by yellow

arrowheads), as well as in fully developed myotubes, which was confirmed by co-

expression of NNMT with MHC (marker for myotubes/myofibers; Fig. 5B). Addition of

NNMTi to confluent myoblasts under differentiation conditions produced a

concentration-related increase in myoblast differentiation (Fig. 5C; F(2, 21) = 10.27, p =

0.0008 for NNMTi treatment factor). 30 µM NNMTi resulted in 18 ± 0.03% MHC-positive

myotube nuclei, representing a 45% increase in the extent of myoblast differentiation

(expressed as %MHC positive myotube nuclei) compared to untreated differentiating

myoblasts that only produced 12 ± 0.4 % MHC-positive myotube nuclei (Fig. 5C; p =

0.0004 versus control).

 C2C12 myoblasts and differentiating myotubes were treated with NNMTi for 24 h

to determine the effects of NNMTi treatment on NAD+ and NADH levels and the

metabolic redox states. NNMTi treatment did not significantly alter NAD+ (Fig. 5D; H(2)

= 4.57, p = 0.067) and NADH (Fig. 5E; H(2) = 3.71, p = 0.2) levels or the redox state

(NAD+/NADH ratio; Fig. 5F) in C2C12 myoblasts. Similarly, NNMTi treatment produced

no significant change in the NAD+ levels (Fig. 5G, F(2, 8) < 1.0, p > 0.05, n.s.) in

differentiated C2C12 myotubes. However, NNMTi treatment produced concentration-

dependent increases in NADH levels (Fig. 5H; F(2, 8) = 7.059, p = 0.0171), as indicated

by 50% higher slopes generated from reaction progress curves for NADH extracted

samples with 30 µM NNMTi treatment suggesting 50% higher NADH concentrations

compared to control untreated myotubes (Fig. 5H; p = 0.0118 versus control). Given the

observed NAD+ and NADH responses to NNMTi treatment, NAD+/NADH ratios

decreased in a concentration-related manner with NNMTi treatment of myotubes (Fig.

5I; F(2, 8) = 11.65, p = 0.0043). The NAD+/NADH ratio was 25% lower with 10 µM NNMTi

(Fig. 5I; p = 0.0443 versus control) and 40% lower with 30 µM NNMTi (Fig. 5I; p =

0.0024 versus control) compared to control.

4. DISCUSSION
To our knowledge, this is the first study to demonstrate that systemic treatment of aged

animals with a small molecule drug-like NNMT inhibitor [38, 39] can rejuvenate muSCs,

thereby robustly promoting muSC activation and fusion which are critical to support de

novo myofiber regeneration and repair following injury [14-16]. At 1-week post-injury,

control aged mice showed poor myofiber growth, minimal muSC activity, and a

compromised skeletal muscle repair profile; these observations were consistent with
impaired muSC regenerative capacity previously reported in skeletal muscles from aged

mice [45, 46]. In contrast, aged mice treated with NNMTi demonstrated dramatically

increased frequency of activated muSCs and greatly enhanced muscle repair via muSC

fusion and subsequent myonuclear accrual. Both the number of activated muSC and

the average size of regenerating myofibers on the injured limb had nearly doubled at 1-

week post-injury with treatment facilitating the development of myofibers with much

larger cross-sectional areas, similar to youthful myogenic growth response [45, 46].

Consistent with increased muscle fiber growth, NNMTi treatment nearly doubled peak

dorsiflexor torque output on the injured limb, suggesting clinically meaningful overall

improvements in muscle strength and function. Additionally, at 3-weeks post-injury,

when the damaged muscles of the control animals had undergone additional recovery,

prolonged NNMTi treatment resulted in further enhancement of myofiber growth

compared to controls. These results suggest that prolonged NNMTi treatment enables a

more complete recovery of the injured aged muscles, as average myofiber sizes were

comparable between injured TA muscles of the treated aged mice and uninjured TA

muscles of similar aged (24-mo-old) mice [18].

Consistent with our findings, Zhang and colleagues recently observed that 6-8-

week treatment of 22-24-mo-old mice with 400 mg/kg/day of NR (an NAD+ precursor

agent) accelerated muscle regeneration following cardiotoxin-induced injury to the TA

muscle [29]. While Zhang et al. observed that NR treatment also increased the absolute

number of muSCs pooled and analyzed from numerous (e.g., gastrocnemius, soleus,

quadriceps, TA) aged muscle tissues [29], NNMTi treatment did not alter the pool of

muSCs analyzed from the TA muscle of aged mice. This difference may be due to the
shorter (2-week) treatment regimen used in the current study compared to the longer (6-

8 weeks) treatment regimen employed with NR [29], or to the different skeletal muscle

groups examined in the previous studies since it has been observed that only a few

mouse muscle groups (e.g., gastrocnemius, soleus) experience age-related declines in

muSC abundance [21].

The molecular mechanisms by which NNMT inhibition promotes muSC activation

and myogenic response are currently unknown, but potentially associates with NNMT’s

central role in regulating the NAD+ salvage pathway, which in turn affects intracellular

NAD+ levels and SIRT1 (NAD-dependent deacetylase sirtuin 1) activity that modulate a

cell's metabolic and transcriptional responses [33, 47]. Since muSCs represent only

about 3-5% of adult muscle fiber nuclei and a small proportion of the cells within the

whole muscle tissue [29, 48], we performed additional experiments in cultured C2C12

myoblasts undergoing programmed differentiation, a system that recapitulates many of

the features of muSC activation and thus can serve as a model to better understand

how NNMTi treatment rejuvenates aged muSC and alters metabolic states of cells

during differentiation [48, 49]. NNMTi treatment enhanced C2C12 myoblast

differentiation and promoted myotube formation, selectivity increasing NADH levels and

reducing NAD+/NADH ratio in differentiating myotubes, without impacting the metabolic

states of myoblasts. These in vitro results suggest that NNMT inhibition enables muSCs

to become increasingly activated and committed to muscle precursor cells with

increased potential to differentiate, which is consistent with the increased differentiation

and fusion of activated muSCs observed in NNMTi-treated aged mice following muscle

injury.
 muSC differentiation-dependent metabolic reprogramming has been previously

demonstrated by Ryall and colleagues [48]. Specifically, activated and differentiation-

committed C2C12 myoblasts were shown to switch to glycolytic metabolism with

marked reductions in NAD+/NADH ratio (mediated by increased NADH levels and

reduced NAD+ levels) and SIRT-1 histone deacetylase activity, and accompanied by

myogenic stimulation and increased MyoD expression (muscle-specific transcription

factor routinely used to denote activated muSCs). In contrast, oxidation-dependent

metabolism marked by higher NAD+ levels and SIRT-1 activity predominate in C2C12

myoblasts that are more equivalent to quiescent muSCs [29, 48, 50]. Thus, our findings

that NNMTi treatment selectively increased NADH levels and reduced NAD+/NADH ratio

in differentiating myotubes, without impacting metabolic states in quiescent myoblasts

are consistent with results from Ryall et al., favoring a switch towards glycolytic

metabolism reprogramming (i.e., increased NAD+ substrate utilization in glycolysis for

meeting muSC differentiation energy demands) that supports enhanced myoblast

differentiation. Taken together, our working model regarding the molecular mechanisms

whereby NNMT inhibition promotes muSC activation and increases myogenic response

is illustrated schematically in Figure 6. As muscles age, NNMT expression and activity

increases in the skeletal muscles, which impairs NAD+ flux through the salvage pathway

and dysregulates SIRT-1 activity, thereby interfering with the capacity of muSC to

maintain quiescence as well as the efficiency to proliferate, differentiate, and promote

efficient muscle regeneration following injury [29, 48]. Given that NNMT appears to be

uniformly expressed in skeletal muscles independent of fiber type distribution (i.e., type

I, IIa, IIb) as previously demonstrated [34], suggests its dynamic involvement in the
regulation of both oxidative and glycolytic metabolism. Future studies will investigate if

similar metabolic changes are observed in muSCs isolated from NNMTi-treated skeletal

muscle ex-vivo, and subsequently investigate the effects of NNMTi treatment on

mitochondrial biogenesis and epigenetic modulations (both via SIRT-1 and SAM in the

methionine pathways) in aged muscles.

In conclusion, the present findings convincingly demonstrate that NNMT inhibition

provides a viable, safe, and non-invasive therapeutic approach to rejuvenate

dysregulated muSCs and restore their regenerative capacity in aged skeletal muscles,

as well as their intrinsic quiescent properties, to support dynamic muscle repair

mechanisms after injury. Significantly, NNMT inhibition enhanced myofiber regeneration

and growth following injury and dramatically improved the strength of the injured

muscle, suggesting overall functional improvements to aged skeletal muscles. These

results support NNMT inhibitors as novel therapeutics to restore a healthy myogenic

response in injured aging skeletal muscle through rejuvenation of muSC activity,

translating into improved muscle function and potentially mitigating the functional

decline which sarcopenic older adults typically experience following muscular injury.

Evaluations on the longitudinal effects of NNMTi treatment on muscle functional outputs

(including measures of strength and endurance), safety/toxicology, and mechanistic

implications of NNMT inhibition on aged muscle physiology will be continued to validate

the clinical translational benefits of NNMTi therapeutics.

ACKNOWLEDGEMENTS

We thank our biostatistician expert Mr. Clark Anderson (The Office of Biostatistics,

Dept. of Preventative Medicine and Community Health, University of Texas Medical

Branch) for support on conducting mixed effect logistic regression analysis on the stem

cell datasets using R statistical software, and discussions of the results and their

interpretations. This work was supported by the Sanofi iAward grant (S.J.W., C.C.F),

University of Texas Medical Branch Technology Commercialization Award (S.J.W, H.N),

T32 training grant T32AG000270 (C.R.B.), NCATS CTSA TL1TR001440 NSRA

Fellowship (T.G.G.), and NIH R01 AR072061 (C.S.F.). The content is solely the

responsibility of the authors and does not necessarily represent the official views of the

National Institutes of Health. The Pax7 (#PAX7) antibody was developed by A.

Kawakami and obtained from the Developmental Studies Hybridoma Bank, created by

the NICHD of the NIH and maintained at The University of Iowa, Department of Biology,

Iowa City, IA 52242.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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TABLE

Table 1. Serum chemistry panel from control and NNMTi-treated (10 mg/kg) aged mice.

p-value calculated using 2-tailed Student's t-test with Welch's correction (populations

not assumed to have same standard deviations).

Serum marker Control Treated p-value*

Creatine kinase (U/L) 648 ± 229 419 ±88 0.39
AST(U/L) 280 ± 84 248 ± 81 0.79
ALT(U/L) 142 ± 61 110 ± 44 0.69
ALP (U/L) 85 ± 15 75 ± 8 0.55
GGT (U/L) <3 <3
GLDH (U/L) 73 ± 46 103 ± 43 0.66
Amylase(U/L)^ 1365 ± 748 1344 ± 554 0.77
Total bilirubin (mg/dL) 0.18 ± 0.03 0.18 ± 0.03 >0.99
BUN (mg/dL) 23 ± 2 43 ± 12 0.17
Glucose (mg/dL) 194 ± 23 173 ± 10 0.43
Cholesterol (mg/dL) 81 ± 3 94 ± 12 0.36
Total protein (g/dL) 4.4 ± 0.2 4.4 ± 0.1 0.91
Albumin (g/dL) 2.4 ± 0.063 2.4 ± 0.048 0.25
Globulin (g/dL) 1.9 ± 0.2 2.2 ± 0.075 0.29
Calcium (mg/dL) 8.7 ± 0.3 8.7 ± 0.2 0.94
Phosphorous (mg/dL) 6.7 ± 0.8 5.6 ± 0.5 0.29
Sodium (mEq/L) 147.5 ± 1.7 153.8 ± 2.8 0.10
Potassium (mEq/L) 5.6 ± 0.5 5.0 ± 0.2 0.30
Chloride (mEq/L) 114.5 ± 1.1 119.5 ± 2.4 0.10

Data represent mean ± SEM; n=4/group

*, p > 0.05; no significant difference

^, elevated levels in both cohorts suggesting age-related dysfunctions

FIGURE LEGENDS

Figure 1. NNMT expression in aged (24 mo) and young (4 mo) TA muscle.

Representative images of (A) aged (top panels) and (B) young (bottom panels) TA

muscle tissues immunolabeled for detection of NNMT protein (red), myofiber membrane

border (marked by wheat germ agglutinin [WGA] in green), and nuclei (marked by DAPI

in blue). (C) Representative immunoblots obtained from replicate aged and young TA

mouse tissue samples. (D) NNMT protein expression normalized to the loading control

β-actin in aged and young TA muscle tissues as analyzed using western blotting. **, p <

0.01 vs. aged TA muscle NNMT expression.

Figure 2. Effects of NNMTi on proliferation and fusion of muSCs into myofibers

following injury of aged TA muscle. (A) Representative image of control aged and

injured TA muscle tissue immunolabeled to detect proliferation and fusion of muSCs

using EdU (red), Pax7 (green), DAPI (blue), and laminin (white/gray) to trace

myofibers.. (B) Representative image of aged and injured TA muscle tissue following 1-

week post-injury treatment with NNMTi immunolabeled as in panel (A). White

arrowheads denote muSCs that have proliferated post-injury (EdU+/Pax7+/DAPI+).

Yellow arrowheads indicate myonuclei fused into damaged myofibers post-injury

(EdU+/Pax7-/DAPI+). (C) Relative number of activated muSCs compared to total

population of muSCs (% muSC EdU+) in NNMTi-treated and control aged TA muscle

tissues following injury (n=10-12 per group). Data represent adjusted mean % muSC

EdU+ ± 95% CI). *, p < 0.05 and ***, p < 0.001 vs. control as determined by Tukey-

adjusted posthoc comparisons. (D) Percentage of myofibers containing EdU+

myonucleus (% fibers with EdU+ myonucleus) in NNMTi-treated and control aged TA

muscle tissues following injury (n=10-12 per group). Data represent adjusted mean %

fibers with EdU+ myonucleus ± 95% CI. Consistent with % muSC EdU+ data (C), an

increase in % fibers with EdU+ myonucleus trend was observed with NNMTi treatment

relative to control, with the high dose of 10 mg/kg treatment showing a near statistically

significant increase (p = 0.069) vs. control.

Figure 3. Effect of NNMTi on damaged TA muscle fiber cross-sectional area (CSA). (A)

Representative images of aged and injured TA muscle tissue following 1-week post-

injury in control (left panel) NNMTi-treated (right panel); immunolabeled with laminin

(indicated in white) to quantify CSA. (B) Mean CSA of damaged TA muscle fibers

analyzed in aged control and NNMTi-treated mice (n=10-12 per group). Data represent

adjusted mean CSA (µm2) ± 95% CI. **, p < 0.01 vs. control; ^, p < 0.05 vs. 5 mg/kg. (C)

Mean CSA of damaged TA muscle fibers analyzed in aged control and treated (10

mg/kg) mice following longer-term (3-week post-injury) treatment to assess more

complete recovery (n=6 per group). Data represent adjusted mean CSA (µM2) ± 95% CI

with individual datapoints represented as a scatter plot. *, p < 0.05 vs. control, with one

datapoint (indicated in filled diamond) in the treated group eliminated from analysis on

the basis of an unusually low (i.e., below threshold) CSA value for complete recovery

analysis (p > 0.05, n.s. with this data point included). (D) Frequency of fibers binned as

a function of CSA area in aged control and NNMTi (high dose, 10 mg/kg)-treated mice

(n=12/group). Data represent mean frequency of fibers per bin ± SEM. *, p < 0.05 vs.

control and ***, p < 0.001 vs. control.

Figure 4. Effect of NNMTi (1-week treatment post-injury) on TA muscle contractile force

in aged mice following TA muscle injury. (A) Maximum torque measured per g of body

mass in vivo in response to maximum force delivered via minimal electric stimulation of

the TA muscle in aged control and NNMT-treated (10 mg/kg, bid) mice (n=6-7 per

group). Data represent mean peak torque per g of body mass (mN*m/g) ± SEM. *, p <

0.05 vs. control. (B) Maximum torque measured normalized to fiber CSA analyzed per

animal in aged control and NNMT-treated mice (n=6-7 per group). Data represent mean

normalized peak torque (mN*m/µM2) ± SEM.

Figure 5. Effects of NNMTi on C2C12 myoblast differentiation and NAD+ salvage

pathway metabolites in C2C12 myoblasts and myotubes. (A) Representative image of

C2C12 myoblasts immunolabeled to highlight NNMT (green) and nuclei (blue). All

myoblast cells appear to express NNMT. (B) Representative image of C2C12

differentiated myotubes immunolabeled to highlight NNMT (green), myosin heavy chain

(MHC; red), and nuclei (blue). Yellow arrowheads represent NNMT expression in

elongated, differentiation-committed myogenic precursor cells (co-labeled with MHC)

and white arrowheads indicate NNMT expression in fully-differentiated myotubes (co-

labeled with MHC marking myofibers). (C) Relative frequency of MHC positive cells in

control and NNMTi -treated myoblasts at 96 h post-treatment in culture and under

differentiation conditions. Data represent mean % MHC positive cells ± SEM measured

in duplicates per condition. ***, p < 0.001 vs. control. (D) NAD+ levels, (E) NADH levels,

and (F) NAD+/NADH ratio in control and NNMTi-treated (24 hr) C2C12 myoblasts. (G)
NAD+levels, (H) NADH levels, and (I) NAD+/NADH ratio in control and NNMTi-treated

(24 hr) differentiated C2C12 myotubes. Data represent mean slopes calculated from

reaction progress curves (D, E, G, H) or ratios of slopes (F, I) obtained from

spectrophotometric enzyme assays, where the slope is proportional to NAD+ and NADH

concentrations (performed in duplicates across two experiments). *, p < 0.05 and **, p <

0.01 vs. control.

Figure 6. Schematic illustration of pathways regulated by NNMT and proposed working

model of mechanisms of action of NNMT inhibition as it relates to muSC activation and

metabolic state in aged muscle tissue. Maintenance of flux through the NAD salvage

pathway by NNMTi is indicated by gradient blue/red arrow;

(i) increase flux of NAD+, activated SIRT-1, and oxidative metabolism that support

quiescent muSCs are indicated by red arrows; (ii) increased NADH (i.e., increased

NAD+ utilization in glycolysis), enhanced glycolysis, and reduced SIRT-1 mediated

histone deacetylation that support muSC activation and differentiation are indicated by

blue arrows. Pathway abbreviations include AOx1 (aldehyde oxidase), NA

(nicotinamide), MNA (1-methylnicotinmaide), NAMPT (nicotinamide

phosphoribosyltransferase), NMN (nicotinamide mononucleoside), NMNAT

(nicotinamide adenylyltransferase), NNMT (nicotinamide N-methyltransferase), NR

(nicotinamide riboside), NRK (nicotinamide riboside kinase), PNP1 (purine nucleoside

phosphorylase), PYR-2/4 (1-methyl-2/4-pyridone-5-carboxamide), SAH (S-adenosyl-L-

homocysteine), SAM (S-adenosylmethionine), MTases (methyl transferases), SIRT1

(NAD-dependent deacetylase sirtuin 1), NAD+/NADH (oxidized Nicotinamide adenine

dinucleotide/reduced form of NAD+), OXPHOS (oxidative phosphorylation),

Histone*protein-Ac/DeAc (Acetylated/deacetylated histones).

Figure 1.
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Figure. 6