Nicotinamide N-methyltransferase inhibition mimics and boosts exercise-mediated improvements in muscle function in aged mice

Human hallmarks of sarcopenia include muscle weakness and a blunted response to exercise. Nicotinamide N-methyltransferase inhibitors (NNMTis) increase strength and promote the regenerative capacity of aged muscle, thus offering a promising treatment for sarcopenia. Since human hallmarks of sarcopenia are recapitulated in aged (24-month-old) mice, we treated mice from 22 to 24 months of age with NNMTi, intensive exercise, or a combination of both, and compared skeletal muscle adaptations, including grip strength, longitudinal running capacity, plantarflexor peak torque, fatigue, and muscle mass, fiber type, cross-sectional area, and intramyocellular lipid (IMCL) content. Exhaustive proteome and metabolome analyses were completed to identify the molecular mechanisms underlying the measured changes in skeletal muscle pathophysiology. Remarkably, NNMTi-treated aged sedentary mice showed ~ 40% greater grip strength than sedentary controls, while aged exercised mice only showed a 20% increase relative to controls. Importantly, the grip strength improvements resulting from NNMTi treatment and exercise were additive, with NNMTi-treated exercised mice developing a 60% increase in grip strength relative to sedentary controls. NNMTi treatment also promoted quantifiable improvements in IMCL content and, in combination with exercise, significantly increased gastrocnemius fiber CSA. Detailed skeletal muscle proteome and metabolome analyses revealed unique molecular mechanisms associated with NNMTi treatment and distinct molecular mechanisms and cellular processes arising from a combination of NNMTi and exercise relative to those given a single intervention. These studies suggest that NNMTi-based drugs, either alone or combined with exercise, will be beneficial in treating sarcopenia and a wide range of age-related myopathies.

Grip strength and physical function measures Mice underwent the study design outlined in Fig. 1 A to assess NNMTi treatment and exercise interventions for age-associated muscle weakness. Both PoWeR cohorts had an ~ 8% decline in body weight (BW) in study week 1, while the Sed groups’ BWs remained essentially unchanged throughout the study (Fig. 1 B, Supplemental Fig. 1 ). Figure 1 Muscle performance is enhanced by NNMTi treatment, exceeding exercise effects in Sed mice and additively improving performance in exercised mice. Twenty-two-month-old mice were randomized to PoWeR or Sed groups, with NNMTi treatment or vehicle control [ A ]. Longitudinal measures of change in body weight (BW) from baseline displayed a significant effect of study cohort (2-way repeated measures ANOVA, main effect of study cohort P = 0.004 [ B ]). Average grip strength, analyzed at the end of week (wk) 6, revealed that both NNMTi treatment and exercise improved grip strength (2-way ANOVA, main effect of NNMTi treatment P < 0.001 and exercise P = 0.042; [ C ]). Similar results were observed when grip strength was normalized to BW (BWN; 2-way ANOVA main effect of NNMTi treatment P = 0.005 and exercise P = 0.002; NNMTi Sed vs Sed, P < 0.06 [ D ]). Results for [ C ] and [ D ] are shown scaled to the Sed cohort (unscaled data shown in Supplemental Fig. 1 ). Relative change in average daily running distance from wk 1 in PoWeR-trained mice revealed a significant effect of group (wk 1 not included in the analysis; Mixed Effects Model: P = 0.0039; main effect of time not significant); posthoc testing showed significant differences between groups from weeks 5–8 [ E ]. Mean + / − SEM; * P < 0.05;a, PoWeR vs Sed; b, PoWeR NNMTi vs PoWeR; c, PoWeR NNMTi group, P < 0.05 vs. Wk 3; d, PoWeR group, P < 0.05 vs. Wk 7–8; e, PoWeR group, P < 0.05 vs. Wk 7 only. Forelimb grip strength was chosen to measure muscle function since it has been repeatedly shown that C57BL/6 mouse forelimb grip strength begins to decline at or before 24 months of age 18 – 20 . Additionally, progressive weighted wheel running has been shown to significantly improve forelimb grip strength in young (< 8-month-old) C57BL/6 mice 21 . Grip strength measured during week 6 of our study showed main effects of both exercise and NNMTi treatment. Notably, the NNMTi-treated Sed and PoWeR cohorts had ~ 40% and 20% greater grip strengths, respectively, relative to the untreated (control) Sed cohort. Moreover, the NNMTi-treated PoWeR cohort had ~ 60% greater grip strength than the Sed cohort (Fig. 1 C, Supplemental Fig. 1 ), suggesting that combining NNMTi and PoWeR treatments produced additive effects. Normalizing grip strength to BW did not dramatically change the observed improvements noted in the NNMTi-treated cohorts relative to the untreated cohorts. However, BW normalization did increase the relative strength in the untreated PoWeR cohort since these animals had the lowest BWs relative to the other cohorts (Fig. 1 D, Supplemental Fig. 1 ). During the first three weeks of our study, both PoWeR cohorts showed increases in their average weekly running distances (Supplemental Fig. 1 ). Given differences in the initial average weekly running distance between the cohorts, data were analyzed as the change in running relative to week 1 (Fig. 1 E, Supplemental Fig. 1 ). By week 3, NNMTi-treated PoWeR mice were running an additional 3.0 km/day compared to week 1 (a 250% increase), whereas the PoWeR cohort only ran an extra 2.3 km/day compared to week 1 (a 170% increase). The NNMTi-treated PoWeR mice largely maintained their improved running capacity through week 8, running ~ 1.8 km/day more in week 8 compared to week 1 (Supplemental Fig. 1 ). In contrast, by weeks 7 and 8, the average daily running distance of the PoWeR cohort had returned to week 1 levels (Fig. 1 E). Importantly, the change in running distance relative to week 1, averaged by week, throughout the study showed a significant difference between treatment group, with the NNMTi-treated PoWeR group running significantly more than the PoWeR group weeks 6–8 (Fig. 1 E). These results suggest that NNMTi treatment improved tolerance to, or recovery from, intensive exercise. Peak plantarflexor torque and BW-normalized (BWN) peak plantarflexor torque (measured at the end of week 8) were not significantly changed by NNMTi or PoWeR (Supplemental Fig. 1 ). Fatigue, measured as the number of contractions ≥ 70% or ≥ 50% of the maximum peak torque observed during a 54-bout test, and cumulative work across the fatigue test, were used to further assess plantarflexor complex (i.e., gastrocnemius, plantaris, and soleus) function. A clear trend emerged for reduced susceptibility to fatigue in NNMTi-treated mice relative to matched controls (Supplemental Fig. 1 ; Supplemental Table 1 ; main effect of NNMTi treatment on repetitions above 70% in the fatigue test, P = 0.098). This may directly relate to the significantly reduced cumul

Global proteomic analysis of the gastrocnemius was completed to uncover molecular mechanisms responsible for the observed differences in muscle function, mass, and composition. A common set of 2,512 proteins were identified, quantified, and analyzed in all samples. Principal component analysis (PCA) of the quantified proteins showed distinct Sed cohort and PoWeR cohort clusters. The NNMTi-treated Sed cohort substantially overlapped across the Sed cluster and PoWeR cluster. The NNMTi-treated PoWeR cluster largely overlapped the PoWeR cluster (Fig. 2 C). We identified 346 differentially expressed proteins (DEPs; 281 upregulated, 65 downregulated; Fig. 2 D) from NNMTi treatment in the Sed context (NNMTi-treated Sed vs. Sed), 288 DEPs (182 upregulated, 106 downregulated; Fig. 2 E) from NNMTi treatment in the exercise context (NNMTi-treated PoWeR vs. PoWeR), and 943 DEPs (734 upregulated, 209 downregulated; Fig. 2 F) from exercise (PoWeR vs. Sed). The number of DEPs between study cohorts aligned with the PCA plot; the comparisons with the largest number of DEPs (PoWeR vs. Sed) had the least PCA plot overlap, while those with fewer DEPs had greater overlap. Several DEPs in the NNMTi-treated Sed cohort vs. the Sed cohort were associated with 60S and 40S ribosomal complexes and were significantly upregulated (Supplemental Table 2 ). Other upregulated DEPs across these cohorts were involved in cytoskeletal structure and regulation ( e.g. , protein flightless-1 homolog, nexilin, coronin-1A, leiomodin-3, obscurin-like protein 1), immune signaling ( e.g. , immunoglobulin [Ig] heavy variable 14–4, Ig kappa variable 4–63, Ig lambda constant 1), and ubiquitination ( e.g. , tripartite motif-containing protein 16, ubiquitin-conjugating enzyme E2 R2, Ubiquitin carboxyl-terminal hydrolase 13). The most upregulated DEPs in NNMTi-treated Sed vs. Sed mice included β-taxilin, desmin, and the leukemia inhibitory factor receptor. The most downregulated DEPs included serine protease inhibitors ( i.e. , murinoglobulin-1 [Mug1], serine protease inhibitor [serpin] A3M) and metallothionein-1 and -2 (MT1, MT2; Supplemental Table 2 ). The most highly upregulated DEPs between NNMTi-treated PoWeR and PoWeR mice included ubiquitin carboxyl-terminal hydrolase 13, β-taxilin, Kelch-like protein 31, smoothelin, nexilin, and protein tyrosine phosphatase type IVA 2. The most downregulated DEPs included MT1 and MT2, peptidyl-prolyl cis–trans isomerase FKBP5, transforming acidic coiled-coil-containing protein 2, and Mug1 (Supplemental Table 2 ). The serpin A1e and A3n proteins were significantly downregulated by NNMTi treatment only in the exercise context, indicating that NNMTi treatment may impact additional unique pathways when combined with exercise. These DEPs and their associated pathways could be responsible for increasing gastrocnemius Type IIb fiber CSA, as was only observed in NNMTi-treated PoWeR mice. They may offer new drug targets for increasing muscle mass and function. NNMTi treatment in the Sed or the exercise context significantly changed the expression levels of only a limited number of proteins (on average, treatment modulated ~ 10% of the > 2,500 proteins analyzed; Supplemental Table 2 ), although many of these DEPs were common to both comparisons. Three proteins, BCL2 interacting protein 3 (Bnip3), MT1, and MT2, significantly downregulated by NNMTi treatment in both the Sed and exercise contexts, have been definitively linked to skeletal muscle atrophy 35 – 39 . Furthermore, each protein’s expression level was significantly and negatively correlated with the gastrocnemius pooled CSA (Spearman’s correlation, P < 0.05 for each, simple linear regression R 2 = 0.331, 0.423, and 0.462 for Bnip3, MT1, and MT2, respectively). Exercise treatment alone (PoWeR vs. Sed) resulted in ~ 38% of all analyzed proteins being differentially expressed (Supplemental Table 2 ). Some mitochondrial proteins ( e.g. , mitochondrial chaperone BCS1, mitochondrial nicotinamide adenine dinucleotide [NAD] transporter SLC25A51), including several involved in β-oxidation ( e.g. , acyl-CoA dehydrogenases), were uniquely upregulated by exercise alone. However, many mitochondrial proteins ( e.g. , hydrogenated NAD [NADH] dehydrogenase subunits, 39S ribosomal proteins) and several immunoglobulins ( e.g. , kappa variable 4–63, lambda constant 1, heavy variable 14–4) were consistently upregulated by exercise and NNMTi treatment of Sed mice. DEPs were mapped to GO-defined molecular functions to elucidate the cellular processes that respond to NNMTi treatment, exercise, or their combination (Fig. 2 G). NNMTi treatment of Sed mice was significantly associated with nucleic acid and ribosomal RNA binding, and ribosome structure. Interestingly, the ribosome structure and nucleic acid binding molecular functions were also enriched for exercise-produced DEPs (PoWeR vs. Sed). NADH dehydrogenase activity was independently enriched for exercise-produced DEPs. Many functio

Resistance or combined resistance and endurance exercise are the current standard of care treatments that attempt to slow age-associated loss of muscle mass and strength 40 , 41 . This study showed that NNMTi treatment could surpass the muscle health benefits of resistance and endurance exercise. NNMTi treatment dramatically increased grip strength in both aged Sed and aged exercised animals, suggesting that treatment prevents age-associated loss of strength and actively rebuilds strength. Importantly, NNMTi treatment almost doubled the increase in muscle strength compared to exercise alone. Moreover, the increases in muscle strength resulting from NNMTi treatment or exercise were additive, with the strength of the NNMTi-treated exercise cohort being increased by ~ 60% compared to the Sed cohort. Grip strength has been associated with glutathione homeostasis 42 , and glutathione regulation appears to be modulated, at least in the NNMTi-treated exercise group relative to the exercise group. Although both grip strength and plantarflexor strength heavily rely on force exertion 43 , the prioritization of the rate of force generation may differ for the muscle groups. Thus, unsurprisingly, peak torque did not correlate with grip strength in any cohort (data not shown). Prior work demonstrates progressive declines in many measures of functional capacity and strength with increasing age, although it is noted that the declines vary substantially and in a non-linear manner 18 , 44 . In response to voluntary wheel running (both weighted and unweighted) in aged mice, improvements in grip strength without concomitant improvements in plantarflexor strength have been observed, similar to our findings 45 – 47 . Thus, incongruency between these strength measures may be expected. NNMTi-treated exercised mice increased their initial running capacity and sustained this increase throughout the study. In contrast, control-treated exercised mice showed a temporary improvement in running capacity that was not sustained throughout the study. This suggests that NNMTi treatment enhanced tolerance to exercise, consistent with the tendency for reduced susceptibility to muscle fatigue observed in NNMTi-treated animals. The sustained running capacity in NNMTi-treated animals may also be tied, in part, to improved recovery from exercise-associated injury, consistent with work in a different muscle injury model 16 . NNMTi treatment and intensive exercise were associated with numerous upregulated proteins involved in protein translation (Fig. 3 , Supplemental Fig. 3 ). This likely accounts for the increased gastrocnemius weight observed with exercise and the increased CSA observed with NNMTi treatment. Protein upregulation may relate to the fact that AMP was upregulated in the exercised and NNMTi-treated Sed cohorts, each relative to the Sed cohort. AMP’s upregulation could drive AMPK activation, which has been implicated in energy-sensing, muscle hypertrophy, and protein translation 48 – 50 . Additionally, high IMCL content correlates with poorer muscle quality/functional performance 51 , increased skeletal muscle insulin resistance 52 , and reduced myofiber contraction velocity, force, and power 53 . NNMTi treatment and intensive exercise independently reduced IMCL content and increased grip strength relative to the Sed cohort, suggesting improved muscle quality with both interventions. Figure 3 –Omics results mapped to potential downstream pathways suggest different NNMTi treatment-mediated mechanisms in exercised and Sed contexts. In Sed mice, NNMTi treatment upregulates several components critical to protein translation. In exercised (PoWeR) mice, NNMTi treatment upregulates transsulfuration pathway components, suggesting increased protection against reactive oxygen species damage. Critical elements of protein translation and β-oxidation are upregulated in exercised mice relative to Sed mice. Together, the data support that downstream mechanisms of NNMTi treatment vary with the cellular context and that some mimic exercise. Gene names corresponding to the proteins are italicized when listed. Other abbreviations are: 1-MNA 1-methylnicotinamide; ADAR adenosine deaminase acting on RNA; CBS cystathionine β-synthase; HGPRT hypoxanthine–guanine phosphoribosyltransferase; IMP inosine monophosphate; IRS-1 insulin receptor substrate 1; NAMPT nicotinamide phosphoribosyltransferase; NMN nicotinamide mononucleotide; NMNAT NMN adenylyltransferase; PNP purine nucleoside phosphorylase; SAH S-adenosyl-L-homocysteine; SAM S-adenosylmethionine. There were significant increases in plantarflexor complex muscle masses and CSA in the exercised cohorts, with the CSA increases occurring mainly in glycolytic (Type IIb, Type IIx) fibers of the gastrocnemius and plantaris and oxidative (Type I, Type IIa) soleus fibers (Table 1 ). These results align with previous work in aged mice 13 , which showed an exercise-mediated increase in the BWN soleus weight and soleu

Animal procedures were performed per all national and local guidelines and regulations, the Guide for the Care and Use of Laboratory Animals, the ARRIVE guidelines, and the approval of the Institutional Animal Care and Use Committee (protocol #2019-3301) at the University of Kentucky. Aged (22-mo) female mice (n = 35), obtained from the US NIH National Institute on Aging (NIA) Aged Rodent Colony, were randomly assigned to one of four groups: Sed (saline; n = 9), Sed NNMTi-treated (10 mg/kg BW; n = 9), progressive weighted wheel running (PoWeR; saline; n = 10), and PoWeR NNMTi-treated (10 mg/kg BW; n = 7). Our published 16 and unpublished studies show this NNMTi dose promotes effective muscle regeneration post-injury in aged mice; Supplemental Fig. 4 shows data from a pharmacokinetic study for NNMTi 5A-1MQ; additional method details are included in Supplemental File 1 . Results suggest rapid and substantial absorption of 5A-1MQ post-subcutaneous dosing, a robust half-life (~ 7 h), and an absence of accumulation with repeat dosing. Female mice were chosen for this study since previous work have shown that they run longer distances than males in this age range 75 , including when the wheel is weighted in the PoWeR model 76 . Since mice were randomized by cage, slight differences across cohorts in BW mean and variation existed at the study start, with the mice assigned to NNMTi treatment roughly 10% heavier than their control counterparts. All mice received daily subcutaneous injections of saline or NNMTi and were weighed weekly to adjust dose volumes. Group-housed Sed cohorts were compared to singly-housed PoWeR cohorts that underwent a 1-week introduction to an unweighted wheel, followed by eight weeks of weighted wheel running under the progression outlined in Fig. 1 . Forelimb grip strength was assessed by a single NNMTi treatment-blinded investigator during week six and averaged across 2–4 trials/mouse. At the end of week 8, the strength of the right limb plantarflexor muscle complex was measured using an in vivo isometric peak tetanic torque technique. Animals were anesthetized using ~ 2.5% isoflurane, and the tibial nerve was stimulated. During this procedure, electrode placement was optimized to minimize antagonistic dorsiflexion. An ideal amperage was defined and maintained during a force-frequency experiment to establish peak tetanic torque. Immediately after, each mouse underwent a fatigue test of 54 submaximal contractions at 60 Hz. The peak torque of each contraction peak torque and the maximum for all contractions were computed. The number of contractions with peak torque values exceeding 50% and 70% of that maximum were defined for each mouse. Cumulative work was calculated by summing the integral of each contraction after correction for baseline force. Data for fatigue and cumulative work calculations were not recorded for three Sed cohort mice; additionally, two Sed cohort mice, one NNMTi-treated Sed mouse, and one NNMTi-treated PoWeR mouse exhibited cumulative workloads that suggested an inability to perform the task. After the fatigue test, mice were euthanized, and tissues were weighed and collected. Of note, hind limb muscles from the right limb (the limb that underwent in vivo isometric peak tetanic torque and fatigue testing) were processed for immunohistochemistry to avoid acute effects of muscle functional testing on the proteome and metabolome. Hind limb muscles from the left limb that did not undergo torque and fatigue testing were flash-frozen for proteome and metabolome analyses. Given the terminal BW differences between the Sed and PoWeR groups and the potential for differences in the sarcopenic state of the animals, BWN values are emphasized. However, raw muscle masses are reported in Supplemental Table 1 for completeness. The solei of one Sed mouse and one NNMTi-treated PoWeR mouse were not collected. Further details on housing conditions and protocols for in vivo and euthanasia methods are in Supplemental File 1 .

To avoid altering molecular signaling in the gastrocnemius muscle right before collection, the contralateral limb (i.e., the limb not used for terminal torque and fatigue measurements) was immediately flash-frozen when the animals were euthanized, and it was this flash-frozen unstressed muscle that was used for -omics sample preparation. The gastrocnemius samples used for -omic analyses were processed in two batches, one with the Sed group samples (n = 8 Sed; n = 8 NNMTi-treated Sed) and the other with the PoWeR group samples (n = 7 PoWeR; n = 7 NNMTi-treated PoWeR). Reagents and equipment used for sample processing are outlined in Supplemental Table 4 , with protocols outlined in Supplemental File 1 . All samples were diluted to 2 mg/mL.

Supplemental File 1 includes additional metabolomic workflow and analysis details; reagents and equipment are in Supplemental Table 4 . In brief, samples from all groups were simultaneously analyzed for metabolites and heavy isotope standards within a single set of mass spectroscopy experiments; mass spectrometrists were blinded to the NNMTi treatment cohort. One PoWeR control sample was excluded for issues during sample preparation of its aliquot. Experiments used the ESI ion spray source in positive and negative ion modes and hydrophilic interaction liquid chromatography. Data were processed by SCIEX MultiQuant (v3.0.3, https://sciex.com/products/software/multiquant-software ) software with relative quantification based on each metabolite’s peak area. As noted in Supplemental Table 3 , the CV was < 25% between quality control samples run at the beginning and end of the positive and negative ion mode mass spectrometric runs, suggesting the system was working correctly. Additionally, the CVs for the heavy isotope standards remained under 15% for both runs, reinforcing that there were no issues throughout the ionization process. The approach to determining DEMs was similar to that for DEPs. DEMs discussed herein were significant before correction for multiple comparisons; FDR-corrected P values are reported in Supplemental Table 3 . Quantifications from the negative and positive polarity column analyses are reported for metabolites; when relevant (i.e., glutathione, S-adenosyl-L-homocysteine), both results are reported since these were processed separately.

Supplementary Figures. Supplementary Tables. Supplementary Information 1. Supplementary Information 2.