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

Abstract

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.

Keywords: Aging; Exercise; Muscle; NNMT; Nicotinamide N-methyltransferase; Proteomics.

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.

Figure 2

Gastrocnemius histology, proteomics, and metabolomics suggest that NNMTi treatment in Sed mice recapitulates many exercise effects, while NNMTi treatment in exercising mice has disparate effects. Gastrocnemius (gastroc) histology of intramyocellular lipid (IMCL) content (representative histology, [A]) revealed that oxidative fibers demonstrate a significant NNMTi x exercise (PoWeR) interaction (2-way ANOVA main effect of interaction: P = 0.002), with significantly greater IMCL content in oxidative fibers of the Sed cohort relative to the NNMTi-treated Sed cohort or PoWeR cohort [B]. Gastroc glycolytic fibers demonstrate a stronger effect, with significant main effects of NNMTi treatment, PoWeR, and an interaction (2-way ANOVA: P = 0.039, P = 0.019, and P < 0.001, respectively); on average, there was greater IMCL content in the Sed cohort than all others [C]. Principal component analysis (PCA) of the gastroc proteome revealed distinct protein expression in the Sed cohort and PoWeR cohort, with the NNMTi-treated Sed cohort substantially overlapping across these cohorts and the NNMTi-treated PoWeR cohort largely overlapping the PoWeR cohort [D]. The differentially expressed proteins (DEPs) revealed that exercise most dramatically changed the proteome. Still, several NNMTi-regulated proteins with large fold changes are relevant to muscle physiology [E–G]. GOrilla analyses exploring the molecular functions enriched (GOrilla-computed FDR-adjusted P < 0.05) for the DEPs supported that many NNMTi-mediated effects in Sed mice impinge upon the same functions as exercise [H]. The 38 DEPs common to all three comparisons are associated with metabolic processes and stress response [I, J]. The metabolomic PCA mostly recapitulates the proteomic PCA but shows a unique separation of the NNMTi-treated PoWeR cohort from the PoWeR cohort [K]. Similarly, metabolomic results suggested that exercise changes the expression of the greatest number of metabolites but that many NNMTi-regulated metabolites with the greatest fold change are relevant to muscle physiology and metabolism [L–N]. Only one differentially expressed metabolite (DEM) was common in all comparisons [O]. Mean + / − SEM; *P and FDR-adjusted P < 0.05; rRNA ribosomal RNA; tRNA transfer RNA.

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.