Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle

Abstract

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.

Figure 1. Characterization of mice deficient for Nampt in skeletal muscle

A, Expression of Nampt protein in hindlimb muscles of skeletal muscle-specific Nampt knockout (mNKO) mice compared to floxed littermates. Gastrocnemius (gast.), tibialis anterior (TA), and quadriceps (quad.) of 3-month-old male mice represented. B, Intramuscular NAD content of hindlimb muscles from female (n=8–10) and C, Male (n=4–6) mice assessed at 3 months of age. D, Intramuscular ATP content (n=4–6). E, NAD content of mitochondria isolated from triceps bracii muscle (n=4–5). F, Respiratory capacity of isolated muscle mitochondria sequentially provided with pyruvate (pyr), malate (mal), and ADP, followed by complex V inhibitor oligomycin (oligo), uncoupler (FCCP), finally complex III inhibitor antimycin A (AntA) (n=4–6). G, Mean oxygen consumption (VO₂) normalized to lean body mass (n=7–9) and H, Voluntary ambulatory activity during light and dark hours (n=8–10). I, 24-hour time course of respiratory exchange ratio (RER)(n=7–9, *p<0.05). J, Treadmill running performance (n=6 at baseline). Dashed line indicates exhaustion threshold. K, Body composition assessed by NMR (n=7–9). L, Mass of isolated hindlimb muscles (n=6–10). M, Twitch force and N, Tetanic force generated by isolated EDL muscle. Dashed lines indicated error bars. Scale bars indicate 100 msec. Inset: maximum force generated per subject (n=4–6). Error bars represent SEM. Significance was determined by Student’s t-test (*p<0.05, **p<0.01, ***p<0.001). Mice were 3-month-old males unless otherwise indicated. See also Figure S1.

Figure 2. Loss of salvage NAD synthesis results in progressive muscle degeneration

A, Histological sections of EDL muscle from 3-month-old mice stained with H+E or WGA (wheat germ agglutinin) and DAPI to highlight fiber morphology. The scale bar represents 50 µm. B, Distribution and descriptive statistics of minimum Feret diameters of EDL fibers from 3-month-old mice. Inset: proportion of centrally nucleated fibers (CNF). At least 2.4×10³ fibers from 3 individuals per group were considered. C, Representative hindlimb musculature of 3-month-old mice 16 hours after intraperitoneal injection of Evans Blue Dye (EBD). Inset: Histological sections of whole TA muscle stained with laminin and exhibiting fluorescence of EBD. D, Growth curves from weaning to adulthood (n=7–12 males, 4–15 females). E, Body composition assessed by NMR at 7 months of age (n=5–10). F, Mass of isolated male hindlimb muscles at 7 months of age (n=6–10). G, Treadmill running performance examined at 7 months of age. Dashed line indicates exhaustion threshold (n=6). H, Twitch force and I, Tetanic force generated by isolated EDL muscle. Dashed lines indicated error bars. Scale bar indicates 100 msec. Inset: maximum force generated per subject (n=5–10). J, Histological sections of EDL muscle from 7-month-old mice stained with H+E or WGA and DAPI. The scale bar represents 50 µm. K, Electron micrographs of EDL muscle taken from female mice aged 7 months. Top left: adjacent fibers in a control muscle. Top middle and top right: A disordered necrotic fiber (yellow asterisk) next to a healthy fiber in an mNKO muscle. Bottom left: Grossly normal fiber morphology in an mNKO muscle. Bottom middle: Pre-necrotic fiber morphology showing swollen sarcoplasmic reticulum (yellow arrows) in an mNKO muscle. Bottom right: Phagocytic cell engulfing debris of a necrotic mNKO fiber (yellow asterisk). Error bars represented SEM. Significance was determined by Student’s t-test (*p<0.05,***p<0.001). Mice were male unless otherwise indicated. See also Figure S2.

Figure 3. Muscle Nampt deficiency induces pro-inflammatory and regenerative transcriptional programs coincident with alterations in glucose metabolism

A, Heatmap of gene expression in mNKO muscle with each genes assigned to one of four groups according to k-means clustering. Colors indicate fold change (FC) normalized to group means. B, Selected gene ontology of differentially regulated clusters in B. C, Coverage of sequencing reads (UCSC browser) for transcripts of the muscle-specific ubiquitin ligases Trim63 and Fbxo32. D, Heat map of differentially expressed genes relating to muscle regeneration in mNKO. E, Heatmaps of commonly differentially expressed genes. Left: Control mice aged 24 months vs. 4 months compared to mNKO vs. age-matched controls. Right: The mdx model of muscular dystrophy vs. age-matched wildtype controls compared to mNKO vs. age-matched controls. See also Figure S3, S4, and Table S1. F, Correlation of metabolite abundance between FK866-treated C2C12 myotubes and mNKO quadriceps muscle. Myotubes received 10 nM FK866 for 24 hours. G, Heatmap indicating relative abundance of metabolites detected in 7-month-old male quadricep muscle (n=5–10) and in C2C12 myotubes treated with 10 nM FK866 and 100 µM NR (n=3), subdivided by metabolic pathway. Grey boxes represent undetected metabolites. H, Schematic representation of metabolomic analysis in G. Colored metabolites were differentially detected to varying degrees in mNKO muscle while unchanged metabolites did not reach statistical significance. Location of the oxidative pentose phosphate pathway (oxPPP) and the catalytic activity of Nampt and GAPDH are indicated. Mice were male aged 7 months. See also Figure S4.

Figure 4. NR ameliorates deficits in mNKO mitochondrial function and fiber morphology

A, Treadmill running performance of 7-month-old mice provided with unsupplemented drinking water (Veh) or NR-supplemented drinking water for 6 weeks and B, Concentration of blood lactate at baseline and at the point of exercise exhaustion (n=4–8). C, Distribution and descriptive statistics of minimum Feret diameters of EDL fibers. Inset: proportion of centrally nucleated fibers (CNF). At least 2.0×10³ fibers from 3 individuals per group were considered. D, Respiratory capacity of muscle mitochondria provided with excess palmitoyl carnitine or E, Excess pyruvate (n=4–6, significant changes relative to mNKO Veh). F, Intramuscular ATP content (n=4–7) in quadriceps muscle following NR treatment. G, Intramuscular NAD content (n=4–7) and H, Mitochondrial NAD I, Relationship between oxygen consumption and NAD content of mitochondria isolated from skeletal muscle (n=4–6). The line indicates a rectangular hyperbolic least squares regression and associated correlation coefficient. J, Relationship between complex I-dependent respiration and basal NAD content of mitochondria isolated from C2C12 myotubes treated for 24 hours with 10 nM FK866 and 0–300 µM NR. The line indicates a rectangular hyperbolic least squares regression and associated correlation coefficient. Error bars represented SEM. Significance was determined by one-way ANOVA with Tukey’s post-hoc test (ns, not significant, *p<0.05,***p<0.001). Mice were female aged 7 months at sacrifice.

Figure 5. NR reverses deficits in muscle mass, strength, and exercise capacity of mNKO mice

A, Treadmill performance of 5.5-month-old mice before beginning NR administration (n=5–6). B, Treadmill performance after 1 week of treatment and C, Change in exhaustion time of individual mice at the point of exhaustion across the first week of treatment (n=5–6). D, Treadmill performance after 5 weeks of NR treatment (n=5). E, Mass of hindlimb muscles isolated after 6 weeks of NR administration (n=5–6). F, Twitch force and G, Tetanic force generated by isolated EDL muscle. Dashed lines indicated error bars. Scale bars indicate 100 msec. Insets: maximum force generated per subject (n=5–6). H, Intramuscular NAD content and I, Intramuscular ATP content assessed in quadriceps after 6 weeks of treatment (n=5–6). Error bars represented SEM. Significance was determined by one-way ANOVA with Tukey’s post-hoc test (ns, not significant, *p<0.05,***p<0.001). Mice were male aged 7 months at sacrifice. See also Figures S5 and S6.

Figure 6. Oral bioavailability of NR to muscle

A, Molecular structure of the NR^M+2 mass isotopologue. Green and red arrows indicate locations of ¹³C and deuterium labels, respectively. B, Schematic of proposed NR^M+2 entry into the NAD salvage pathway (dashed lines, ATP omitted) and the subsequent separation of the isotopes on ribose and NAM moieties. Basal metabolic redox processes do not affect the arrangement of the labels. C, Incorporation pattern of heavy isotopes into intermediates of the NAD salvage pathway in skeletal muscle and D, Liver tissue 100 minutes after oral gavage with 200 mg/kg NR^M+2 (n=3). E, Detection of NR isotopologues in quadriceps muscle and F, Liver tissue 100 minutes after oral gavage with 200 mg/kg NR^M+2. Some isotopologues were not detected (n.d.) in muscle (n=3). G, Appearance of blood metabolites after oral gavage with 200 mg/kg NR^M+2 and H, Fractional labeling in blood after 100 minutes (n=3 per genotype, pooled). NR was detected in trace amounts. Error bars represented SEM. Significance was determined by one-way ANOVA with Tukey’s post-hoc test (ns, not significant, *p<0.05,**p<0.01). Mice were female aged 7 months.

Figure 7. Lifelong elevation of muscle Nampt expression increases exercise capacity of aged mice

A, Expression of exogenous Nampt protein persists in muscle until 24 months of age. B, Age-related alterations in intramuscular NAD content assessed in gastrocnemius muscle of 4-month-old (4 mo) and 24-month-old (24 mo) mice with one (mNTG^het) or two (mNTG) copies of the Nampt transgene (n=4–11). C, Body weights of old mice were moderately affected by the transgene (n=7–14). D, Treadmill performance in 24-month-old mice. Horizontal dashed line indicates the exhaustion threshold. The blue dashed line indicates the performance of young control littermates. Inset: time elapsed at the point of exhaustion (n=7–14). E, Histogram and descriptive statistics of stimuli received by group and binned by running distance at the point of exhaustion, as described in D. Vertical dashed lines indicate group means. The blue arrow indicates the mean running distance of young control littermates. F, Correlation plot representing RNAseq gene expression profiling in quadricep muscle in 24-month-old mice (n=3–5). Labeled red dots indicate significant differences in expression (Padj, adjusted p-value) with associated gene names indicated. G, Heatmap of relative gene expression reveals negligible impact of sustained NAD salvage on transcriptional programs of muscle with age. Genes in mNTG are presented based on significant differences in expression between controls aged 4 vs. 24 months. H, Overlap in differentially regulated genes of control and mNTG muscle between 4 and 24 months of age. There were no significantly opposing transcripts between the genotypes. Young control data are reproduced from previously published results (Frederick et al., 2015). Error bars represented SEM. Significance was determined by one-way ANOVA with Tukey’s post-hoc test (ns, not significant, *p<0.05,***p<0.001). Mice were male of respective ages. See also Table S2.