A combination of metformin and galantamine exhibits synergistic benefits in the treatment of sarcopenia

Abstract

Age-associated sarcopenia, characterized by a progressive loss in muscle mass and strength, is the largest cause of frailty and disability in the elderly worldwide. Current treatments involve nonpharmacological guidelines that few subjects can abide by, highlighting the need for effective drugs. Preclinical models were employed to test the benefits of RJx-01, a combination drug composed of metformin and galantamine, on sarcopenia. In worms, RJx-01 treatment improved lifespan, locomotion, pharyngeal pumping, and muscle fiber organization. The synergistic effects of RJx-01 were recapitulated in a transgenic mouse model that displays an exacerbated aging phenotype (Opa1-/-). In these mice, RJx-01 ameliorated physical performance, muscle mass and force, neuromuscular junction stability, and systemic inflammation. RJx-01 also improved physical performance and muscle strength in 22-month-old WT mice and also improved skeletal muscle ultrastructure, mitochondrial morphology, autophagy, lysosomal function, and satellite cell content. Denervation and myofiber damage were decreased in RJx-01-treated animals compared with controls. RJx-01 improved muscle quality rather than quantity, indicating that the improvement in quality underlies the beneficial effects of the combination drug. The studies herein indicate synergistic beneficial effects of RJx-01 in the treatment of sarcopenia and support the pursuit of RJx-01 in a human clinical trial as a therapeutic intervention for sarcopenia.

Keywords: Aging; Drug therapy; Muscle Biology; Skeletal muscle.

Figure 1. RJx-01 synergistically extends lifespan and improves fitness in C. elegans.

(A) The manual lifespan of worms treated with 25 mM Met, 100 μM Gal, the combination drug RJx-01 (25 mM Met and 100 μM of Gal), or with vehicle (water). For additional data, see Supplemental Table 3. (B–E) Locomotion of worms at day 7 of adulthood in worms treated with 25 mM Met, 100 μM Gal, the combination drug RJx-01, or vehicle (n = 60–80 per group). (F) Pharyngeal pumping at day 7 of adulthood in worms treated with 25 mM Met, 100 μM Gal, the combination drug RJx-01, or vehicle (water) (n = 10). (G) Representative images of muscle morphology at day 14 of adulthood of RW1596 worms treated with the combination drug RJx-01 or vehicle. Scale bar: 10 μm. Data are representative of at least 3 independent experiments. Data are shown as mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001. In A, Kaplan-Meier curves were generated, and statistical significance was calculated using the log-rank test followed by Benjamini-Hochberg. One-way ANOVA followed by Holm step-down method (2-tailed Student’s t test) was performed in B–F.

Figure 2. RJx-01 treatment suppresses muscle mass and quality loss and enhances functional outcomes in muscle-specific Opa1^–/– mice.

(A) Percentage of lean mass of Opa1^–/– mice fed a control diet or a diet supplemented with RJx-01 (Opa1^–/^–, n = 11; Opa1^–/– RJx-01, n = 8). (B) Weights of gastrocnemius muscles of Opa1^–/– mice were fed a control diet, a control diet supplemented with metformin (Met), a control diet supplemented with galantamine (Gal), and a control diet supplemented with RJx-01 (Opa1^–/^–, n = 11; Opa1^–/– Met, n = 6; Opa1^–/– Gal, n = 9; Opa1^–/– RJx-01, n = 8). (C) Myofibers cross-sectional area analysis (Opa1^–/^–, n=11; Opa1^–/– Met, n = 6; Opa1^–/– Gal, n = 9; Opa1^–/– RJx-01, n = 8). Scale bar: 50 μm. (D) Representative images of immunostaining for NCAM expression, and quantification of denervated NCAM⁺ fibers in the respective groups (Opa1^–/^–, n = 10; Opa1^–/– Met, n = 6; Opa1^–/– Gal, n = 8; Opa1^–/– RJx-01, n = 8). (E) Exercise performance on the treadmill expressed as running time (Opa1^–/^–, n = 11; Opa1^–/– Met, n = 5; Opa1^–/– Gal, n = 8; Opa1^–/– RJx-01, n = 8). (F) Force-frequency curves were performed in vivo on gastrocnemius muscles. Absolute force and maximal specific force generated during tetanic contraction in the respective groups (Opa1^–/^–, n = 6; Opa1^–/– Met, n = 3; Opa1^–/– Gal, n = 5; Opa1^–/– RJx-01, n = 5). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ^####P < 0.0001 compared with Opa1^–/– RJx-01. In A and F, a linear mixed-effects model was used, and 1-way ANOVA followed by Holm step-down method (2-tailed Student’s t test) was performed in B–E.

Figure 3. RJx-01 treatment reduces systemic and muscular inflammatory levels.

(A and B) Inflammatory (IL-6, IL-1α, and IL-1β) levels in serum (Opa1^–/^–, n = 7; Opa1^–/– Met, n = 3; Opa1^–/– Gal, n = 4; Opa1^–/– RJx-01, n = 6) (A) and tibialis anterior muscles of Opa1^–/– mice (B) fed a control diet or a diet supplemented with metformin (Met), a control diet supplemented with galantamine (Gal), a control diet supplemented with RJx-01 (Opa1^–/–, n = 5; Opa1^–/– Met, n = 3; Opa1^–/– Gal, n = 4; Opa1^–/– RJx-01, n = 3). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA followed by Holm step-down method (2-tailed Student’s t test).

Figure 4. RJx-01 increases physical performance, without modifying food intake and body weight in aged mice.

(A) Food consumption of aged mice fed a control diet (untreated) and a diet supplemented with RJx-01 (n = 3 cages, 5 mice per cage). (B) Percentage body weight expressed as a ratio over basal body weight (Untreated, n = 10; RJx-01, n = 6). (C) Running time following 18 weeks treatment (Untreated, n = 10; RJx-01, n = 6). Data are shown as mean ± SEM. **P < 0.01 by 2-tailed Student’s t test.

Figure 5. RJx-01 increases muscle strength and reduces the number of denervated fibers in skeletal muscle of aged mice.

(A) Grip test in aged mice treated with and without RJx-01 for 18 weeks (Untreated, n = 10; RJx-01, n = 6). (B) Labeled presynaptic (neurofilament [2H3]; synaptic vesicle 2 [SV2]; green) and postsynaptic (bungarotoxin [BTX]; red) NMJ components in EDL from aged mice supplemented with and without RJx-01 for 18 weeks. Scale bar: 20 μm. At least 200 NMJs were analyzed per mouse. (C) Percentage of denervated fibers in the EDL muscle of untreated and RJx-01–treated mice (n = 3 per group). Data are shown as mean ± SEM. **P < 0.01; ***P < 0.001 by 2-tailed Student’s t test.

Figure 6. RJx-01 protects the skeletal muscle ultrastructure and mitochondrial morphology in aged mice.

(A) Oxygen consumption (VO₂), carbon dioxide production (VCO₂), and energy expenditure (EE) in aged mice treated with RJx-01 for 10 weeks (Untreated, n = 8; RJx-01, n = 4). (B–D) Representative electron micrographs of EDL muscles of aged mice treated with and without RJx-01. (B) Arrows represent sarcomere misalignment. Scale bar: 2 µm. (C) The arrow denotes less electron-dense matrix mitochondria. (D) The arrow points to a swollen mitochondrion. Different magnifications are shown. Scale bars: 500 nm. (E) Number of damaged mitochondria in EDL muscle of untreated and RJx-01–treated mice (number of mitochondria analyzed per mouse, 209–288) (Untreated, n = 3; RJx-01, n = 4). (F) Serum FGF21 and urine GDF15 levels (Untreated, n = 9; RJx-01, n = 6). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by 2-tailed Student’s t test.

Figure 7. RJx-01 increases autophagy in the skeletal muscle of aged mice.

(A) qPCR analysis of transcriptional levels relative to the target genes involved in the autophagic process (Untreated, n = 3; RJx-01, n = 6). (B) Representative Western blot of LC3I/II, P62, and GAPDH of whole protein muscle extract. (C) The relative quantification of B (Untreated, n = 3; RJx-01, n = 6). (D) Representative electron micrographs of EDL muscles of aged mice treated with and without RJx-01. Representative image of an enlarged multivesicular body (MVB). Different magnifications are shown, and scale bar sizes are indicated. Scale bar: 1 µm (left image); 500 nm (middle and right images). (E) Number of MVBs in the muscle of untreated and RJx-01–treated mice (Untreated, n = 6; RJx-01, n = 6). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 by 2-tailed Student’s t test.

Figure 8. RJx-01 reduces the damage in myofibers and increases the number of Pax7⁺ satellite cells in the skeletal muscle of aged mice.

(A) Representative H&E images of the gastrocnemius cross-sections. Arrows show central nuclei. Scale bar: 100 μm. (B) Quantification of myofibers with central nuclei in gastrocnemius muscles as a percentage of total myofibers (Untreated, n = 8; RJx-01, n = 6). (C) Representative images of Pax7 staining. Scale bar: 50 μm. Arrows indicate the nuclei positive for Pax7. (D) Quantification of the percentage of Pax7⁺ cells in the gastrocnemius skeletal muscle of old mice treated with and without RJx-01 (Untreated, n = 5; RJx-01, n = 6). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01 by 2-tailed Student’s t test.