NAD+ enhanced mesenchymal stromal cells effect on muscle atrophy by improving SIRT1/PGC-1α-mediated mitochondrial function via NAMPT

Background: Sarcopenia contributes to all-cause mortality in the elderly; however, there is no specific treatment. Mesenchymal stromal cells (MSCs) ameliorate age-related muscle loss and dysfunction, and are potential therapeutic candidates for sarcopenia. However, their activity is easily affected by the surrounding environment and they are prone to replicative senescence during in vitro culture. Therefore, a drug that delays aging and enhances its function is required. Here, we investigated whether nicotinamide adenine dinucleotide (NAD+) pretreatment enhances the therapeutic efficacy of MSCs on skeletal muscle atrophy and its underlying mechanism in a D-gal-induced aging model. Methods: C57BL/6J mice and C2C12-differentiated myotubes exposed to D-gal were used to explore the effects of MSCs/NAD+-MSCs on muscle atrophy. MSCs/NAD+-MSCs were injected into the skeletal muscles of the hind limbs every 7 days for six cycles. Treadmill running and grip strength tests were used to evaluate the muscle strength. Muscle weight and fiber cross-sectional area (CSA) were used to measure muscle mass. Multiomics analysis of quadriceps and NAD+-pretreated MSCs (NAD+-MSCs), western blotting of muscle atrophy signaling, including Atrogin 1 and MuRF1, the mitochondrial complex, fatty acid oxidation indicators, and Seahorse analysis were performed to explore the underlying mechanisms. Results: MSCs increased grip strength (p=0.0005), running endurance (p=0.0006), and muscle mass (p=0.0165 for tibialis anterior (TA) muscle, p=0.0049 for soleus (SO) muscle) in D-gal-treated mice, with elevated muscle fiber CSA (p<0.0001), and reduced Atrogin1 (p=0.0242) and MuRF1 expression (p=0.0009). NAD+ pretreatment increased the effect of MSCs on muscle atrophy (p=0.0009 for grip strength, p=0.0169 for running endurance, p=0.0506 for TA muscle weight, p=0.0238 for SO muscle weight, p=0.0002 for muscle fiber CSA, p=0.0005 for Atrogin1 expression and p=0.0223 for MuRF1 expression). MSCs/NAD+-MSCs activated the SIRT1/PGC-1α signaling, enhanced mitochondrial function and fatty acid oxidation in D-gal-induced mice and C2C12 myotubes. SIRT1 knockdown weakened the beneficial effects of MSCs/NAD+-MSCs on muscle atrophy. RNA-seq of MSCs/NAD+-MSCs and proteomic analysis of their supernatants revealed that NAD+ enhanced the therapeutic effect of MSCs by promoting NAMPT secretion. Conclusions: NAD+ enhances the therapeutic effect of MSCs on age-related muscle atrophy by promoting NAMPT secretion, which acts on the SIRT1 signaling pathway, and improves mitochondrial function and fatty acid oxidation in skeletal muscles. This study provides new insights and a theoretical basis for the clinical treatment of sarcopenia.