Nicotinamide N-methyltransferase inhibition improves limb function in experimental peripheral artery disease

Peripheral artery disease (PAD) impairs limb perfusion, walking ability, and increases the risk of amputation. Although current therapies reduce cardiovascular events, few interventions improve skeletal muscle function in PAD. Nicotinamide adenine dinucleotide (NAD⁺) metabolism is disrupted in PAD. Thus, it was hypothesized that inhibition of nicotinamide N-methyltransferase (NNMT), an enzyme that diverts precursors from the NAD⁺ salvage pathway, would improve ischemic limb function. We analyzed NAD⁺ pathway expression in gastrocnemius muscle from patients with and without PAD using RNA sequencing and proteomics datasets. Single-cell RNA sequencing data were used to assess NNMT expression in muscle stem cells (MuSCs) from BALB/cJ and C57BL/6J mice following hindlimb ischemia (HLI). Male BALB/cJ mice (n = 24) were randomized to either placebo or a NNMT inhibitor (NNMTi) delivered 3 h prior to HLI and daily thereafter. Functional assessments included laser Doppler perfusion imaging, muscle contractility, and a 6-min limb function test. Histological analyses were used to assess myofiber area and capillary density. NNMT mRNA and protein levels were significantly elevated in skeletal muscle from patients with PAD and were persistently elevated in MuSCs from BALB/cJ mice after HLI. NNMTi treatment did not affect limb perfusion recovery or capillary density but trended toward reduced necrosis severity (p = 0.08). Muscle mass and myofiber size were unchanged by treatment; however, NNMTi significantly improved muscle strength (p < 0.0001), power (p = 0.0305), and total work (p = 0.0367) in ischemic limbs compared to placebo. Inhibition of NNMT enhanced ischemic muscle strength and performance in a preclinical model of PAD independent of changes in perfusion.