Heme Metabolism-Derived Carbon Monoxide Regulates Skeletal Muscle Function

Heme oxygenases, HO-1 (Hmox1) and HO-2 (Hmox2), regulate skeletal muscle homeostasis by degrading heme and generating carbon monoxide (CO), a bioactive signalling molecule. Although HO-1 is known to influence muscle fibre composition and mitochondrial function, the role of HO-2 in activity-dependent neuromuscular plasticity remains poorly understood. This study aimed to define the distinct contributions of each isoform and test whether CO could restore muscle function in HO-deficient states.

We generated Hmox1/2 double-knockout mice (Hmox1/2^-/-) and compared their skeletal muscle phenotype with that of single HO-1 or HO-2 knockouts and wild-type (WT) controls under sedentary and exercised conditions. We evaluated endurance capacity using treadmill running (n = 8-12 per group), assessed fibre-type distribution and neuromuscular junction (NMJ) morphology via immunohistochemistry and measured mitochondrial function using high-resolution respirometry. Primary neuronal cultures were analysed using multielectrode array recordings to assess firing dynamics. Inhaled CO was administered to test its capacity to rescue muscle phenotype and performance.

HO-1 deficiency led to a significant reduction in oxidative fibres (Type I and IIa), decreased mitochondrial respiratory capacity (reduced by ~30%, p < 0.01) and diminished treadmill endurance (-40% running time vs. WT, p < 0.001). Hmox2 deficiency was associated with NMJ remodelling, increased acetylcholine receptor expression, reduced Sox2 transcription and heightened burst firing. The double deletion of HO-1/HO-2 produced an additive phenotype characterized by severe mitochondrial dysfunction, increased glycolytic fibre content and NMJ remodelling. We identify CO, a by-product of HO-1, as a crucial modulator of skeletal muscle adaptation, capable of compensating for HO deficiency. Treatment with CO in Hmox1/2^-/- mice restored fibre-type distribution toward oxidative fibres (increased by 25%, p < 0.01), improved mitochondrial respiratory parameters and doubled endurance performance (p < 0.001). CO also normalized mitochondrial protein expression and modulated key metabolic pathways, including nucleotide metabolism, the TCA cycle and redox balance.

HO-1 and HO-2 have distinct roles in regulating muscle phenotype and metabolic adaptation. HO-1 modulates mitochondrial content and muscle plasticity, whereas Hmox2 regulates, in part, activity-dependent neuromuscular plasticity and responsiveness to exercise. Exogenous CO effectively restores mitochondrial and functional deficits in HO-deficient muscle, mimicking endurance exercise adaptations. These findings support the therapeutic potential of CO in conditions of muscle disuse, aging or disease where exercise is limited or not feasible.