Journal Article

Heme Metabolism‐Derived Carbon Monoxide Regulates Skeletal Muscle Function

Harbison, James, Otterbein, Leo E., Alves de Souza, Rodrigo W., In Kim, Hyo, Shankar, Sidharth, Alves, Paula Ketilly Nascimento, Rasmussen, Ashlee, Voltarelli, Vanessa Azevedo, da Paixão, Ailma Oliveira

2026

Overview

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.

• Publisher: John Wiley & Sons, Inc,Wiley
• Subject: Aerobics / Animals / Carbon Monoxide - metabolism / Enzymes / Exercise / Fitness equipment / Fitness training programs / Heme - metabolism / Heme Oxygenase (Decyclizing) - genetics / Heme Oxygenase (Decyclizing) - metabolism / Heme Oxygenase-1 - genetics / Heme Oxygenase-1 - metabolism / Histology / Metabolism / Mice / Mice, Knockout / Muscle, Skeletal - metabolism / Muscle, Skeletal - physiology / Musculoskeletal system / Neuromuscular Junction - metabolism / Original / Physical fitness / Physiology / Protocol / Running / Workloads