Myoglobin-derived iron causes wound enlargement and impaired regeneration in pressure injuries of muscle

Abstract

Pressure injuries are classified as chronic wounds, but the reasons for poor healing are not well understood, such as whether impairments are due to comorbidities and bacteria. Chronic vascular ulcers have pathologies caused by extracellular release of hemoglobin, so we questioned whether muscle pressure injuries (mPI) would have intrinsic pathologies due to extracellular myoglobin (Mb), and whether iron chelation therapy could improve them. Healthy mice in specific-pathogen-free conditions received pressure injuries. Cohorts were Mb-/- versus Mb+/+ in elderly 20-month-olds, or adult Mb+/+ 5-month-olds treated with saline versus iron chelator (subcutaneous deferoxamine, DFO). For comparison, healthy control regeneration was created using cardiotoxin injection. Unlike acute injuries, the wound bed of mPI displayed failure of phagocytosis at day 3, accompanied by high accumulation of iron, oxidative damage, and a lack of viable immune cells. The necrotic tissue was later expelled as slough (eschar). Mb-knockout mPI had undetectable levels of iron, low oxidative stress (p<0.05), greater immune infiltration (230%, p<0.05), 4-fold decreased marker for extracellular traps (p<0.01), and 50% smaller area of tissue death. Similar to knockout, injecting DFO after wounding caused improvement in oxidative stress (2-fold, p<0.05), extracellular trap marker (4-fold, p<0.01), and diameter of tissue killed (35%, p<0.05). In other words, tissue margins could be protected from dying. At later timepoints, DFO-treated wounds had higher levels of viable immune infiltrate (p<0.05), greater extent of muscle regeneration (46% smaller diameter of gap, p<0.01), and superior myofiber morphology (p<0.0001). We conclude that myoglobin iron from mPI causes oxidative stress and delayed infiltration, despite a lack of infection. Remarkably, tissue viability could be salvaged by post-injury treatment with subcutaneous injections of an FDA-approved iron chelator. This work also sheds light on how a hostile wound microenvironment impairs myogenesis and morphogenesis.

Contributions of individual satellite cells to muscle regeneration assessed using a confetti mouse model

“…Animal experiments were approved by the institutional animal care and use committee of the National University of Singapore. To conditionally label the Pax7 expressing cells, we crossed the Pax7-Cre-ER T2 with the R26R-Confetti mouse, similar to our earlier study (Nasir et al, 2023). The Cre-ER T2 fusion protein is located downstream of the Pax7 stop codon and will only be expressed in cells expressing Pax7.…”

“…The transgenic mouse model (Tierney et al, 2018;Nasir et al, 2023) was validated (Supplemental Text 1, Fig. S1) and we measured the background rate of muscle repair by counting the number of fluorescent fibers at 25 days after the last tamoxifen injection.…”

“…To track the contributions of individual SCs during muscle regeneration, we used a mouse model in which the satellite cell lineage is traced by inducible expression of four fluorescent "confetti" colors (Tierney et al, 2018;Snippert et al, 2010). We then use this mouse model to study 16-day regeneration after a single-dose injection of cardiotoxin in adult mice, similar to our earlier study looking at acute cardiotoxin injury in the panniculus carnosus muscle (Nasir et al, 2023). The single injection of cardiotoxin generates localized injury, giving us the opportunity to study central, peripheral, and intermediate regions of the injury.…”