Myoglobin knockout mice experience less damage and more immune infiltration in muscle pressure injury

• Published in: The FASEB journal Vol. 36; no. S1
• Main Authors: Nasir, Nurul Jannah M., Heemskerk, Johannes A., So, Peter T., Tucker‐Kellogg, Lisa
• Format: Journal Article
• Language: English
• Published: United States The Federation of American Societies for Experimental Biology 01-05-2022
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.0R548

Introduction
Muscle pressure ulcers are slow to heal and classified as “chronic” wounds, but the reasons for slow healing are not well understood. Elevated levels of extracellular hemoglobin have been implicated in the pathogenesis and impaired healing of sickle cell and vascular ulcers. We question whether extracellular release of myoglobin after muscle pressure injury might contribute to a hostile wound environment, analogous to hemoglobin. We hypothesise that knockout of myoglobin will decrease tissue damage and improve the microenvironment of muscle pressure ulcers.
Methods
We developed germline Myoglobin knockout (Mb–/–) mice via CRISPR deletion. Similar to previous studies, our adult Mb–/– mice show no phenotypic differences to Mb‐wildtype (Mb+/+) mice. Pressure injuries were created in mice by applying a pair of 12 mm magnets to the dorsal skinfold and panniculus carnosus muscle, in two intervals of 12 hours. We compared pressure ulcers in elderly 20‐month‐old mice carrying Mb–/– or Mb+/+. Ex vivo wound tissues were analysed at day 3 after injury.
Results
Mb‐wildtype mice had high levels of ferric iron in the wound localized to the extracellular space and infiltrating cells (presumably macrophages). In knockout mice, iron was undetectable. The knockout mice had smaller pressure ulcers post‐injury and 50% less tissue damage at the wound centre (p < 0.01), compared to control Mb+/+ mice. In addition, the wounds of Mb–/– mice show a marked decrease in protein nitration (2.7‐fold decrease in nitrotyrosine levels; p < 0.0001) compared to Mb+/+ mice. Furthermore, Mb‐knockout wounds exhibited an almost 21‐fold decrease in DNA oxidation, measured by oxidised guanine (8‐oxoguanine; p < 0.0001) compared to Myoglobin+/+. We found 3.5‐fold greater infiltration of immune cells (p < 0.01) and higher CCL7 chemokine expression (p < 0.01) in Mb‐knockout tissues than Mb‐wildtype. Furthermore, Mb–/– wounds displayed a four‐fold decrease in citrullinated histone 3 (p < 0.0001), a marker of extracellular traps, compared to Mb+/+.
Conclusion
In the absence of myoglobin, pressure‐injured tissues displayed less oxidative and nitrosative damage. Interestingly, they also showed higher immune infiltration and decreased extracellular traps, meaning that increased immune infiltration co‐occurred with decreased oxidative stress. We conclude that extracellular myoglobin contributes to oxidative stress and immune dysfunction in muscle pressure ulcers, and its removal causes less tissue death. This has important implications for the use of iron chelation therapy in the treatment of muscle pressure ulcers.