Genetic deletion of skeletal muscle Cox6a2 (Cytochrome C Oxidase Subunit 6a polypeptide 2) delays regeneration in preclinical model of Peripheral Artery Disease
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Date
2023-12-04
Access
2025-12-01
Authors
Kolasa, Makenzie
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Publisher
East Carolina University
Abstract
Chronic limb threatening ischemia (CLTI) is the most severe clinical manifestation of peripheral artery disease (PAD). There are no current therapeutic options for CLTI outside of surgical intervention and it remains the leading cause of limb amputation. There is a dire need for therapeutic innovation for these patients. Mitochondrial insufficiency is a hallmark of the clinical CLTI presentation and an exciting avenue for therapeutic development. Previously, we identified unique CLTI patient deficits in Cox6a2 protein abundance, a muscle specific binding subunit in Cytochrome C Oxidase. We hypothesize that skeletal muscle Cox6a2 is necessary for tissue regeneration after hindlimb ischemia (HLI) due to its integral role in the bioenergetics of skeletal muscle. To test this hypothesis, we generated Pax7-Cre+;Cox6a2fl/fl mice, which genetically possess a non-inducible (lifelong) deletion of Cox6a2 in the muscle progenitor cells and mature skeletal muscle myofibers. At baseline, we observed similar oxygen consumption rates under physiologically relevant ranges of ATP free energy yet severe reductions in Complex IV-linked respiration under maximal energetic demand in the Cox6a2 knockout (KO; n=10) group compared to the wildtype control group (Pax7-Cre-;Cox6a2fl/fl or fl/- (WT); n=13). Proteomics profiling of mitochondria isolated from KO animals indicated significant decreases in the complex IV proteome. We then subjected these mice to hindlimb ischemia (HLI; n= 35 KO, n=31 WT). We observed similar blood flow and vascular recovery and no differences in muscle contractility between KO and WT ischemic limbs 7 days post-HLI. Decreased mitochondrial Complex IV-specific maximal respiration was observed in KO ischemic limbs, though no differences in respiration in a physiologically relevant range of ATP free energy occurred between groups. At d7, the KO ischemic mitochondrial proteome revealed major downregulations of complex IV proteins and upregulations of complex I and V, indicating a compensatory shift in expression due to the loss of cox6a2 in ischemic conditions. Extended (28-day) HLI (n=16 KO, n=19 WT) revealed delays in regeneration indicated by increased centrally located nuclei and decreased myofiber size in KO limbs. Fiber type, density of total and perfused vessels, and muscle contractility remained similar between KO and WT groups. Together, these data indicate that Cox6a2 is involved in efficient muscle regeneration during prolonged ischemic events.