Skeletal muscle proteome and mitochondrial health distinguishes the failing and salvageable ischemic limb

Abstract

INTRODUCTION Chronic limb threatening ischemia (CLTI) is the most severe manifestation of peripheral vascular disease. Despite improved surgical approaches for CLTI, amputation rates remain high and the factors contributing to the failing ischemic limb remain unclear. In this study, we explored potential differences in the proteome and mitochondrial health of muscle specimens obtained from control and CLTI patients at the time of surgical intervention or limb amputation. METHODS AND RESULTS Gastrocnemius muscle specimens were obtained from non-ischemic controls (n = 10), CLTI patients undergoing surgical revascularization procedures (n = 15), and CLTI patients undergoing limb amputation (n = 15). Muscle specimens were processed for isobaric TMT-labeled proteomics analyses to identify potential differences in the cellular proteome. Half of the proteins that were significantly less abundant in limb amputation muscle were related to the mitochondrion, whereas ~41% of those upregulated were associated with the extracellular matrix. Notably, pre-surgery specimens displayed enhanced abundance of numerous mitochondrial proteins compare with both control and amputation muscles, despite the presence of a mild functional deficit in mitochondrial energetics. Functional analysis of mitochondria using permeabilized myofibers preparations demonstrate a severe respiration deficit in amputation specimens which also corresponded with significantly greater hydrogen peroxide production. Moreover, we establish potential causal links between the differential muscle proteome and mitochondrial physiology. CONCLUSIONS This is the first study to explore the muscle proteome in the failing ischemic limb of CLTI patients undergoing amputation. A severe deficit in muscle mitochondrial function and the corresponding mitochondrial proteome was present at the time of amputation but not in pre-surgery specimens. Future work is warranted to determine if therapeutically targeting mitochondrial health during/after surgical intervention could improve patient outcomes.