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. 2025 May 12;14(5):537.
doi: 10.3390/biology14050537.

Deciphering the Role of Ferroptosis in the Pathogenesis of Peripheral Artery Disease Myopathy

Trevor Wilkinson  1 , Emma Fletcher  2 , Andrew Ring  1 , Cassandra Bradley  1 , Evlampia Papoutsi  2 , Dimitrios Miserlis  3 , Robert S Smith  4 , William T Bohannon  4 , Iraklis I Pipinos  5 , Panagiotis Koutakis  1   2   5
Affiliations

Deciphering the Role of Ferroptosis in the Pathogenesis of Peripheral Artery Disease Myopathy

Trevor Wilkinson et al. Biology (Basel). .

Abstract

This study investigates ferroptosis in the context of peripheral artery disease (PAD), a vascular disease characterized by atherosclerosis of the lower extremities. Muscle atrophy and increased oxidative stress are hallmarks of PAD and correlate with worse clinical outcomes. Given ferroptosis' association with oxidative stress, we explored its role in PAD myopathy by examining gene and protein markers related to metabolic pathways implicated in ferroptosis using both human PAD patients and cultured myotubes. Intermittent claudication (IC) PAD patients, critical limb ischemia (CLI) PAD patients, and non-PAD controls were recruited for this study. Calf muscle biopsies were analyzed for gene expression using qPCR, and protein levels were determined by Western blotting. Cultured myotubes treated with the ferroptosis inducer erastin provided an in vitro comparison. Results demonstrated upregulation of ferroptosis markers such as lipid peroxidation and PTGS2 gene expression in the muscle of CLI PAD patients compared to controls. Increased expression of ferroptosis-related genes HMOX1, ACSL4, ELAVL1, and Beclin-1 was also observed. Protein analysis showed trends consistent with gene expression in some ferroptosis markers. The increase in ferroptosis markers in CLI PAD patients, particularly in iron metabolism and autophagy pathways, suggests ferroptosis contributes to PAD myopathy.

Keywords: autophagy; ferroptosis; iron metabolism; lipid peroxidation; oxidative stress; peripheral artery disease.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
RNA expression of major markers of ferroptosis in the muscle tissue of IC and CLI PAD patients. Graphs show the normalized relative expression of (A) HMOX1, (B) TFRC, (C) FTH1, (D) SLC40A1, (E) NCOA4, (F) GPX4, (G) SLC7A11, (H) ACSL4, (I) PTGS2, (J) ELAVL1, (K) Beclin-1, and (L) STAT3 RNA. Control n = 7, IC n = 10, CLI n = 10 for all graphs. * p < 0.05, ** p < 0.01, **** p < 0.0001.
Figure 2
Figure 2
RNA expression of ferroptosis markers in cultured myotubes treated with and without erastin. Graphs show the normalized relative expression of (A) HMOX1, (B) TFRC, (C) FTH1, (D) SLC40A1, (E) NCOA4, (F) GPX4, (G) SLC7A11, (H) ACSL4, (I) PTGS2, (J) ELAVL1, (K) Beclin-1, and (L) STAT3 RNA. Control n = 7, IC n = 10, CLI n = 10 for all graphs. * p < 0.05, ** p < 0.01, **** p < 0.0001.
Figure 3
Figure 3
Protein expression in the muscle tissue of IC and CLI PAD patients. (A) Western blot results for HMOX1, GPX4, SLC7A11, ACSL4, PTGS2, ELAVL1, and Beclin-1, with a representative Ponceau S stain. Graphs depict the fold change relative to control for (B) HMOX1, (C) GPX4, (D) SLC7A11, (E) ACSL4, (F) PTGS2, (G) ELAVL1, and (H) Beclin-1 protein expression. Control n = 7, IC n = 9, CLI n = 9 for all graphs. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
Protein expression in cultured myotubes treated with either FBS or erastin. (A) Western blot results for GPX4, SLC7A11, PTGS2, ELAVL1, and Beclin-1, with a representative Ponceau S stain. Graphs depict the fold change relative to control for (B) GPX4, (C) SLC7A11, (D) PTGS2, (E) ELAVL1, and (F) Beclin-1 protein expression. FBS n = 3, erastin n = 3 for all graphs. * p < 0.05.
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