Peripheral artery disease (PAD) is caused by atherosclerosis in the lower extremities, which leads to a spectrum of life-altering symptomatology, including claudication, ischemic rest pain, and gangrene requiring limb amputation. Current treatments for PAD are focused primarily on re-establishing blood flow to the ischemic tissue, implying that blood flow is the decisive factor that determines whether or not the tissue survives. Unfortunately, failure rates of endovascular and revascularization procedures remain unacceptably high and numerous cell- and gene-based vascular therapies have failed to demonstrate efficacy in clinical trials. The low success of vascular-focused therapies implies that non-vascular tissues, such as skeletal muscle and oxidative stress, may substantially contribute to PAD pathobiology. Clues toward the importance of skeletal muscle in PAD pathobiology stem from clinical observations that muscle function is a strong predictor of mortality. Mitochondrial impairments in muscle have been documented in PAD patients, although its potential role in clinical pathology is incompletely understood. In this review, we discuss the underlying mechanisms causing mitochondrial dysfunction in ischemic skeletal muscle, including causal evidence in rodent studies, and highlight emerging mitochondrial-targeted therapies that have potential to improve PAD outcomes. Particularly, we will analyze literature data on reactive oxygen species production and potential counteracting endogenous and exogenous antioxidants.

Background: Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD. Methods: AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHR ^mKO (skeletal muscle–specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD. Results: PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes ( Cyp1a1 , Cyp1b1 , and Aldh3a1 ) when compared with either muscle from the PAD condition with normal renal function ( P <0.05 for all 3 genes) or nonischemic controls. AHR ^mKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle–specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. Conclusions: These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions.

BACKGROUND: Lower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle. METHODS: We obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas. RESULTS: Single-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD. CONCLUSIONS: This reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition.

Chronic limb threatening ischemia (CLTI) is the most severe manifestation of peripheral atherosclerosis. Patients with CLTI have poor muscle quality and function and are at high risk for limb amputation and death. The objective of this study was to interrogate the metabolome of limb muscle from CLTI patients. To accomplish this, a prospective cohort of CLTI patients undergoing either a surgical intervention (CLTI Pre-surgery) or limb amputation (CLTI Amputation), as well as non-peripheral arterial disease (non-PAD) controls were enrolled. Gastrocnemius muscle biopsy specimens were obtained and processed for nuclear magnetic resonance (NMR)-based metabolomics analyses using solution state NMR on extracted aqueous and organic phases and ¹H high-resolution magic angle spinning (HR-MAS) on intact muscle specimens. CLTI Amputation specimens displayed classical features of ischemic/hypoxic metabolism including accumulation of succinate, fumarate, lactate, alanine, and a significant decrease in the pyruvate/lactate ratio. CLTI Amputation muscle also featured aberrant amino acid metabolism marked by elevated branched chain amino acids. Finally, both Pre-surgery and Amputation CLTI muscles exhibited pronounced accumulation of lipids, suggesting the presence of myosteatosis, including cholesterol, triglycerides, and saturated fatty acids. Taken together, these metabolite differences add to a growing body of literature that have characterized profound metabolic disturbance’s in the failing ischemic limb of CLTI patients.

• scAAV9 gene therapies can be delivered to, and produce robust transgene expression in, the critically ischemic mouse limb without adverse impact on perfusion recovery or muscle health/function. • Treatment of the ischemic limb with scAAV9–IGF-1 significantly improved muscle size and strength in male and female mice with hindlimb ischemia. • Differences in the efficacy of scAAV9–IGF-1 were observed, with female mice displaying greater improvements in muscle contractile function than male mice. • IGF-1-mediated improvements in muscle function occurred without changes in limb perfusion recovery or capillary density.

BACKGROUND: Chronic kidney disease (CKD) has emerged as a significant risk factor that accelerates atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the modulators underlying this exacerbated pathobiology are ill-defined. Recent work has demonstrated that uremic toxins are associated with limb amputation in PAD and have pathological effects in both the limb muscle and vasculature. Herein, we use multiomics to identify novel modulators of disease pathobiology in patients with PAD and CKD. METHODS: A cross-sectional study enrolled 4 groups of participants: controls without PAD or CKD (n=28), patients with PAD only (n=46), patients with CKD only (n=31), and patients with both PAD and CKD (n=18). Both targeted (uremic toxins) and nontargeted metabolomics in plasma were performed using mass spectrometry. Calf muscle biopsies were used to measure histopathology, perform bulk and single-nucleus RNA sequencing, and assess mitochondrial function. Differential gene and metabolite analyses, as well as pathway and gene set enrichment analyses, were performed. RESULTS: Patients with both PAD and CKD exhibited significantly lower calf muscle strength and smaller muscle fiber areas compared with controls and those with only PAD. Compared with controls, mitochondrial function was impaired in patients with CKD, with or without PAD, but not in PAD patients without CKD. Plasma metabolomics revealed substantial alterations in the metabolome of patients with CKD, with significant correlations observed between uremic toxins (eg, kynurenine and indoxyl sulfate) and both muscle strength and mitochondrial function. RNA sequencing analyses identified downregulation of mitochondrial genes and pathways associated with protein translation in patients with both PAD and CKD. Single-nucleus RNA sequencing further highlighted a mitochondrial deficiency in muscle fibers along with unique remodeling of fibro-adipogenic progenitor cells in patients with both PAD and CKD, with an increase in adipogenic cell populations. CONCLUSIONS: CKD significantly exacerbates ischemic muscle pathology in PAD, as evidenced by diminished muscle strength, reduced mitochondrial function, and altered transcriptome profiles. The correlation between uremic toxins and muscle dysfunction suggests that targeting these metabolites may offer therapeutic potential for improving muscle health in PAD patients with CKD.