Extensive skeletal muscle cell mitochondriopathy distinguishes critical limb ischemia patients from claudicants

Abstract

The most severe manifestation of peripheral arterial disease (PAD) is critical limb ischemia (CLI). CLI patients suffer high rates of amputation and mortality; accordingly, there remains a clear need both to better understand CLI and to develop more effective treatments. Gastrocnemius muscle was obtained from 32 older (51-84 years) non-PAD controls, 27 claudicating PAD patients (ankle-brachial index [ABI] 0.65 ± 0.21 SD), and 19 CLI patients (ABI 0.35 ± 0.30 SD) for whole transcriptome sequencing and comprehensive mitochondrial phenotyping. Comparable permeabilized myofiber mitochondrial function was paralleled by both similar mitochondrial content and related mRNA expression profiles in non-PAD control and claudicating patient tissues. Tissues from CLI patients, despite being histologically intact and harboring equivalent mitochondrial content, presented a unique bioenergetic signature. This signature was defined by deficits in permeabilized myofiber mitochondrial function and a unique pattern of both nuclear and mitochondrial encoded gene suppression. Moreover, isolated muscle progenitor cells retained both mitochondrial functional deficits and gene suppression observed in the tissue. These findings indicate that muscle tissues from claudicating patients and non-PAD controls were similar in both their bioenergetics profile and mitochondrial phenotypes. In contrast, CLI patient limb skeletal muscles harbor a unique skeletal muscle mitochondriopathy that represents a potentially novel therapeutic site for intervention.

Keywords: Atherosclerosis; Cardiovascular disease; Metabolism; Skeletal muscle.

Figure 1. Flowchart of sample processing for experimental procedures.

Descriptive flowchart of how human specimens were processed for data collection including analyses and sample sizes for each experiment performed.

Figure 2. Histological assessment of skeletal muscle specimens.

Skeletal muscle biopsy specimens were obtained from the gastrocnemius of healthy adults (HA), intermittent claudicants (IC), and critical limb ischemia (CLI) patients. (A) Histological (H&E staining) assessment and immunofluorescent staining for dystrophin confirms that samples obtained were not from necrotic regions within the limb (note: color differences in IC samples are due to paraffin embedding). Small white arrows indicate evidence of small, irregularly shaped myofibers in IC and CLI patients. (B) Distribution plots of myofiber cross-sectional area from each patient group (N = 3 for HA, N = 8 for IC, N = 6 for CLI). (C) Quantification of mean myofiber cross-sectional area (N = 3 for HA, N = 8 for IC, N = 6 for CLI). (D) Representative immunofluorescence images stained for myosin heavy chain (MyHC) type I (slow twitch myofibers). (E) Quantification of the percentage of type I myofibers in each group (N = 3 for HA, N = 7 for IC, N = 6 for CLI). **P < 0.01 using ANOVA with Tukey’s multiple comparison test. NS, not significant. Data are presented as the mean ± SEM.

Figure 3. RNA-sequencing in muscle biopsy specimens.

(A) Schematic diagram of tissue acquisition and RNA-sequencing analysis. (B) Principal component analysis (PCA) of the samples. (C) Volcano plot representing differential gene expression between HA and IC. (D) Heatmap of all differentially expressed genes from panel C. (E) Volcano plot representing differential gene expression between HA and IC. (F) Heatmap of all differentially expressed genes from panel E. (G) Volcano plot representing differential gene expression between HA and IC. (H) Heatmap of all differentially expressed genes from panel G. N = 15 for HA, N = 20 for IC, N = 16 for CLI.

Figure 4. Gene expression signature is unique to CLI.

Gene expression profiles were determined by whole genome sequencing of RNA isolated from muscle biopsy samples of the gastrocnemius. (A) Gene ontology (GO) enrichment analysis indicating the most significant gene expression changes between groups. (B) A heatmap of gene expression differences (log₂[fold change from HA]) for the GO terms and genes associated with mitochondrial function. (C) mRNA changes were validated by qRT-PCR of selected genes. ***P < 0.001, ****P < 0.0001 using ANOVA with Tukey’s multiple comparison test. NS, not significant. Sample sizes for RNA-sequencing and qRT-PCR were N = 15 for HA, N = 20 for IC, N = 16 for CLI. Data are presented as the mean ± SEM.

Figure 5. Comprehensive mitochondrial phenotyping.

Skeletal muscle mitochondrial respiratory function was measured in permeabilized myofiber samples (representative image in panel A). (A) CLI patients displayed decreased complex I₃– (state 3), I+II–, II–, and IV–supported oxygen consumption compared with both IC and HA patients (N = 26 for HA, N = 7 for IC, N = 19 for CLI). Mitochondrial content was assessed by citrate synthase activity (B) (N = 17 for HA, N = 14 for IC, N = 16 for CLI), cardiolipin content (C) (N = 10/group), and mtDNA/nDNA ratio (D) (N = 6/group), indicating that mitochondrial content was not different between IC and CLI. (E) Biochemical enzyme assays of muscle lysates were performed to further dissect changes in the mitochondrial electron transport system. These assays indicate decreased specific activities (normalized for citrate synthase activity) in complexes I, III, and IV in CLI patients (N = 17 for HA, N = 14 for IC, N = 16 for CLI). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using ANOVA with Tukey’s multiple comparison test. NS, not significant. Data are presented as the mean ± SEM (note: error bars in panel B are 95% CI).

Figure 6. Mitochondrial protein changes in CLI.

Patient muscle specimens were analyzed by Western blotting. (A) Whole blot images are shown using chemiluminescence. (B) Selected proteins (panel A) were quantified by standard densitometry. *P < 0.05, **P < 0.01, ****P < 0.0001 using ANOVA with Tukey’s multiple comparison test (N = 8/group). NS, not significant. Data are presented as the mean ± SEM.

Figure 7. Correlational analyses of mitochondrial outcomes and ABI.

Pearson correlation coefficients were performed for mitochondrial function outcomes and ABI in both IC and CLI patients. Permeabilized myofiber oxygen consumption rate (OCR, pmol/s/mg), complex-specific enzyme activity (mU/U citrate synthase [CS]), protein abundance by Western blotting, and ABI were used. Heatmaps were generated from these Pearson correlation matrixes for both IC (A) and CLI (B) patients.

Figure 8. Skeletal muscle stem cells from CLI patients display a unique bioenergetics gene expression signature.

Primary muscle progenitor cells (satellite cells) were isolated from muscle biopsies. Isolated myoblasts were differentiated into myotubes by serum withdrawal (A). (B) Cellular respiration was assessed using a Seahorse XF analyzer. Oligo, oligomycin; AmA, antimycin A; Asc, ascorbate; OCR, oxygen consumption rate. (C) Quantification of cellular respiration under different substrate/inhibitor combinations indicates impaired basal, maximal, and complex IV–linked respiration in cells from CLI patients (N = 8 for HA, N = 7 for IC, and N = 8 for CLI). (D) Citrate synthase activity was not different in cells (N = 4 for HA, N = 6 for IC, and N = 4 for CLI). (E) Quantification of mitochondrial volume from Z-stack confocal imaging of fluorescently labeled mitochondria in MPCs (N = 4 for HA, N = 4 for IC, and N = 4 for CLI). (F) Representative images of labeled mitochondria (TOMM20, green). Counterstaining was performed with DAPI (blue, nuclei) and phalloidin (red, actin). (G) Gene expression analysis by qRT-PCR indicated a unique gene signature related to mitochondrial metabolism. All sample sizes indicate independent MPC isolations from different patients. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using ANOVA with Tukey’s multiple comparison test. NS, not significant. Data are presented as the mean ± SEM.