Skeletal muscle MiR-210 expression is associated with mitochondrial function in peripheral artery disease patients

Abstract

Previous studies have demonstrated that circulating microRNA (miR)-210 levels are elevated in peripheral artery disease (PAD) patients. MiR-210 is known to be a negative regulator of mitochondrial respiration; however, the relationship between miR-210 and mitochondrial function has yet to be studied in PAD. We aimed to compare skeletal muscle miR-210 expression of PAD patients to non-PAD controls (CON) and to examine the relationship between miR-210 expression and mitochondrial function. Skeletal muscle biopsies from CON (n = 20), intermittent claudication (IC) patients (n = 20), and critical limb ischemia (CLI) patients (n = 20) were analyzed by high-resolution respirometry to measure mitochondrial respiration of permeabilized fibers. Samples were also analyzed for miR-210 expression by real-time PCR. MiR-210 expression was significantly elevated in IC and CLI muscle compared to CON (P = 0.008 and P < 0.001, respectively). Mitochondrial respiration of electron transport chain (ETC) Complexes II (P = 0.001) and IV (P < 0.001) were significantly reduced in IC patients. Further, CLI patients demonstrated significant reductions in respiration during Complexes I (state 2: P = 0.04, state 3: P = 0.003), combined I and II (P < 0.001), II (P < 0.001), and IV (P < 0.001). The expression of the miR-210 targets, cytochrome c oxidase assembly factor heme A: farnesyltransferase (COX10), and iron-sulfur cluster assembly enzyme (ISCU) were down-regulated in PAD muscle. MiR-210 may play a role in the cellular adaptation to hypoxia and may be involved in the metabolic myopathy associated with PAD.

Figure 1.. Skeletal muscle mitochondrial respiration is reduced in PAD patients.

Oxygen consumption rate (JO2) in permeabilized muscle fibers from non-PAD control (CON) patients (n=20), intermittent claudication (IC) patients (n=20), and critical limb ischemia (CLI) patients (n=20). JO2 measured during (A) Complex I, state 2 respiration (CI.2), (B) Complex I, state 3 respiration (CI.3), C, combined Complex I and II respiration (CI+II), (D) Complex II respiration (CII), and (E) Complex IV respiration (CIV). (F) Mitochondrial content was assessed by citrate synthase (CS) activity. One-way ANCOVA with post-hoc Bonferroni test used for analysis, ns represents no significant difference between groups, ns represents no significant difference between groups, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

Figure 2.. MiR-210 expression is elevated in PAD muscle.

(A) Relative miR-210 levels in gastrocnemius muscle of non-PAD control (CON) (n=10), intermittent claudication (IC) patients (n=10), and critical limb ischemia (CLI) patients (n=10) and (B) relative miR-210 levels in serum. One-way ANCOVA with post-hoc Bonferroni test used for analysis, ** represents p<0.01, and *** represents p<0.001. (C) Association between circulating levels of miR-210 and skeletal muscle levels of miR-210, correlation tested by Pearson correlation, coefficient of determination (R²) and p-value for relationship shown.

Figure 3.. Skeletal muscle miR-210 expression is associated with mitochondrial respiration and walking performance.

(A) Association between combined Complex I and II (CI+II) respiration and skeletal muscle miR-210 relative expression in non-PAD control (CON), intermittent claudication patients, and critical limb ischemia (CLI) patients, (B) association between Complex IV (CIV) respiration and muscle miR-210 relative expression, and (C) association between 6-minute walk distance (6MWD) and muscle miR-210 relative expression. Correlations tested by Pearson correlation, coefficient of determination (R²) and p-values for relationships shown.

Figure 4.. Skeletal muscle miR-210 target expression in PAD.

(A) Heatmap analysis of gastrocnemius tissue expression of miR-210 target genes, color legend represents calculated relative expression of intermittent claudication (IC) (n=6) and critical limb ischemia (CLI) patients (n=3) compared to non-PAD controls (CON) (n=3), (B) Volcano plot identifying significant gene expression changes in IC and (C) in CLI, by plotting the log2 of the fold changes in gene expression on the x-axis versus their statistical significance on the y-axis. The center vertical line indicates unchanged gene expression, while the two outer vertical lines indicate the selected fold regulation threshold. The horizontal line indicates the selected p-value threshold. Genes with data points in the far upper left (down-regulated) and far upper right (up-regulated) sections meet the selected fold regulation and p-value thresholds. By combining the fold change results with the p-value statistical test results, genes with both large and small expression changes that are statistically significant are easily visualized.

Figure 5.. Skeletal muscle COX10 and ISCU expression are reduced in PAD.

(A) Relative fold change of mRNA expression detected by real-time PCR. COX10 and ISCU gene expression measured in skeletal muscle homogenates of intermittent claudication (IC) (n=10), critical limb ischemia (CLI) (n=10) patients, and non-PAD controls (CON) (n=10). (B) Western blot of COX10 and ISCU, GAPDH used as loading control, (C) fold change of COX10 and ISCU protein expression. One-way ANCOVA with post-hoc Bonferroni test used for analyses, * represents p<0.05 and *** represents p<0.001.

Figure 6.. Potential mechanisms of miR-210 mediated mitochondrial dysfunction in PAD.

In ischemic conditions, hypoxia-inducible factor 1-alpha (HIF-1α) binds to the hypoxia response element (HRE) on the miR-210 promoter, increasing the transcription of pri-miR-210. After cleavage of excess base pairs by RNAse III Drosha, pre-miR-210 is exported from the nucleus. RNAse III Dicer further cleaves the loop structure, separating the 2 strands of RNA, leaving a single stranded mature miR-210. The mature miR-210-RNA-induced silencing complex (RISC) complexes can target and repress iron-sulfur cluster scaffold homolog (ISCU) and cytochrome c oxidase assembly protein (COX10) genes. The downregulation of these genes can lead to reduced activity of mitochondrial electron transport chain Complex I and reduced expression of functional cytochrome c oxidase protein.