Associations of Peripheral Artery Disease With Calf Skeletal Muscle Mitochondrial DNA Heteroplasmy

Abstract

Background Patients with peripheral artery disease (PAD) undergo frequent episodes of ischemia-reperfusion in lower extremity muscles that may negatively affect mitochondrial health and are associated with impaired mobility. We hypothesized that skeletal muscle from PAD patients will show high mitochondrial DNA heteroplasmy, especially in regions more susceptible to oxidative damage, such as the displacement loop, and that the degree of heteroplasmy will be correlated with the severity of ischemia and mobility impairment. Methods and Results Mitochondrial mutations and deletions and their relative abundance were identified by targeted mitochondrial DNA sequencing in biopsy specimens of gastrocnemius muscle from 33 PAD (ankle brachial index <0.9) and 9 non-PAD (ankle brachial index >0.9) subjects aged ≥60 years. The probability of heteroplasmy per DNA base was significantly higher for PAD subjects than non-PAD within each region. In adjusted models, PAD was associated with higher heteroplasmy than non-PAD (P=0.003), but the association was limited to microheteroplasmy, that is heteroplasmy found in 1% to 5% of all mitochondrial genomes (P=0.004). Heteroplasmy in the displacement loop and coding regions were significantly higher for PAD than non-PAD subjects after adjustment for age, sex, race, and diabetes mellitus (P=0.037 and 0.004, respectively). Low mitochondrial damage, defined by both low mitochondrial DNA copy number and low microheteroplasmy, was associated with better walking performance. Conclusions People with PAD have higher "low frequency" heteroplasmy in gastrocnemius muscle compared with people without PAD. Among people with PAD, those who had evidence of least mitochondrial damage, had better walking performance than those with more mitochondrial damage. Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02246660.

Keywords: D‐loop; ankle brachial index; heteroplasmy; mtDNA; mtDNA copy number; peripheral artery disease.

Figures

Figure 1. Distribution of heteroplasmic variants by region and frequency in the whole study sample (n=42).

A, Rate of heteroplasmy (heteroplasmy count per base) by mitochondrial DNA region among all participants; (B) proportion of heteroplasmic variants categorized as low (microheteroplasmy), mid, or high frequency. Parentheses indicate the number of bases used to calculate rate for each region. D‐loop indicates displacement loop; and mtDNA, mitochondrial DNA.

Figure 2. Heteroplasmy count by mitochondrial DNA region and peripheral artery disease status (n=42, darker boxes=non‐peripheral artery disease; lighter boxes=peripheral artery disease; *P<0.05 and # P<0.01).

mtDNA indicates mitochondrial DNA; and PAD, peripheral artery disease.

Figure 3. Distribution of ankle brachial index (top panel) and normal 4‐m walking speed (bottom panel) by categories of heteroplasmy count and copy number among participants with peripheral artery disease (n=33).

Participants were cross‐classified by heteroplasmy count ≤ vs >36 (median level) and mitochondrial DNA copy number ≤ vs >3041 (median level). ABI indicates ankle brachial index.