Abstract
Diabetes Mellitus (DM) is a major risk factor for developing peripheral arterial disease (PAD) and individuals with DM have worse PAD outcomes but the molecular mechanisms involved are poorly understood. Previously, in a hind limb ischemia (HLI) model of PAD, we identified a disintegrin and metalloproteinase gene 12 (ADAM12) as a key genetic modifier of post-ischemic perfusion recovery. Moreover, we showed that expression of ADAM12 in mouse and human tissue is regulated by miR29a. In non-diabetic mice, miR29a expression is downregulated after HLI that allows increased expression of ADAM12. However, upon HLI in high fat diet feed (HFD) mice, a model of type 2 diabetes, miR29a expression remains elevated that prevents ADAM12 increase and results in poor reperfusion recovery, increased skeletal muscle injury and decreased muscle function. Hence, we hypothesized that inhibition of miR29a or augmenting ADAM12 would improve these functional outcomes.
Mice (male, 26–28 weeks old) were randomized into 3 treatment groups and their hind limbs were treated with saline (grp1), ADAM12 cDNA (grp 2) or mir29a-inhibitor (grp3), through targeted micro-bubble delivery. Mice were treated at -3 days and -1 pre-surgery, followed by post-surgery weekly boosting. HLI was achieved by unilateral ligation and excision of the femoral artery of the left hind limb. The right hind limb served as non-ischemic control. Gene expression analysis in the hind limbs 3 days post HLI showed decreased miR29a expression in normal chow fed B6, but elevated miR29a expression in HFD (B6 vs HFD; 0.5730±0.01 vs.1.02 ± 0.06, n=3–4, p= 0.001). Treatment with miR29a inhibitor decreased miR29a expression in HFD and increased ADAM12 expression compared to control untreated HFD mice (miR29a INH vs Control HFD: 0.70±0.06 vs 1.02±0.06, n= 4–5, p= 0.004) ADAM12 expression (miR29A INH vs Control: HFD 208.62±24.52 vs 11.75±4.94, n= 3–4 P<0.01). Although ADAM12 cDNA improved ADAM12 expression, miR29a inhibition increased ADAM12 expression to a greater extent (HFD vs ADAM12 vs miR29aINH; 11.75±4.94 vs 20.71±2.98 vs 208.62±24.52, n3-4, p=< 0.001). Accordingly, miR29a inhibition and ADAM12 augmentation decreased skeletal muscle injury assessed by the number of centralized nuclei/muscle fibre (Control vs ADAM12 vs miR29aINH: 0.252±0.043, vs 0.139±0.041 vs 0.040±0.012 n=4, p= 0.05), and improved skeletal muscle function assessed as maximum muscle contraction (Control vs ADAM12 vs miR29aINH: 0.17±0.06 vs 0.26±0.06, vs 0.54±0.08, n=6–7, p<0.01). It also improved perfusion recovery, (% ischemic to non-ischemic limb, control vs ADAM12 vs miR29aINH: 42.52±5.35, vs 58.45±4.87, vs 97.59±6.14, n= 5–10, p<0.01). Thus, our results show augmentation of ADAM12 and Inhibition of MiR29a improves outcomes in experimental PAD in diabetic mice but inhibiting miR29a is a more effective strategy.
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Impaired Redox Capacity, Muscle Injury, and Microtubule Alterations Conspire to Impact Skeletal Muscle Function
