Abstract Body: Impairment of wound healing and blood flow in diabetes, a serious complication of types 1 and 2 diabetes, is associated with delayed muscle regeneration, atrophy, and, in severe cases, limb amputation or death. In type 1 diabetic Akita mice, hind limb ischemia (HLI) induced by femoral artery ligation (FAL) results in loss of body and muscle mass as a consequence of a failed capacity of the myoblast progenitors to proliferate during homeostasis and injury.
Mesenchymal progenitor cells (MPCs), including adult myocyte progenitor myoblasts and the supporting fibroadipogenic progenitors (FAPs), and their maladaptive inter-cellular communications, contribute to the impairment of muscle maintenance and regeneration in diabetes. To discover the underlying mechanisms and develop muscle-directed therapeutic strategies, we performed single cell RNA sequencing of diabetic and non-diabetic muscle at baseline and post-unilateral FAL in mice. We found that skeletal muscle (gastrocnemius) of diabetic mice, but not non-diabetic muscle, displayed a reduction in myoblast cell cycle progression in FAPs at 3 day post-FAL vs. baseline. Cell-cell interaction analysis at day 3 post-FAL revealed a reduction in adaptive FAP-myoblast interactions in diabetic vs. non-diabetic muscle. Strikingly, we observed increased fat infiltration and fibrosis at day 7 post-FAL only in diabetic mice, which was sustained at least through day 35 post-FAL. Single cell RNAsequencing uncovered the novel observation of attenuation of the Wnt signaling pathway, with low ligand levels of Wnt9a and high levels of TGFB2 in both myoblasts and FAPs in diabetic mice, particularly on day 3 post-FAL, which was verified at the mRNA and protein levels. To test if forced expression of Wnt9a could rescue and improve regeneration in diabetic mice, immediately after FAL (day 0), the gastrocnemius muscle of diabetic mice was injected with either AAV9-tMCK-mWnt9a.GFP vector vs. control vector and blood flow recovery was monitored. Compared to control vector, injection of Wnt9s-vector significantly improved blood flow on days 3, 7 and 14 (N=5/group): day 3, 16.9% vs. 29.5% (p<0.01); day 7, 42.6% vs. 59.9% (p<0.05); and day 14, 51.9% vs. 86.4% (p<0.01), respectively. Additional histology and molecular studies are pending. In conclusion, expression of Wnt9a in diabetic mice muscle post-FAL may be a novel therapeutic strategy to rescue impaired recovery post-ischemia in the peripheral vasculature.