AAV-mediated expression of PFKFB3 in myofibers, but not endothelial cells, improves ischemic muscle function in mice with critical limb ischemia.

Abstract

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a powerful driver of angiogenesis through its modulation of glycolytic metabolism within endothelial cells. Recent work has demonstrated that PFKFB3 modulates the response to muscle ischemia, however the cell specificity of these effects is not fully understood. In this study, we tested the impact of viral mediated expression of PFKFB3, driven by gene promoters specific for myofibers or endothelial cells, on ischemic hindlimb revascularization and muscle function. We hypothesized that both endothelium- and muscle-specific expression of PFKFB3 would attenuate limb pathology following femoral artery ligation. Male and female BALB/cJ mice were injected with adeno-associated virus encoding the either a green fluorescent protein (GFP) or PFKFB3 driven by either the human skeletal actin (ACTA1) or cadherin-5 (Cdh5) promoters. Four weeks after AAV treatment, mice were subjected to unilateral femoral artery ligation and limb perfusion and muscle function were assessed. Both endothelium- and muscle-specific PFKFB3 expression resulted in significantly more perfused capillaries within the ischemic limb muscle, but neither changed myofiber size/area. Muscle-specific, but not endothelium-specific, PFKFB3 expression significantly improved maximal force production in ischemic muscle (P = 0.0005). Notably, there was a significant effect of sex on maximal force levels in both cohorts of mice (P = 0.0075 and P = 0.0481), indicating that female mice had higher ischemic muscle strength compared with male mice, regardless of treatment group. Taken together, these data demonstrate that although both muscle- and endothelium-specific expression of PFKFB3 enhanced ischemic revascularization, only muscle-specific PFKFB3 expression improved muscle function.NEW & NOTEWORTHY Critical limb ischemia (CLI) carries a significant risk for limb amputation, and treatment options remain limited. We tested the impact of expression of PFKFB3 in myofibers or endothelial cells on limb pathology in mice with CLI. Although both muscle and endothelium-specific PFKFB3 expression increased perfused capillary density, only muscle-specific PFKFB3 expression improve contractile function. Regardless of treatment, female mice demonstrated better recovery from limb ischemic compared with male mice.