Lower extremity peripheral artery disease (PAD) significantly affects aging populations and results in functional
impairment. Although the clinical importance of finding efficacious interventions for PAD is well-recognized, few
medical therapies are currently available. PAD is diagnosed using the ankle brachial index (ABI), a measure of
blood flow to the lower extremities. Lower ABI is associated with worse function; however, low ABI alone
cannot fully explain functional impairments in PAD. Small studies have reported oxidative stress, mitochondrial
dysfunction and/or fiber damage in gastrocnemius muscle biopsies from PAD patients, suggesting skeletal
muscle perturbations may contribute to functional decline. We reported highly variable fiber type composition
and fiber type grouping in a small cohort of PAD patients, and observed lack of intermyofibrillar mitochondria
(IMFM-) in oxidative, myosin heavy chain (MyHC) type I fibers. We have provocative new preliminary data
suggesting variability in response to ongoing denervation, and in fiber type and mitochondrial adaptations, with
PAD. The purpose of this study is to define specific characteristics of muscle in PAD associated with impaired
walking performance through detailed immunohistochemical analyses of 400 baseline gastrocnemius muscle
biopsies stored in the Northwestern biorepository, collected from 9 different clinical trials. This biorepository of
muscle from PAD patients is one-of-a-kind and is associated with detailed clinical and functional characteristics
of the donors. We hypothesize that variability in fiber size, fiber type and mitochondrial adaptations in response
to ischemia-reperfusion damage and denervation in individuals with PAD will have value in predicting walking
impairment. In Aim 1, we will quantify the proportion of IMFM- areas in type I fibers with normal type I MyHC
abundance, or accumulation of type IIX MyHC and/or LC3, a marker of autophagy, and determine associations
with fiber type composition and fiber size, as well as relationships of muscle features to walking performance in
PAD. We hypothesize that LC3 will co-localize with IIX MyHC in IMFM- areas, suggesting both incomplete
autophagic clearance of IIX MyHC and mitochondrial biogenesis during fiber transition from type IIX to type I
as a result of denervation and reinnervation. In Aim 2 we will quantify denervated, NCAM+ fibers and fibers
with elevated oxidative damage markers by fiber type. We hypothesize that denervation in PAD will
preferentially affect fibers expressing IIX MyHC and that only IMFM- areas that accumulate IIX MyHC will be
NCAM+. In Aim 3 we will perform predictive modeling of PAD disease severity and functional impairment using
morphological characteristics of muscle quantified in Aims 1 and 2 as biomarkers in conjunction with
supervised classification approaches. In Aim 4 we will test the hypothesis that baseline muscle characteristics
will predict longitudinal functional outcomes at 6-month follow up. This model will provide a powerful tool to
aide in identifying biologic processes for targeted interventions and to assess the mechanism of action and
effectiveness of current pharmacological and exercise interventions in ongoing PAD clinical trials.
