Identification of Fiber Type Characteristics Associated with Altered Mitochondrial Activity within PAD Patient Gastronemius Muscle

Peripheral artery disease (PAD) results from atherosclerotic artery narrowing leading to muscle damage and mobility loss. The hemodynamic origins of PAD are undisputed; however, blood flow reductions cannot fully explain mobility impairments with PAD. Muscle perturbations, particularly within the gastrocnemius (calf) muscle, have been documented with PAD for over 40 years; yet specific contributions of muscle changes to disease severity and mobility loss remain unclear. Mitochondrial dysfunction is reported within PAD patient calf muscles. In healthy muscles, mitochondrial activity is tightly associated with fiber type, whereby oxidative type I fibers display the most mitochondrial activity and glycolytic type IIx fibers display the least. The association between mitochondrial activity and fiber type is paramount to the function of a muscle. In a study from 26 PAD patients, we observed type I fibers lacking intermyofibrillar mitochondria (IMFM-). Fiber type composition was highly variable across PAD patients but IMFM- fibers were correlated to type I fiber abundance. These surprising results suggest mitochondria within type I fibers may be preferentially disrupted, which may in turn affect fiber type composition within PAD calf muscles. This study aims to determine if fiber type-specific mitochondrial activity is altered in PAD patient calf muscle compared to healthy control muscle, and to identify relationships among fiber type-specific mitochondrial activity, fiber size, fiber shape and capillary density. Importantly, we propose to perform these analyses on tissue from the same muscle samples Saini et al are using to characterize calf muscle specific microRNAs unique to PAD. We believe the morphological analyses proposed here will complement findings from the Saini study providing potential cellular targets of microRNAs unique to PAD calf muscle. In Aim 1, we will quantify mitochondrial activity by fiber type, using succinate dehyrodgenase (SDH) histochemistry. In healthy control muscles, we expect SDH to label type I fibers darkest and to appear uniform throughout each fiber. In contrast, we expect IMFM- areas within type I fibers in PAD patient muscles. In Aim 2 we will quantify fiber size and shape (angularity, circularity, roundness, aspect ratio and perimeter), and determine associations of these features with fiber type-specific mitochondrial activity. We expect calf muscle fiber size will be variable across PAD individuals and will not correspond to fiber type or mitochondrial activity. We also expect calf muscle fibers from PAD patients will display altered shape characteristics compared to healthy controls. In Aim 3 capillary density (capillary number/fiber number) will be quantified and relationships to the fiber characteristics measured in Aims 1 & 2 will be determined. Identification of specific muscle fiber characteristics (size, shape and capillary density) related to altered mitochondrial activity in PAD patient calf muscles will provide important insight into cellular changes with PAD that may affect muscle function. Additionally, associations among in situ morphological features and unique microRNAs within PAD patient calf muscles will strengthen findings from both studies and may provide novel information underlying fiber type-specific regulation of adaptive responses to PAD.