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P&F Grant Awards

Year 6


Grant # 13

Preclinical Studies to Assess Autophagic Flux in Human Skeletal Muscle

PI: Conrad Weihl, PhD, MD

 

Specific Aims

Protein degradation occurs via two principal degradative pathways, the ubiquitin proteasome system (UPS) and autophagy [1]. The importance of autophagic protein degradation in normal cellular metabolism and its dysfunction in disease has become increasingly clear over the past five years [2]. Alterations in autophagic activity have been a proposed pathogenic mechanism in many musculoskeletal disorders including Duchenne Muscular Dystrophy [3], osteoarthritis [4], Paget’s disease of the bone [5], bone and muscle related malignancy (e.g. osteosarcoma and rhabdomyosarcoma) [6] and even sarcopenia [7]. More importantly, correction of the aberrant autophagic activity is therapeutic in some mouse models of these same human diseases [3, 4, 8-10]. Our laboratory is interested in autophagic protein degradation in skeletal muscle. Skeletal muscle is the largest reservoir of free amino acids in the body. Autophagic protein degradation in skeletal muscle is modulated by diverse stimuli including nutritional state, exercise and atrophic signaling pathways [2, 11]. Moreover, many FDA approved drugs have been repurposed as autophagy modulating therapies that may have efficacy in skeletal muscle [12]. However, whether skeletal muscle autophagy is similarly regulated and dysregulated in humans and human disease states is not known.

The greatest limitation to understanding the regulation of autophagic processes in human skeletal muscle and muscle disease is the lack of a reliable analytical method for quantifying autophagic degradation in vivo.

Proteolysis (the degradation of proteins and subsequent liberation of free amino acids) is tightly regulated in cells and tissue; occurring via the UPS and autophagy. Unfortunately, measuring total proteolysis within a tissue cannot distinguish between these two pathways. Therefore, one must identify a protein that is exclusively degraded via the autophagic pathway. Our preliminary studies have identified the autophagic adaptor protein p62/SQSTM1 as a likely candidate autophagic substrate in skeletal muscle. However, static levels of p62 measured via immunohistochemistry or immunoblot, do not accurately correlate with autophagic protein degradation since p62 is both simultaneously synthesized and degraded when autophagy is stimulated.

To circumvent this inherent difficulty in interpreting the degradation of any autophagic substrate, we propose to quantify the in vivo rate of p62 degradation in human skeletal muscle using stable isotope labeling tandem mass spectrometry (MS). Stable isotope labeling has been utilized to quantify skeletal muscle actin, myosin, and mitochondrial protein synthesis and degradation rates [13, 14]. However unlike those highly abundant muscle proteins, most proteins within biologic samples are present at concentrations less than one nM. This concentration approaches the detection limit for gas chromatography-MS necessitating the utilization of newer technologies. Our methodology can detect and quantitate labeling in low abundant (femtomolar) proteins. More importantly, it can be used on small tissue samples such as skeletal muscle needle biopsy specimens from human patients.

This pilot application is a collaboration between Dr. Weihl (a neuromuscular physician interested in protein degradation pathways in skeletal muscle), Dr. Yarasheski (a pioneer in stable isotope labeling and MS analysis of human skeletal muscle proteins), and Dr. Bateman (the developer of human stable isotope labeling technology for Aβ kinetics). The infrastructure for this proposal is in place at Washington University School of Medicine within the Department of Neurology and we are uniquely positioned to address this question.

We hypothesize that the half-life (t½) of p62 in skeletal muscle represents a quantitative biomarker of autophagic kinetic activity in skeletal muscle.

Aim: Define the t½ of p62 and correlate p62 t½ with autophagic activity in mouse skeletal muscle. Our preliminary data demonstrates our ability to detect labeled and unlabeled p62 in normal mice. We will A) define the synthesis rate of p62 in normal mouse skeletal muscle; B) define the t½ of p62 in normal mouse skeletal muscle and C) manipulate and quantify the t½ of p62 after modulating skeletal muscle autophagy using nutrient deprivation, pharmacologic compounds and transgenic mouse lines.

Upon completion of these studies, we will have validated a novel method to quantify the in vivo t½ of p62 in skeletal muscle and have proof of concept that changes in p62 t½ reflect autophagic activity. We have identified p62 via tandem-MS in human skeletal muscle and begun to orally label humans subjects with stable isotopes. The current proposal will be translated to human patients allowing us to be able to quantify the kinetics of autophagic dysfunction in human muscle disease and normal aging.