Acute Effects of Contractile Activity on Skeletal Muscle Exosomes

Introduction: Skeletal muscle functions as an endocrine organ, and a key way to accomplish that is through exosomes. The internal budding of a multivesicular body (MVB) forms exosomes and contains mRNA, miRNA, and proteins. Through a multifaceted approach, the following three experiments were designed to better understand the effect of contractile activity on skeletal muscle MVB/exosome biogenesis and release. Study 1: Exosome-related pathway response to acute exercise. Sedentary, lean, healthy male subjects (n=12) performed 45 min cycling (AEx), followed by single leg knee extensor exercise (A+REx). A biopsy was obtained prior to exercise and 1 hr post-exercise from each leg. Stimulation of MVB biogenesis occurred in the A+REx group, with miRNA biogenesis activated by both AEx and A+REx. Overall, there appeared to be no additive effect of REx for angiogenesis or mitochondrial improvements following acute exercise. Our data displays no total protein changes for known exosome surface markers. Increased MVB biogenesis and release indicate exciting possibilities for exercise stimulated exosome release. Study 2: Effects of obesity and acute resistance exercise on skeletal muscle intercellular communication pathways. To more fully determine the effect of resistance exercise (REx) alone on MVB/exosome signaling and obesity on skeletal muscle angiogenesis, the following study was performed: Lean (LN) and obese (OB) (n=8 each group) sedentary human subjects performed single leg knee extension exercise at 80% 1-RM, 3 sets, 8-12 reps. Muscle biopsies were obtained at rest, 15 min, and 3 hr post-exercise. With OB, baseline anti-angiogenic miRNA and diminished MVB biogenesis were displayed, along with an increased anti-angiogenic factor thrombospondin-1 in response to acute REx, indicating plausible pathways to explain diminished capillary density of Type II muscle fibers. No total changes were observed in VEGF between LN and OB, though a trend for reduced VEGF existed in the OB group compared to LN. Study 3: Role of CaMKII in skeletal muscle exosome release. To determine a candidate pathway linked with calcium signaling, muscle contraction, and exosome release, specific attention was given to the role of calcium-calmodulin kinase II (CaMKII). Skeletal muscle biopsies were obtained from lean, healthy, male college-aged subjects (n=8) and satellite cells isolated for use in cell culture (SkMCC). To ensure proper differentiation, SkMCC had markers Pax7, Myogenin (MyoG), and myosin heavy chain (MHC) measured via Western blot on days 0, 2, 4, 6, and 8. SkMCC were stimulated for 30 min with electrical pulse stimulation (EPS), an exercise mimetic, to validate activation of in vivo exercise pathways adenosine monophosphate kinase (AMPK), mitogen activated protein kinase (MAPK) and CaMKII. SkMCC underwent 24 h of EPS with and without the addition of KN-93, a chemical inhibitor of CaMKII. Quantification of isolated exosomes following EPS was accomplished by use of a NanoSight, RT-PCR, and Western blot analysis. The main finding, despite an identifiable change in MHC and formation of myotubes, minimized contractile activity in vitro leading to inadequate activation of metabolic pathways and MVB release. Strong trends for the phosphorylation of AMPK, interaction of CaMKII with clathrin gene expression, reduced gene expression of PDK4, and reduced total nanoparticles in response to EPS indicate that changes occurred, though not to the anticipated levels based on preliminary data