Metabolic Stress is Not Sufficient to Increase Skeletal Muscle Tissue Extracellular Vesicle Secretion in an Ex Vivo System

Abstract

Skeletal muscle (SkM) is a metabolically active tissue and accounts for ~35% of total human body mass. Exercise imposes acute metabolic stress on SkM myofibers, and contractile proteins require energy sustained through both glycolytic and oxidative metabolism. Increased metabolic demand during contraction promotes intercellular communication through secreted factors that include extracellular vesicles (EVs). However, the mechanisms linking metabolic flux and EV secretion are currently unknown. We hypothesized that mimicing metabolic stress by blocking glycolysis with 2-deoxyglucose (2-DG), or oxidative phosphorylation with BAM15, a mitochondrial uncoupler, would increase EV secretion in SkM. Additionally, we aimed to characterize the effects of contractile inhibition with blebbistatin (BLEB) to better understand the role of contraction on EV release. We tested our hypothesis using an ex vivo EV secretion assay. Vastus medialis muscle (VM) from male C57Bl/6J mice (n=6) and soleus (SOL) and plantaris (PLA) muscle from female mT/mG mice (n=6) was cut into ~5 mg pieces and added to 12 well plates (~50 mg/well) filled with 750mL of serum-free DMEM and placed in a cell culture incubator at 37°C for 24 hours. The effect of 2-DG on VM EV secretion was measured during a log dose response (0.1nM – 100mM). SOL and PLA from female mice were treated with either 10mM of BLEB, or 10mM of BAM15. After incubation, tissue mass was measured and conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. EVs were isolated using ExoQuick-TC. NTA was performed on isolated EVs using a Horiba Viewsizer 3000. EV secretion was normalized to tissue mass and culture media volume and reported as ([Particle]/mL/mg tissue). Statistical comparisons for 2-DG experiments were made using a repeated measures 1-way ANOVA with Bonferroni post-hoc testing. BLEB (n=6) and BAM15 (n=4) experiments were analyzed using a paired Student's t-test. No effect of 2-DG was observed in regard to EV abundance (p = .07) or diameter (p = 0.37). BAM15 did not affect EV abundance in SOL (p = 0.64) or PLA (p = 0.78). BLEB did not alter EV abundance (p = 0.69; CON: 1.32E+09 ± 4.86E+08 vs BLEB 1.67E+09 ± 5.87E+08), but significantly reduced EV mean diameter (p = 0.007; DMSO: 114.9 ± 5.1 vs. BLEB: 103.9 ± 4.2). Contrary to our hypothesis, inducing metabolic stress did not significantly increase SkM EV secretion. Mitochondrial uncoupling did not increase EV secretion in oxidative SkM such as the soleus, which is predominately comprised of fibers with high mitochondrial density. Inhibition of glycolysis with 2-DG did not increase SkM EV secretion. In the presence of BLEB, EV mean diameter was reduced but not EV abundance, suggesting that inhibiting contractile function may affect specific pathways of EV release. Collectively, our data demonstrate that metabolic stress may not be sufficient to increase EV secretion from SkM in an ex vivo system. Future studies might explore the specific role of contraction or extracellular factors as mediators of SkM EV secretion.