Effects of Oxidative Stress on Exosome Release in Human Skeletal Muscle Satellite Cells

Zachary R Hettinger, Purdue University

Abstract

In aging, cellular senescence contributes to the impaired capacity of progenitor cells to repair tissue. Decreased regenerative capabilities in skeletal muscle increase with age, where senescence is postulated to contribute to this phenotype. Activation of senescence is accompanied by a secretory network termed the senescence-associated secretory phenotype (SASP) that propagates tissue remodeling and senescent signaling. In skeletal muscle, there are currently no reports of secretory activity following activation of senescence. We hypothesized that by isolating satellite cells from human skeletal muscle and activating senescence in vitro, we could measure the secretory activity of skeletal muscle satellite cells (SkMSCs). Specifically, we hypothesized an increase secretory activity of small paracrine communicating molecules termed exosomes (30–150 nM), along with reported SASP markers of Interleukin-1α, -6, -8 and Transforming Growth Factor-β (i.e. IL-1a, IL-6, IL-8 and TGF-β) compared to controls. Six lean men and women (26.6 4.6 yr) had vastus lateralis muscle biopsies performed where satellite cells were isolated and cultured. SkMSCs were exposed to 200 µM hydrogen peroxide (H2O2) for 24 hours to induce senescence and collected after 4 days. Following treatment, outcomes of proliferation, markers of senescence and SASP activity, exosome release and exosome counts were analyzed. Cell proliferation was decreased by H 2O2 as evidenced by population doubling levels (PDLs) (H 2O2: 1.86 vs. Con. 1.23, PDLs, *p < 0.05) and DNA replication stain, 5-ethynyl-2’-deoxyuridine (EdU), compared to controls (H 2O2: 3.52% vs. Con. 24.53%, %EdU Positive Cells). Senescence marker p21 mRNA (H2O2: p21 mRNA 1.53 vs. Con: 1.00, AU) and protein (H2O2: p21 protein 1.49 vs. Con: 1.00, AU) were increased in response to H2O2. Exosome release (30–150 nM) was increased from senescent cells compared to controls (H2O2: 74.11 vs. Con: 10.93, particles/cell) evidenced by Nanosight micrscopy. Senescence increased gene expression of the multivesciuclar body (MVB) biogenesis pathway for STAM (H2O2: 1.47 vs. Con: 1.00, AU), TSG-101 (H2O2: 1.30 vs. Con: 1.00, AU), CD-63 (H2O2: 1.28 vs. Con: 1.00, AU) and CHMP4C (H 2O2: 2.12 vs. Con. 1.00, AU). Senescence increased gene expression of exosome secretion markers Rab27a (H2O2: 1.49 vs. Con: 1.00, AU) and Rab35 (H2O2: 1.32 vs. Con: 1.00, AU). Activation of senescence trended towards increased total and phosphorylated NF-kB (p = 0.06), where increased downstream SASP factors of TGF-β mRNA (H2O2: 1.39 vs. Con: 1.00, AU) and IL-8 mRNA (H 2O2: 1.61 vs. Con: 1.00, AU) were observed compared to controls. The results of the present study suggest H2O2- induced senescence increases exosomes release (30–150 nM) from senescent SkMSCs, evidenced by increased MVB biogenesis and exosome secretion pathways. Also, H2O2 increased skeletal muscle satellite cell TGF-β and IL-8 mRNA, known factors of SASP. The present study suggests that senescence in primary human skeletal muscle satellite cells increases the release of exosomes and activates SASP, which may be important for impaired cellular communication in aging skeletal muscle. Future studies are needed to study the consequence of increased exosome release and the activation of satellite cell SASP in contribution to the aged phenotype of skeletal muscle.

Degree

M.S.

Advisors

Gavin, Purdue University.

Subject Area

Molecular biology|Cellular biology|Kinesiology

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