Mitchell, A. D. | bioRxiv Science

bioRxiv2020

Satellite cell-derived extracellular vesicles reverse peroxide-induced mitochondrial dysfunction in myotubes

Satellite cells (SCs) are muscle-specific stem cells that have a central role in muscle remodeling. Despite their therapeutic potential, SC-based therapies have been met with numerous logistical challenges, limiting their ability to effectively treat systemic muscle diseases, such as Duchenne muscular dystrophy (DMD). Delivery of SC-derived extracellular vesicles (SC-EVs) may unlock the potential offered by SCs and overcome their numerous limitations. PurposeThe purpose of this investigation was to determine the extent to which SC-EVs could restore mitochondrial function in cultured myotubes following oxidative injury. MethodsSC-EVs were isolated from cultured SCs from C57 mice and quantified using nanoparticle tracking analysis (NTA). C2C12 myotubes were cultured and divided into four treatment groups: untreated control, treated for 24 h with SC-EV, 24 h exposure to 50 M H2O2 followed by a 24 h recovery period with no treatment, or 24 h exposure to 50 M H2O2 followed by a 24 h treatment with SC-EV. Inter-group differences in mitochondrial function were assessed via one-way ANOVA with Tukey post hoc analysis (p<0.05). ResultsGiven the seeding density used, we calculated that each SC releases approximately 2.35 x 105 {+/-} 3.10 x 104 EVs per 24 h. Further, using fluorescent microscopy, we verified SC-EVs deliver cargo into myotubes, some of which was localized to the mitochondria. H2O2 exposure resulted in a 42% decline in peak mitochondrial respiration (p=0.0243) as well as a 46% reduction in spare respiratory capacity (p=0.0185) relative to the untreated control group. Subsequent treatment with SC-EVs (3.12x108 SC-EV; 24 h) following H2O2 exposure restored 76% of peak mitochondrial respiration (p=0.0187) and 84% of spare respiratory capacity in the damaged myotubes (p=0.0198). SC-EVs did not affect mitochondrial function in the undamaged myotubes. ConclusionCollectively, these data demonstrate SC-EVs may represent a novel therapeutic approach for treatment of myopathies associated with mitochondrial dysfunction.

physiology↗

bioRxiv2018

An RNAi screen in human cell lines reveals conserved DNA damage repair pathways that mitigate formaldehyde sensitivity

Formaldehyde is a ubiquitous DNA damaging agent, with human exposures occuring from both exogenous and endogenous sources. Formaldehyde can also form DNA-protein crosslinks and is representative of other such DNA damaging agents including ionizing radiation, metals, aldehydes, chemotherapeutics, and cigarette smoke. In order to identify genetic determinants of cell proliferation in response to continuous formaldehyde exposure, we quantified cell proliferation after siRNA-depletion of a comprehensive array of over 300 genes representing all of the major DNA damage response pathways. Three unrelated human cell lines (SW480, U-2 OS and GM00639) were used to identify common or cell line-specific mechanisms. Four cellular pathways were determined to mitigate formaldehyde toxicity in all three cell lines: homologous recombination, double-strand break repair, ionizing radiation response, and DNA replication. Differences between cell lines were further investigated by using exome sequencing and Cancer Cell Line Encyclopedia genomic data. Our results reveal major genetic determinants of formaldehyde toxicity in human cells and provide evidence for the conservation of these formaldehyde responses between human and budding yeast.

cell biology↗