Biology subjects

Souza, T. F. G.

Publications and source records attributed to Souza, T. F. G..

3 recordsLinked to original sources

bioRxiv2024

Identification of novel myokines and putative protein targets that mediate functional adaptations in response to chronic contractile activity induced skeletal muscle-extracellular vesicle treatment

We have previously shown that skeletal muscle-derived extracellular vesicles (EVs) released post-chronic contractile activity (CCA) increased mitochondrial biogenesis in murine myoblasts, and decreased cell viability and induced apoptosis and senescence in non-small cell lung cancer cells. While the underlying mechanisms are unknown, the effects perpetuated were dependent on membrane-bound proteins. Here, we performed an extensive LC-MS/MS proteomic analysis on EVs from control and CCA myotubes. A total of 2900 proteins were identified in CON-EVs and CCA-EVs, including EV-associated proteins such as TSG101, tetraspanins (CD9, CD81, and CD63), flotillin-1, and annexins. Of these, 856 proteins are novel and not listed in EV databases (ExoCarta and Vesiclepedia), indicating that myotube-EVs harbor proteins not yet identified in EVs of different origin. Additionally, we identified 2062 unique proteins that have not yet been previously reported in myotube-EVs to date. Remarkably, of the 2900 total proteins identified, we observed 46 upregulated, and 25 downregulated differentially expressed proteins (DEPs) in CCA-EVs vs. control-EVs. Most of upregulated DEPs include EV-associated proteins. Comparing the 71 DEPs with proteins expressed in skeletal muscle indicated 61 of these as potential myokines. We identified actin cytoskeleton signaling, integrin signaling and muscle contraction as the most enriched pathways among the DEPs using different databases/software including FunRich, KEGG, STRING and Ingenuity Pathway Analysis. Using a relevance score that prioritized membrane-bound proteins with known function in mitochondrial biogenesis and inhibition of cancer growth, we identified top-scoring highly enriched DEPs of interest: IGF1R, ATP7A, PFN1, GJA1, PRKCA and ITGA6. We confirmed upregulation of these targets in EVs using immunoblotting. Among these top-scoring DEPs, PFN1, and ITGA6 are associated with EVs, with expression upregulated following acute exercise. In summary, we report the first comprehensive analysis of skeletal muscle-EV proteome following CCA, with identification of putative protein targets and signaling pathways that may execute the pro-metabolic and anti-tumorigenic effects of CCA-EVs.

cell biology↗

bioRxiv2024

The pro-apoptotic effect of chronic contractile activity-induced extracellular vesicles on Lewis Lung Carcinoma cells

Regular exercise reduces tumor growth in vivo and in vitro, but the exact mechanisms have yet to be fully elucidated. We have previously shown that chronic contractile activity (CCA) increases the concentration of skeletal muscle-derived EVs, and these in turn increased mitochondrial biogenesis in myoblasts. Here, we hypothesized that skeletal muscle-EVs derived post-CCA will mediate the anti-tumorigenic effects associated with chronic exercise. C2C12 myoblasts were differentiated into myotubes, electrically paced, and EVs isolated from conditioned media from control and CCA myotubes using differential ultracentrifugation. Lewis lung carcinoma (LLC) cells were treated with the total number of control-EVs or CCA-EVs isolated after each day of contractile activity for 4 days. Permeabilized CCA-EVs with or without proteinase K before co-culture with LLC cells were used as controls. Effect of EV treatment on cell count, viability, apoptosis, senescence, migration, and mitochondrial content was measured. CCA-EV treatment reduced cell count by 18% and cell viability by 6% vs. control-EVs. CCA-EVs increased the incidence of apoptotic hallmarks: DNA fragmentation by 13%, Annexin V+/PI+ cells by 21%, and the expression of pro-apoptotic Bax (by 25%) and Bax/Bcl-2 ratio (by 60%) vs. control-EVs. CCA-EVs increased number of senescent cells by 29%, and senescence markers, HMGB1 (by 49%) and p16 (by 92%) vs. control-EVs. When CCA-EVs were pretreated with Triton X-100 with or without proteinase-K, the increase in apoptosis and senescence was abrogated, confirming the effect is due to intact EVs and likely through EV membrane-proteins. CCA-EVs did not have any effect on cell migration and mitochondrial content vs. control-EVs. This study illustrates for the first time the potential of CCA-induced skeletal muscle-EVs in mediating anti-tumorigenic effects traditionally linked with chronic exercise.

cancer biology↗

bioRxiv2024

Chronic contractile activity induced skeletal muscle-derived extracellular vesicles increase mitochondrial biogenesis in recipient myocytes via transmembrane or peripheral membrane proteins

The effect of chronic contractile activity (CCA) on the biophysical properties and functional activity of skeletal muscle extracellular vesicles (Skm-EVs) is poorly understood due to challenges in distinguishing Skm-EVs originating from exercising muscle in vivo. To address this, myoblasts were differentiated into myotubes, and electrically paced (3 h/day, 4 days @ 14 V). CCA evoked an increase in mitochondrial biogenesis in stimulated vs. non-stimulated (CON) myotubes as expected. EVs were isolated from conditioned media from control and stimulated myotubes using differential ultracentrifugation and characterized biophysically using tunable resistive pulse sensing (TRPS, Exoid), TEM and western blotting. TEM images confirmed isolated round-shaped vesicles of about 30 - 150 nm with an intact lipid bilayer. The mean size of EVs ranged from 98 -138 nm, and was not altered by CCA. Zeta potential and total EV protein yield remained unchanged between groups, and total EV secretion increased after 4 days of CCA. Concomitant analysis of EVs after each day of CCA also demonstrated a progressive increase in CCA-EV concentration, while size and zeta potential remained unaltered, and EV protein yield increased in both CON-EVs and CCA groups. CCA-EVs were enriched with small-EVs vs. CON-EVs, concomitant with higher expression of small-EV markers CD81, Tsg101 and HSP70. In whole cell lysates, CD63 and ApoA1 were reduced with CCA in myotubes, whereas CD81, Tsg101, Flotillin-1 and HSP70 levels remained unchanged. To evaluate the functional effect of EVs secreted post-CCA, we treated C2C12 myoblasts with all EVs isolated from CON or CCA myotubes after each day of stimulation, and measured cell count, cell viability, protein yield and mitochondrial biogenesis in recipient cells. There was no effect on cell count, viability and protein yield. Myoblasts treated with CCA-EVs exhibited increased mitochondrial biogenesis as indicated by enhanced MitoTracker Red staining, cytochrome c oxidase activity, and protein expression of electron transport chain subunit, CIV-MTCO1. Further, CCA-EV treatment enhanced maximal oxygen consumption rates (OCR), and ATP production in treated myoblasts. This increase in maximal OCR was abrogated when CCA-EVs pre-treated with proteinase K were co-cultured with myoblasts, indicating the pro-metabolic effect was likely mediated by transmembrane or peripheral membrane proteins in CCA-EVs. Our data highlight the novel effect of Skm-EVs isolated post-CCA in mediating pro-metabolic effects in recipient cells and thereby transmitting the effects associated with traditional exercise. Further investigation to interrogate the underlying mechanisms involved in downstream cellular metabolic adaptations is warranted.

cell biology↗