Human primary skeletal muscle cells coming from triceps (type II) or soleus (type I) were treated under control or TNFα condition for 24 h, and conditioned media was collected for exosomes isolation. Several months were required to reach the desired number of material needed (more than two hundred flasks for each condition), as primary human culture is very sensible to contamination and low proliferation rate. Some of the characterization approaches have been first validated in a rat muscle cell line (C2C12), because of it higher proliferation rate.
1. We have observed that soleus- and triceps-derived myotubes secrete EVs at the same concentration. However, EVs concentration released from soleus and triceps myotubes from healthy donnors are differentially affected by TNFα. There are significantly more exosomes in Triceps-TNFα samples vs Triceps-control, while Soleus-TNFα derived exosomes were undetectable (Figure 1).
2. SEM images showed vesicles of around 100 nm diameter in both control and TNFα-treated triceps-derived EVs (Figure 2). We can observe a positive expression of CD63 in control and TNFα-treated samples by TEM images (Figure 3). However, we observe cluster of CD63 in the control condition which are not observables in the TNFα condition. Thus, suggesting a different population of EVs in response to TNFα.
3. We have analysed the mRNA and miRNA expression on different muscle type (soleus vs triceps vs vastus lateralis) cells in the presence or absence of TNFα. We found that each muscle type has a unique expression signature for mRNA. Genes which are highly expressed (in red) in one muscle type had, on the contrary, a low expression (in green), showing a “mirror” expression pattern between muscle types (Figure 4A). When we compared the most differentially expressed genes between muscles, we obtained 22 genes that are different among them (Figure 4B and 4C). Those genes are implicated in different biological process, components and functions (Figure 4D). Furthermore, we obtained a different expression of miRNA among different muscle cell types (Figure 5A). At least 5 miRNA from the top differentially expressed miRNA were different the different muscle types. Those 5 miRNA are implicated in different metabolic functions (Figure 5B).
4. We studied the lipid concentration and distribution in MPs and exosomes obtained from C2C12 cells. Cholesterol concentration was lower in exosomes but higher in MPs isolated from TNFα-treated C2C12 cells in comparison to control (Figure 4). For ceramides, a diminution in TNFα-MPs and an increased in TNFα-exosomes was observed when compared to control (Figure 6). Regarding phospholipids (phosphatidylcholines [PC], phosphatidylethanolamines [PE] and phosphatidylserine [PS]), a decreased of PC in TNFα-exosomes and an increased in TNFα-MPs is observed when compared to control exosomes and MPs, respectively (Figure 6), while PE and PS the opposite was observed.
5. C2C12 treated with MPs from TNFα C2C12 showed a significant decrease of AKT phosphorylation compared to control-MPs treated cells (Figure 7B). However, when treated with exosomes from TNFα C2C12, no changes were observed in AKT phosphorylation in healthy cells (Figure 7A).
6. Beta-cells glucose-stimulated insulin secretion showed a tendency to decrease when treated with MPs from control and TNFα-treated C2C12 cells, when compared to untreated beta-cells (Figure 8A). However, when treated with exosomes from control and TNFα-treated C2C12 cells, we observed and increase in the basal insulin secretion compared to untreated beta-cells, indicating a potential effect on the insulin granules trafficking (Figire 8A). No effect was observed in cell death in any condition (Figure 8B and C)
Dissemination and publications
The ExoDia project has been disseminated at national and international congress and scientific publications, giving always visibility to the EU funding and logo. Furthermore, the laureate has participated in many abroad public events as diabetes screenings, running races, national science day and lab visits of high school students. Several General public Press articles related to the Marie Curie Fellowship were also published.
