Circulating Tetraspanins: From Markers to Mechanisms Driving Systemic Exercise Adaptation

Exercise impr ov es cardiometa bolic health thr ough a range of systemic [ie , bey ond w orking skeletal muscle (SkM)] mec ha-nisms typically attributed to small molecules and peptide hormones. Recent discoveries have shown that the abundance and cargo of circulating small extracellular vesicles (sEVs) like exosomes ar e alter ed by exer cise , but linking these c hanges to SkM-deri v ed systemic exercise adaptations has been challenging. A key barrier to linking SkM sEVs to exercise adaptations is determining which of the hundreds of molecules that may be transported by SkM sEVs have functional relevance in the context of exer cise . One surprisingly untested str ate gy is to start with the most abundant sEV car go . Tetraspanins like CD81 are tr ansmembr ane protein hallmarks of sEVs. To date, CD81 has only been described as an sEV marker, not an instrument of sEV function. However, ∼ 30 yr of resear c h has established CD81 as a tr ansmembr ane adaptor protein that influences a variety of cellular functions by altering the organization of r ece ptor pr oteins within membranes. Multiple groups

Exercise impr ov es cardiometa bolic health thr ough a range of systemic [ie , bey ond w orking skeletal muscle (SkM)] mec hanisms typically attributed to small molecules and peptide hormones.Recent discoveries have shown that the abundance and cargo of circulating small extracellular vesicles (sEVs) like exosomes ar e alter ed by exer cise , but linking these c hanges to SkM-deri v ed systemic exercise adaptations has been challenging.A key barrier to linking SkM sEVs to exercise adaptations is determining which of the hundreds of molecules that may be transported by SkM sEVs have functional relevance in the context of exer cise .One surprisingly untested str ate gy is to start with the most abundant sEV car go .Tetraspanins like CD81 are tr ansmembr ane protein hallmarks of sEVs.To date, CD81 has only been described as an sEV marker, not an instrument of sEV function.However, ∼30 yr of resear c h has established CD81 as a tr ansmembr ane adaptor protein that influences a variety of cellular functions by altering the organization of r ece ptor pr oteins within membranes.Multiple groups hav e r ecentl y shown that exercise incr eases the a bundance of circulating sEVs containing CD81 1 , 2 (CD81 + sEVs), which suggests that exercise may increase the delivery of CD81 to recipient cells.Another recent discovery has shown that CD81 supports the pr eserv ation of adipose tissue metabolic health during obesity, 3 but it is not known to what extent CD81 + sEVs contribute to this phenomenon.Mor eov er, no studies to date have described the impact of obesity on the abundance of circulating CD81 + sEVs or what effects circulating CD81 protein may have on recipient cells.In this Opinion, w e combine contempor ary evidence on exer cise-induced circulating sEVs and the metabolic effects of CD81 in adipocytes with historical evidence on cellular CD81 function to propose SkM-to-adipose tissue tr affic king of CD81 in sEVs as a novel mechanism driving exercise adaptations.
What evidence links CD81 + EVs to the physiology of exercise?The study of sEVs and their cargo during exercise is a popular but conv oluted ar ea of inv estigation.Inconsistent isolation methods, various exercise modalities, and different model systems hav e hinder ed pr ogr ess and pr ecluded consensus in the field.This convolution is worsened by a lack of tools to study celltype-specific (eg, SkM) sEV populations in vi v o.Despite these challenges, some of the strongest evidence linking sEVs to exercise is through CD81.Both progressive 1 and high-intensity interval training 2 exercises acutel y incr ease the circulating abundance of CD81 + sEVs of human subjects ( Figure 1 A).Brahmer et al. 1 measured CD81 protein from isolated plasma sEVs from young, healthy, and acti v e men at three time points of an incremental cycling exercise protocol: baseline (immediately prior to exercise), during exercise once r espirator y quotient (RQ) exceeded 0.9 (which the authors equated to a "moder ate" exer cise intensity), and immediately after ( < 2 min) cessation of exercise at maximal workload.The authors found that both moderate (RQ ∼ 0.9) and maximal intensity exercises incr ease CD81 pr otein a bundance in circulating plasma sEVs.
A mor e r ecent study fr om MclIv enna et al. 2 quantified individual circulating plasma CD81 + sEVs during exer cise .In this study young, acti v e, healthy males and females performed high intensity interval training (HIIT) exercise (4 × 30 s of cycling at 200% of each participant's predetermined maximal power output).Plasma was collected immediately prior to exercise and < 30 s after the final exercise interval.CD81 + sEVs were captured on a microfluidics chip using a CD81-specific antibody and dir ectl y quantified using fluorescence detection.Using this more  rigor ous and dir ect appr oach to quantify pr otein-specific sEV populations, the authors found that HIIT increases the number of circulating CD81 + sEVs.To build upon these promising findings, future studies could explore how long CD81 + sEVs are present in the circulation after exercise, identify the cells (ie , SkM m y ofibers) that secrete CD81 + sEVs during exercise, and discover what function(s) they have on recipient cells.
It is intuiti v e and conv enient to attribute the effects of exercise on circulating sEVs to SkM myofibers, but there is limited direct evidence to support (or refute) this idea.This knowledge gap exists because there is no known sEV cargo that is exclusi v e to SkM m y ofibers, and ther efor e esta b lishing the mere existence of SkM sEVs in the circulation has been challenging.To address this knowledge gap, our group used a fundamentally different str ate gy.Instead of trying to find an endogenous SkMspecific sEV car go , we used Cr e-de pendent expr ession of a fluor escent r e porter pr otein in mice to tr ac k SkM m y ofiber-deri v ed sEVs. 4With this approach, we found that SkM secretes CD81 + sEVs, which reach the circulation.It is not yet known if, and to what extent, exercise increases circulating SkM CD81 + sEVs or where they might accumulate .How ever, recent work by Ismaeel et al. found that sEVs containing the muscle (SkM and cardiac)specific miRNA, miR-1, accumulate in white adipose tissue. 5hese data show that exercise increases circulating CD81 + sEVs, some of which may originate from SkM myofibers, and suggest that white adipose tissue may be a tar get or gan of SkM sEVs secreted during exer cise .
How might SkM EVs reach adipose tissue?It is not yet clear how sEVs travel from tissues to the blood, but the known anatomical features of the body offer important clues.That is, sEVs ar e m uch larger (50-200 nm diameter) than tight junctions of the SkM capillary endothelium (2-3 nm diameter), which should not allow for the passi v e transendothelial flux of sEVs from the SkM interstitium dir ectl y into the cir culation ( F igur e 1 B).Mor eover, Starling forces dictate that an intact cardiovascular system forces fluid from the capillary endothelium into the interstitial space and subsequently into the lymphatic circulation.Therefore , w e believe it is most likely that SkM sEVs reach the blood through the lymphatic circulation.There is experimental evidence for both lymphatic and transendothelial flux of e xo genous sEVs, but no studies to date have examined SkM sEV trafficking or esta b lished a mechanism of endogenous sEV tr affic king from tissues to the blood.Establishing how sEVs from SkM m y ofibers (and other tissue resident cell types) reach the blood may impr ov e the design and dev elopment of tissue-deri v ed sEVs as disease biomarkers and therapeutics.
The mec hanisms re gulating sEV uptake into cells are also incompletel y defined.Pr evious work has sho wn that adipoc ytes uptake endothelial cell-deri v ed sEVs in vitr o and in vi v o and this process is impaired by overnutrition. 6In vitro studies have shown that CD81 protein delivered to cultured cells via sEVs is functional, 7 , 8 whic h suggests that inter cellular tr ansfer of CD81 + sEVs in vi v o allows r ecipient cells to "adopt" functions attributa b le to CD81.Future studies are needed to firmly establish the physiological function(s) of CD81 + sEVs on metabolic health and disease outcomes.
What are the cellular functions of CD81?CD81 is a transmembrane protein that indirectly enhances adhesion r ece ptor-mediated cellular processes via molecular "webs" [or tetr aspanin-enric hed microdomains (TEMs)] that a ggr egate r ece ptors at the cell surface 9 ( Figure 1 C).The most thoroughly described mechanism of CD81 action is its ability to lower the thr eshold to acti v ation of B cells and T cells through physical interactions with CD19/CD21 and CD4/CD8, r especti v el y.CD81 is also known to interact with integrin r ece ptors that not only regulate cellular behavior, but are also involved in metabolic disease etiology.Recent discoveries by Oguri et al. have demonstrated that CD81 pr eserv es cardiometa bolic health via integrin-dependent signaling in white adipose tissue. 3n their study, the authors demonstrated that CD81 is both necessary and sufficient for de novo beige adipogenesis, 3 an adaptation that increases energy expenditure and may be a treatment for obesity and other metabolic diseases.In this same study, the authors discov er ed that whole-body deletion of CD81 in mice worsened the consequences of diet-induced obesity in white adipose tissue (eg, fibrosis, inflammation, and insulin resistance) by disrupting integrin signaling.Finally, the authors demonstrated that a low abundance of CD81 + adipocyte progenitor cells within resident white adipose tissue depots is associated with impaired glucose homeostasis, adiposity, and hypertension in human subjects.The authors did not examine a role for CD81 + sEVs in this study, and it is not yet known how metabolic disease influences the circulating abundance and tr affic king of CD81 + sEVs.However, the acute exercise-induced increase in circulating CD81 + sEVs may cause greater delivery of CD81 protein to adipose tissue (and other tissues), thereby contributing to exercise adaptations.Future studies could seek to define the ability of CD81 + sEVs to positi v el y modulate cell signaling pathways necessary for cardiometabolic health.
As the body of literature on sEVs continues to grow, it is important to recognize that the systemic effects of sEVs and their cargo are the product of tr affic king, abundance , and intracellular function(s).Because CD81 is such an abundant sEV protein, it is possible that its systemic physiological impact when tr affic ked in sEVs could meet, or exceed, the effects of miRNAs and other scarce sEV cargo that have more tractable molecular mechanisms.The ability of CD81 to enhance the sensitivity of cell signaling pathways makes it a particularly interesting target for cardiometabolic diseases that are c har acterized by resistance to second messenger signaling.Based on the evidence pr ovided her e , w e hypothesize that sEVs contribute to systemic adaptations to exercise via SkM-to-adipose tr affic king of CD81.This hypothesis could be tested using emerging transgenic "EV r e porter" mouse models 10 and careful study and manipulation of sEV populations in vi v o.If CD81 + sEVs are indeed therapeutic, industrial-scale manufacturing of sEVs is already being utilized for n umer ous clinical trials, so CD81-enriched sEVs could be developed from "bench to bedside" rapidly to improve human metabolic health.

Figure 1 .
Figure 1.Evidence linking the cellular functions of CD81 to sEVs during exer cise .(A) A single bout of continuous aerobic or high-intensity interval training increases CD81 + sEVs in the circulation in human subjects.In mice, SkM myofibers are a major source of CD81 + sEVs reaching the circulation.Endogenous SkM sEVs accumulate in adipose tissue.(B) sEVs are too large to cross endothelial tight junctions, and Starling forces across the capillary endothelium push plasma into the interstitial space.SkM EVs may be taken up by lymphatic capillaries that collect interstitial fluid (which contains sEVs) and transported back to the blood.(C) In cells, CD81 modulates signaling pathwa ys b y forming "webs" consisting of CD81 and a variety of cell surface r ece ptors, including integrins (shown).CD81 is necessar y and sufficient for de novo beig e adipog enesis, which has health-promoting effects on energy expenditure.Future studies could examine whether CD81 + sEVs are capable of regulating signaling pathways and physiological functions already attributed to cell-intrinsic CD81. Figure created with Biorender.com .