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. 2022 Aug 25:13:937899.
doi: 10.3389/fphys.2022.937899. eCollection 2022.

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

Christopher G Vann  1 , Xin Zhang  1   2 , Alastair Khodabukus  3 , Melissa C Orenduff  1 , Yu-Hsiu Chen  1 , David L Corcoran  4 , George A Truskey  3 , Nenad Bursac  3 , Virginia B Kraus  1   2   5
Affiliations

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

Christopher G Vann et al. Front Physiol. .

Abstract

Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle ("myobundles"). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p < 0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo.

Keywords: engineered tissue; extracellular vescicles; miRNA sequencing; microRNA; skeletal muscle.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Study design. Abbreviations: CTL, control; CLFS, chronic low frequency stimulation; IHFS, intermittent high frequency stimulation.
FIGURE 2
FIGURE 2
Differential miR Expression Between Population and Within Treatment Condition. Abbreviations: EV, extracellular vesicles; MB, myobundle; CTL, control; CLFS, chronic low frequency stimulation; IHFS, intermittent high frequency stimulation. Legend: data for Panel (A) is presented as mean normalized count. Panel (B) represents overlap of miRs for analyses completed. Panel (C–E) are volcano plots with blue data points depicting p < 0.05 and red data points depicting p < 0.05 and logFC of >1.5 or < -1.5.
FIGURE 3
FIGURE 3
Relationships of Differentially Expressed miRs. Abbreviations: EV, extracellular vesicles; MB, myobundle; CTL, control; CLFS, chronic low frequency stimulation; IHFS, intermittent high frequency stimulation. Legend: data for panels (A–C) are presented as mean Z-Score with nodes depicting relationships between population and treatment.
FIGURE 4
FIGURE 4
Top 10 Overrepresented KEGG Pathways Based on Observed miRs. Abbreviations: PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; MAPK, mitogen activated protein kinase; FoxO, forkhead box, Rap-1, ras-related protein 1. Legend: pathways are presented by dot size (observed miR count) and color (expected miR count) and plotted in order based on the observed count.
FIGURE 5
FIGURE 5
Differential Myobundle miR Expression Between Treatments. Abbreviations: EV, extracellular vesicles; MB, myobundle; CTL, control; CLFS, chronic low frequency stimulation; IHFS, intermittent high frequency stimulation. Legend: data for Panel (A) is presented as mean normalized count. Panel (B) represents overlap of miRs for analyses completed. Panels (C–E) are volcano plots with blue data points depicting p < 0.05 and red data points depicting p < 0.05 and logFC of >1.5 or < -1.5.
FIGURE 6
FIGURE 6
Differential Extracellular Vesicle miR Expression Between Treatments. Abbreviations: EV, extracellular vesicles; MB, myobundle; CTL, control; CLFS, chronic low frequency stimulation; IHFS, intermittent high frequency stimulation. Legend: data for Panel (A) is presented as mean normalized count. Panel (B) represents overlap of miRs for analyses completed. Panels (C–E) are volcano plots with blue data points depicting p < 0.05 and red data points depicting p < 0.05 and logFC of >1.5 or < -1.5.
FIGURE 7
FIGURE 7
Myobundle and Extracellular Vesicle Enriched miRs. Abbreviations: EV, extracellular vesicles; MB, myobundle; CTL, control; CLFS, chronic low frequency stimulation; IHFS, intermittent high frequency stimulation. Legend: Panel (A) represents the overlap of the overall data set used for between population within treatment analyses and the data sets used for the individual myobundle and EV analyses between treatments. Panel (B) represents n = 27 miRs enriched within the myobundle. Panel c represents n = 21 miRs enriched in EVs. Data for panels (B,C) are presented as mean raw count.
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