In vitro exercise model using contractile human and mouse hybrid myotubes

Abstract

Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses ( i . e . myokine secretion). We herein describe an “ in vitro exercise model”, using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the “ in vitro exercise model”.

Histone deacetylase 5 regulates interleukin 6 secretion and insulin action in skeletal muscle

“…We further investigated HDAC5- and contraction-dependent regulation of IL-6 and GLUT4 in the myotubes by applying EPS in vitro . In line with previous reports [ [44] , [45] , [46] ], EPS substantially increased transcription of Il6 and secretion of the hormone into the medium, thus validating the system. Consistent with previous studies, we also found increased phosphorylation of AKT in the C2C12 myotubes in response to EPS that has been attributed to activation of Rac1 [ 47 , 48 ].…”

The Relationship of IL-8 and IL-10 Myokines and Performance in Male Marathon Runners Presenting Exercise-Induced Bronchoconstriction

“…In relation to physical exercise, it has been reported that the IL-6 released by strenuous exercise precedes the release of IL-10, in order to regulate the systemic inflammation induced by the exercise [33]. Chen et al [47] reported remarkable increases in the secretions of myokines, such as IL-8 and IL-10 derived from contraction of cultured myotubes, characterizing IL-10 as a myokine [47,48]. At basal condition, the secretion of myokines, such as IL-6 and IL-8, was also observed in a circadian manner; however, this was not the case with IL-10 levels [49].…”

Topography-Mediated Myotube and Endothelial Alignment, Differentiation, and Extracellular Matrix Organization for Skeletal Muscle Engineering

“…Using murine myoblasts both in vitro and in vivo for extrapolating in vitro data of human myoblasts to in vivo situations are not likely to provide insights due to the intrinsic difference between murine and human myoblasts [44]. It has been shown that after differentiation of human myoblasts less contractile properties are observed than murine myoblasts and that various surface markers and certain proteins (such as Myostatin) are differently expressed upon differentiation and formation of myofibers [44][45][46]. Even though we see the use of human myoblasts as a strong point for investigating tissue engineering approaches with new biofabrication techniques such as the use of specialized topography substrates, clinical translation with high validity remains a point of concern.…”