Cell Density and Joint microRNA-133a and microRNA-696 Inhibition Enhance Differentiation and Contractile Function of Engineered Human Skeletal Muscle Tissues

Abstract

To utilize three-dimensional (3D) engineered human skeletal muscle tissue for translational studies and in vitro studies of drug toxicity, there is a need to promote differentiation and functional behavior. In this study, we identified conditions to promote contraction of engineered human skeletal muscle bundles and examined the effects of transient inhibition of microRNAs (miRs) on myogenic differentiation and function of two-dimensional (2D) and 3D cultures of human myotubes. In 2D cultures, simultaneously inhibiting both miR-133a, which promotes myoblast proliferation, and miR-696, which represses oxidative metabolism, resulted in an increase in sarcomeric a-actinin protein and the metabolic coactivator PGC-1a protein compared to transfection with a scrambled miR sequence (negative control). Although PGC-1a was elevated following joint inhibition of miRs 133a and 696, there was no difference in myosin heavy chain (MHC) protein isoforms. 3D engineered human skeletal muscle myobundles seeded with 5 · 10 6 human skeletal myoblasts (HSkM)/mL and cultured for 2 weeks after onset of differentiation consistently did not contract when stimulated electrically, whereas those seeded with myoblasts at 10 · 10 6 HSkM/mL or higher did contract. When HSkM were transfected with both anti-miRs and seeded into fibrin hydrogels and cultured for 2 weeks under static conditions, twitch and tetanic specific forces after electrical stimulation were greater than for myobundles prepared with HSkM transfected with scrambled sequences. Immunofluorescence and Western blots of 3D myobundles indicate that anti-miR-133a or anti-miR-696 treatment led to modest increases in slow MHC, but no consistent increase in fast MHC. Similar to results in 2D, only myobundles prepared with myoblasts treated with anti-miR-133a and anti-miR-696 produced an increase in PGC-1a mRNA. PGC-1a targets were differentially affected by the treatment. HIF-2a mRNA showed an expression pattern similar to that of PGC-1a mRNA, but COXII mRNA levels were not affected by the anti-miRs. Overall, joint inhibition of miR-133a and miR-696 accelerated differentiation, elevated the metabolic coactivator PGC-1a, and increased the contractile force in 3D engineered human skeletal muscle bundles.

MiR-696 Regulates C2C12 Cell Proliferation and Differentiation by Targeting CNTFRα

“…High expression of miR-696 in skeletal muscle suggests that it may play a role in skeletal muscle myogenesis. Previous research has shown that anti-miR-696 expression could lead to a modest increase in slow MyHC 48. However, no studies have so far been performed to determine the function of miR-696 in skeletal myoblast proliferation and its mechanism in regulating muscle cell differentiation.…”

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

“…[102,106] Whether one of the routes may be better suited for clinical translation remains to be elucidated, although in situ transfecting scaffolds hold greater potential to exist as "off-the-shelf" products. [107] Many distinct scaffold types have been tuned to serve the purpose of miRNA delivery, including several hydrogels, [58,92,106,[108][109][110][111][112][113][114] electrospun fibers, [63,[115][116][117][118] and more prolifically porous or spongy scaffolds. [46,47,50,54,55,96,112,[119][120][121][122][123][124][125][126][127][128][129][130] Before reviewing the details of their applications, summarized in Table 2 and described further in the next section, we will discuss the key characteristics making these materials amenable to miRNA delivery.…”

“…[133] In order to specifically adapt any scaffold to miRNA delivery, the material must retain the miRNA complexes while facilitating their sufficient exposure to the infiltrating cells. [134] Reciprocally, the presence of miRNA Enhanced expression of PGC-1a, and contractile force 2 weeks myotubes culture [111] Muscle complexes in the scaffold structure must not negatively affect its mechanical and structural features or deter cellular retention and attachment to the scaffold. [135] Depending on the nature of their components, biomaterials are typically classified as synthetic, natural, ceramics, or composites, that is, combinations of these.…”

“…[138] To diminish these undesired effects, it is frequently combined with natural polymers such as hyaluronic acid (HyA), as in the commercial HyStem-HP and Glycosan HyStem. Other natural polymers included in miRNA eluting hydrogels are alginate, [112] silk fibrin, [111] fibrinogen, thrombin, and Matrigel. [92,110] Of note, hydrogels are the first biomaterials used successfully for the delivery of naked miRNA therapeutics, [106,108,109] that is, without the addition of a delivery vector.…”

“…Recently, Cheng et al [111] have been able to stimulate contraction of engineered 3D myobundles consisting of a fibrin hydrogel and human skeletal myoblasts (HSkM). The study examined the effects of siPORT NeoFxTM inhibition of miR133a and miR-696 on myogenic differentiation and function in 2D and 3D cultures of engineered myotubes when cultured under static conditions for 2 weeks.…”