Fig. 5

Genetic implications of myoneurovascular triculture. A qRT-PCR analysis of gene expression related to myosin heavy chain isoforms was performed on various culture combinations after 21 days. All the MYH gene isoforms analyzed are represented as colored grouped stack showing the predominance of MYH3 (orange) and MYH 8 (blue), whereas other isoforms were expressed at significantly lower levels. The presence of endothelial cells and motor neurons increased expression of embryonic MYH3, whereas motor neurons alone significantly upregulated expression of neonatal MYH8 gene. Endothelial cells independently (MYO + EC) and in combination with motor neurons (MYO + MN + EC) lead to upregulation genes related to fast type II muscle fibers (MYH1, MYH2, and MYH4). B Innervation alone (i.e., MYO + MN) was found to upregulate genes pertaining to myocyte structural protein titin (TTN) as well as slow-type skeletal muscle-specific troponin T1 (TNNT1), and addition of endothelial cells on top of neuromuscular cocultures (MYO + MN + EC) did not significantly alter the expression of these genes. Further, endothelial cells downregulated expression of type 1 glucose transporter as evidenced by lower SLC2A1 gene expression in MYO + EC and MYO + MN + EC groups. Interestingly, motor neurons and endothelial cells had an additive effect in upregulation of insulin-specific glucose transporter gene (SLC2A4) with MYO + MN + EC group exhibiting highest expression. Ordinary one-way ANOVA analysis with Tukey’s multiple comparison test was performed with n = 3 replicates per group, and p < 0.05 was considered for statistical significance