Abstract 213: Sarcolemma Genes Related to the Transverse Tubule Structure Are Mostly Not Expressed in Differentiating Human iPSC-derived Cardiomyocytes

Abstract

Background: Transverse tubules (TT) are tunnel-like extensions of sarcolemma studded with ion channels coupling excitation, through the cytoplasm, to contraction in the sarcomere of matured cardiomyocytes (CMs). Expression timing of sub-cellular TT-related genes (TT-rgs) in individual human iPSC-derived CMs (hiPSC-CMs) has not yet been reported. Objective: Map out the gene program of TT-rgs by sub-cellular locations during hiPSC-CM differentiation using single-cell transcriptomics (scRNA Seq.). Methods: hiPSC-CMs were differentiated from 2 commercially available lines using sequential GSK3 and Wnt signaling inhibition. The %CMs were assayed by flow cytometry for sarcomeric myosin heavy chain protein, a CM biomarker. Baseline cells (iPSCs, n=24) and day 14 (n=45), 30 (n=64), and 60 (n=5) post differentiation cells were sampled for scRNA Seq. using the Fluidigm C1 platform. We categorized 73 TT-rgs by cellular location of the coded protein (i.e. sarcomere, cytoplasm or sarcolemma). The expression pattern for each location was categorized as induced, repressed, or neither based on the median transcript per million for each individual cell normalized to iPSCs. Results: CMs increased from <1% in iPSCs to 21%, 37% and 59% at D14, D30 and D60 post differentiation (adj. p <0.03 by ANOVA), respectively. 33 genes (45%) had no detectable RNA while 21 (29%) were induced to D60. See table. Conclusion: Single-cell transcriptomes in differentiating hiPSC-CMs revealed a discordance between sarcolemma (mostly not expressed) and sarcomeric genes being induced. Defining timing and within cell variability of TT sub-cellular genes will be critical to understand human CM maturation.