Abstract 212: Adrenergic Signaling Genes Are Not All Expressed During Myogenesis in Individual Human iPSC-derived Cardiomyocytes

Abstract

Background: Adrenergic receptors (AR) in an individual cardiomyocyte (CM) are not uniformly expressed at the single-cell level (Myagmar et al., 2017). The timing and cellular distribution of AR signaling (ARS) genes in individual human iPSC-derived CMs (hiPSC-CMs) have not yet been reported. Objective: To map out the transcription program of ARS genes during hiPSC-CM myogenesis using single-cell transcriptomics (scRNA Seq.). Methods: 132 ARS in CM genes curated by the Kyoto Encyclopedia of Genes and Genomes were studied. The CMs were derived from 2 commercially available hiPSC lines using sequential GSK3 and Wnt signaling inhibition. The CMs were assayed by flow cytometry with sarcomeric myosin heavy chain (MYHC6/7) protein, a biomarker of myogenesis. 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. Data from 10 somatic cell preparations and iPSCs defined the signal specificity and biological noise in the system. The expression patterns were categorized as induced, repressed, or neither based on the median transcript per million for individual cells normalized to iPSCs. A p -value of <0.05 by ANOVA was used for significance. Results: The CMs had spontaneous contractions by D14 and increased from <1% in iPSCs to 21%, 37% and 59% at D14, D30 and D60 post differentiation (adj. p <0.03) respectively. None of the iPSCs had MYH6/7 transcripts, while >90% of differentiating cells did. MYH6/7 median expression increased 15-fold from D14 to D30 (p<0.0001) and 60-fold to D60 (p<0.06). Of 132 ARS genes, 60 (44%) had no detectable expression out to D60. Of the 72 genes with expression, 18 (25%) were induced in parallel to MYH6/7, while 22 (31%) were repressed. The remaining 32 genes (44%) had no distinctive pattern. From 18 induced genes (14% of all), 8 were sarcomeric genes that preceded expression to most ARS genes without induction (86% of all). Conclusion: Single-cell transcriptomes in derived hiPSC-CMs revealed a lack of uniformity between upstream ARS genes and early sarcomeric genes. Defining the timing and cell-to-cell variability of the ARS gene program will be critical to understanding its mechanistic relationship to the sarcomeric gene program in humans.