An Unbiased Proteomics Method to Assess the Maturation of Human Pluripotent Stem Cell–Derived Cardiomyocytes

Abstract

Human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) exhibit the properties of fetal CMs, which limits their applications. Various methods have been used to promote maturation of hPSC-CMs; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-CMs. We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-CM maturation. Utilizing hPSC-CMs from early- and late-stage two-dimensional monolayer culture and three-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications (PTMs). This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein PTMs as promising new markers of hPSC-CMs maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-CM maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca 2+ homeostasis. In particular, up-regulation of myomesin-1 and transmembrane 65 were associated with hPSC-CM maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-CMs. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-CMs, and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-CMs.