Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms

Abstract

Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development.

Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells

“…In addition, we identified 3563 correlated proteins all analyzed in triplicate experiments (Figure B). The number of proteins used in the bioinformatics analysis was similar to that of the previous study . The quantified proteins were distributed in the membrane, 36% in the cytosol, 27% in the nucleus, 11% in the mitochondria, and 8% in the extracellular (Figure S2, Supporting Information).…”

“…The cardiomyogenic differentiation efficiency was also confirmed by comparative proteomic data (Table S1, Supporting Information). Protein ratios of two markers were monitored, OCT4 for hiPSCs and MYL7 for CMs . OCT4 levels decreased by 0.33 and 0.14‐fold in CPCs and CMs, respectively.…”

“…We performed enrichment analysis for the KEGG pathway by calculating Fisher's exact test p ‐values to characterize the predicted roles of highly altered proteins during differentiation (Figure ). Cardiomyocyte‐specific mechanisms, including cardiac muscle contraction, pyruvate metabolism, TCA cycle, and OXPHOS, were present in cluster 3 . In addition, cluster 6 contained proteins upregulated related to pyruvate metabolism and BCAA degradation (Figure S5A, Supporting Information).…”

“…In contrast, cardiomyogenic differentiation is accompanied by dramatic changes in the dimension of the mitochondrial network and a shift from glycolysis to oxidative phosphorylation (OXPHOS) . Quantitative proteome analysis of hiPSCs and CMs by label‐free quantification and the SILAC method led to the identification of new regulators of cardiomyogenesis …”

“…However, previous studies have primarily focused on the identification of cardiomyocyte markers during cardiomyogenesis and the major mechanisms such as energy metabolism and Wnt signaling pathway. For example, the flux through the canonical Wnt pathway was reduced, whereas the tricarboxylic acid (TCA) cycle and muscle contraction mechanisms were induced in CMs . As the heart requires surplus energy for pumping blood throughout the body, various routes of energy production must participate in cardiac energy metabolism.…”