title: 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.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Konze
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Werneburg
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Oberbeck
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Olmer
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Kempf
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Monica
authors.initials: M
authors.lastname: Jara-Avaca
authors.affiliation: Institute of Toxicology, Core Facility Proteomics, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Zweigerdt
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School , 30625 Hannover, Germany.|REBIRTH Cluster of Excellence, Hannover Medical School , 30625 Hannover, Germany.
authors.forename: Falk F R
authors.identifier: 0000-0002-8468-1223
authors.initials: FF
authors.lastname: Buettner
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004734:Energy Metabolism; D004798:Enzymes / Q000378:metabolism*; D005786:Gene Expression Regulation; D006019:Glycolysis; D000072576:Homeobox Protein Nkx-2.5 / Q000032:analysis; D006801:Humans; D007553:Isotope Labeling; D060588:LIM Domain Proteins / Q000737:chemistry / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D032383:Myocytes, Cardiac / Q000166:cytology*; D010085:Oxidative Phosphorylation; D039904:Pluripotent Stem Cells / Q000166:cytology*; D020033:Protein Isoforms / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D044968:Regenerative Medicine
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004798:Enzymes; D000072576:Homeobox Protein Nkx-2.5; D060588:LIM Domain Proteins; C512485:PDLIM5 protein, human; D020033:Protein Isoforms
keywords: PDLIM5; SILAC; cardiomyocytes; human pluripotent stem cells; proteomics