Abstract
The heart is the first functional organ to develop during embryogenesis, yet its cellular and molecular mechanisms remain incompletely understood. Proteomic studies of in vitro cardiomyogenesis have provided valuable insights, but most have focused on late-stage processes leading to cardiomyocyte specification. In this study, we profiled the proteome of differentiating cells at the mesendoderm (ME), cardiac mesoderm (CME), cardiac progenitor cell (CPC), and cardiomyocyte (C) stages using in-depth quantitative analysis via multiplexed tandem mass tag-based mass spectrometry. Unsupervised clustering of 4417 differentially expressed proteins revealed six main clusters corresponding to key biological processes at each differentiation stage. Transitional-specific proteins (TSPs), identified through pairwise comparisons of consecutive stages, were most abundant in CME, with 325 differentially expressed TSPs. Pathway enrichment analysis highlighted ferroptosis as a key process in CME specification, whereas sirtuin signaling was implicated in driving cardiomyocyte fate. This proteomic profiling expands the repertoire of potential biomarkers for each developmental stage toward cardiomyocyte specification. Overall, our stepwise proteome analysis of in vitro cardiomyocyte differentiation uncovers novel molecular mechanisms underlying heart development.