Top-down proteomics for assessing the maturation of stem cell-derived cardiomyocytes

Background: Recent advancements in human pluripotent stem cell (hPSC) technology and cardiomyocyte (CM) differentiation have empowered research in cardiac development and diseases. However, the relative immaturity of stem cell-derived CMs is well documented. Since the expression of sarcomeric protein isoforms, as well as their post-translational modifications (PTMs), change as CMs mature, we sought to develop a method utilizing powerful top-down high-resolution mass spectrometry (MS) to assess the relative maturity of CMs focusing on the expression of sarcomeric protein isoforms and their PTMs. Methods: hPSCs were differentiated to CMs using a small molecule directed protocol and cultured in either 2D monolayer or 3D engineered cardiac tissue (ECT). Sarcomeric proteins were extracted from the CMs and analyzed using a top-down proteomics platform coupling reverse phase chromatography to high-resolution MS. Results: We identified and quantified the major sarcomeric protein isoforms and their PTMs in CMs from 2D monolayer or 3D ECT culture. Slow skeletal troponin I (ssTnI) was highly expressed while cardiac troponin I (cTnI) was under detection limit in 2D-CMs at both day 30 and 60, indicating the relative immaturity of 2D-CMs by day 60. However, the relative expression of the ventricular regulatory light chain versus the atrial isoform in CMs increased by about 7-fold at day 60 compared to the 2D-CMs at day 30, confirming that CMs exhibit maturation with longer 2D culture as anticipated. Tropomyosin phosphorylation decreased in CMs with longer 2D culture, which may serve as a novel marker for assessing CM maturation. CMs cultured in 3D ECT for 42 days after 30 days in 2D culture exhibited a 2-fold increase in cTnI expression relative to ssTnI, compared to day-72 CMs maintained in 2D culture, suggesting that 3D culture can promote CM maturation. These results demonstrate the power of top-down proteomics to assess the maturation of hPSC-derived CMs.