Switching in the expression pattern of actin isoforms marks the onset of contractility and distinct mechanodynamic behavior during cardiomyocyte differentiation.

Differentiation of cardiac progenitor cells (CPC) into cardiomyocytes is a fundamental step in cardiogenesis, which is marked by changes in gene expression responsible for remodeling of the cytoskeleton and in altering the mechanical properties of cells. Here we have induced the differentiation of CPC derived from human pluripotent stem cells into immature cardiomyocytes (iCM) which we compare with more differentiated cardiomyocytes (mCM). Using atomic force microscopy and real-time deformability cytometry, we describe the mechanodynamic changes that occur during the differentiation process and link our findings to protein expression data of cytoskeletal proteins. Increased levels of cardiac-specific markers as well as evolution of cytoskeletal morphology and contractility parameters correlated with the expected extent of cell differentiation that was accompanied by hypertrophic growth of cells. These changes were associated with switching in the balance of the different actin isoforms where β-actin is predominantly found in CPC, smooth muscle α-actin is dominant in iCM cells and sarcomeric α-actin is found in significantly higher levels in mCM. We link these cytoskeletal changes to differences in mechano-dynamic behavior of cells that translate to changes in Young's modulus that depend on the cell adherence. Our results demonstrate that the intracellular balance of actin isoform expression can be used as a sensitive ruler to determine the stage of differentiation during early phases of cardiomyocyte differentiation that correlates with an increased expression of sarcomeric proteins and is accompanied by changes in cellular elasticity.