Maturation Towards Pure β-Myosin Protein Expression and Corresponding Functional Properties of Individual hESC-Cardiomyocytes

Human embryonic stem cell derived cardiomyocytes (hESC-CMs) represent a powerful tool for analyzing pathogenesis of cardiac diseases such as hypertrophic or dilative cardiomyopathies. Several studies revealed an immature state of CMs after differentiation from pluripotent stem cells. This immature state is indicated, for example, by the high expression level of the fast atrial cardiac myosin heavy chain (α-MyHC). In human ventricular cardiomyocytes, however, the slow β-MyHC predominates.Here we show maturation of hESC-CMs towards exclusive expression of β-MyHC protein in individual cardiomyocytes and their functional characterization.After growing hESC-CMs in cardiac bodies vs. plated on laminin-coated coverslips, myosin heavy chain isoform was determined using a specific antibody against ventricular β-MyHC and a newly generated anti-atrial α-MyHC-specific antibody. A single-cell mapping technique was established to relate functional characteristics of individual cardiomyocytes to their expression of α- vs. β-MyHC-protein isoforms.Cardiomyocytes grown in cardiac bodies for a maximum of 110 days mostly contained a mixture of α- and β-MyHC. Only a minority of about 10% of cardiomyocytes expressed β-MyHC exclusively. However, cardiomyocytes plated on laminin-coated coverslips shifted MyHC-expression towards 66% and 87% of all cardiomyocytes expressing exclusively β-MyHC after 35 and 75 days, respectively. This isoform switch was accompanied by morphological changes towards more elongated cardiomyocytes with highly organized sarcomeres. Surprisingly, twitch kinetics and calcium transients were found unaffected by the MyHC-isoform in the sarcomeres while cardiomyocytes grown on laminin-coated coverslips in general displayed faster twitch kinetics and calcium transients.We conclude that cultivating conditions of hESC-CMs during maturation severely affect sarcomeric protein isoform expression in individual cardiomyocytes up to pure β-MyHC expression, thus changing the functional phenotype of the CMs, which is crucial for tissue engineering and cardiac disease models with functional assessment up to single cell level.