<p>To fully exploit the potential of human pluripotent stem cell-derived cardiomyocytes, ideally they should acquire a mature, adult ventricular-like phenotype. Predominant expression of the β-isoform of myosin heavy chain (β-MyHC) and the ventricular isoform of myosin regulatory light chain 2 (MLC2v) is a marker of human adult cardiac ventricle. Yet predominant co-expression of these isoforms is rarely reported by current culture protocols. Here, we assessed the impact of different substrates on β-MyHC and MLC2v expression in single human embryonic stem cell-derived CMs (hESC-CMs). As substrates, surface materials with differing stiffness as defined by Young’s modulus were combined with either laminin, a single-component coating, or Matrigel, a multi-component coating including growth factors. Semi-quantitative single-cell immunofluorescence analysis demonstrated that surfaces with supraphysiological stiffness in combination with laminin are sufficient for promotion of predominant β-MyHC expression, but not for predominant MLC2v expression in hESC-CMs. Accordingly, mechanical stimuli likely promote expression of β-MyHC in these cultures. Culture on matrices with a lower stiffness than glass in combination with growth factor-containing Matrigel led to only moderate increases in MLC2v expression, possibly more dependent on growth factors, suggesting different regulation of expression. Integrin-related downstream signal transducers, integrin-linked and cardiac troponin I-interacting kinase, as well as modulation of intracellular Ca²⁺-concentration and epigenetic signaling did not affect MyHC/MLC2 isoform expression. The data indicate that expression of adult ventricular markers β-MyHC and MLC2v depends on different stimuli like substrate stiffness and growth factors. To conclude, multiple stimuli appear to be necessary to promote an adult ventricular phenotype.</p>