Human embryonic stem cells (hESC) represent an essential source of cardiomyocytes for cardiac regeneration. Nuclear protein import (NPI) is critical in regulating cell differentiation and hypertrophy. The aim of this study was to determine if exposure of adult and hESC-derived cardiomyocytes (hESC-CM) to hypertrophic factor phenylephrine (PE) affects hypertrophy by altering NPI in comparison with adult failing rat and human cardiomyocytes. hESC-CM were generated by exposure of H7 hESCs to ActivinA and BMP-4. Failing cardiomyocytes were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. hESC-CM and adult rat CM were exposed to PE for 48 h. The effects of P38 MAPK inhibitor, HDAC inhibitor, exportin-1 (CRM-1) inhibitor, and GSK-3β inhibitor were investigated by automated microscopy. Cell and nuclear sizes were increased in PE treated-adult rat cardiomyocytes (by 141±12.7% p<0.05 and 130±6.28% p<0.001, respectively) and PE-treated hESC-CM and were much higher in the adult failing rat and human cardiomyocytes compared to normal controls. In younger (<3-month-old) hESC-CM, PE induced an increase in the fluorescence intensity of nucleoporin p62, cytoplasmic Ranbp1 and importins nuclear translocation. In contrast, these parameters were decreased in older PE-treated hESC-CM and PE treated-adult rat cardiomyocytes as well as in adult failing rat and human cardiomyocytes compared to normal controls. These effects were P38MAPK, HDAC and CRM-1 dependent but GSK-3β independent in rat cardiomyocytes. Our results show that alterations in NPI occur in failing cardiomyocytes as well as in adult and hESC-derived cardiomyocytes under pathological stimuli. (Supported by a Royal Society Newton International Fellowship).