HUMAN PEDIATRIC AND ADULT VENTRICULAR CARDIOMYOCYTES IN CULTURE, ASSESSMENT OF PHENOTYPIC CHANGES WITH PASSAGING

Objectives: The purpose of this study was to assess morphologically an d biochemically the phenotypic changes which occur in vitro with passa ging of human pediatric and adult ventricular cardiomyocytes. Methods: Human ventricular cardiomyocytes from 3 children (1 to 2 years of age ) and an adult patient (65 years of age) undergoing open heart surgery and an adult heart transplant patient (55 years of age) were isolated , cultured, purified, and passaged. Growth curves and H-3-thymidine up take studies were performed. Characterization of the cells was done by light microscopy, transmission electron microscopy, immunofluorescent staining for myoglobin, CK-MB, and cardiac-specific troponin I isofor m, human ventricular myosin heavy chain (HVMHC) and light chain I (HVM LCl), Northern blot analysis of HVMHC, and CK-MB activity and mass mea surements. Passage 3 cardiomyocyte and pediatric myocardial phospholip ids were analysed by gas chromatography. Results: Pediatric cells were smaller (P < 0.01) and divided faster (P < 0.001, ANOCOVA) than adult cells. The cardiomyocytes showed phenotypic changes in primary cultur e with essentially complete loss of sarcomeres by 10 days and a gradua l loss of myofilaments with passaging, The cells were identified as ca rdiomyocytes by immunohistochemistry for myoglobin, CK-MB, cardiac-spe cific troponin I isoform, HVMHC and HVMLCl, and by Northern blot analy sis for the 3'-end of HVMHC mRNA. The composition of phospholipid fatt y acids in the cultured pediatric cells was similar to that found in t he pediatric myocardium. CK-MB activity and mass could be measured in the cardiomyocytes. The adult cardiomyocytes were more difficult to ma intain than the pediatric cells which could be cultured for as long as 6 months. Conclusions: Primary cultures of human pediatric and adult partially differentiated ventricular cardiomyocytes can be passaged. A lthough rapid disorganization of the myofibrils occurs, the non-contra ctile cells can be identified as cardiomyocytes by morphological appea rance, immunofluorescent staining, Northern blot analysis for HVMHC, a nd CK-MB activity.