Human pediatric and adult ventricular cardiomyocytes in culture, assessment of phenotypic changes with passaging

Objectives: The purpose of this study was to assess morphologically and biochemically the phenotypic changes which occur in vitro with passaging 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 uptake 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 isoform, human ventricular myosin heavy chain (HVMHC) and light chain I (HVMLCl), Northern blot analysis of HVMHC, and CK-MB activity and mass measurements. Passage 3 cardiomyocyte and pediatric myocardial phospholipids 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 culture with essentially complete loss of sarcomeres by 10 days and a gradual loss of myofilaments with passaging, The cells were identified as cardiomyocytes by immunohistochemistry for myoglobin, CK-MB, cardiac-specific troponin I isoform, HVMHC and HVMLCl, and by Northern blot analysis for the 3'-end of HVMHC mRNA. The composition of phospholipid fatty acids in the cultured pediatric cells was similar to that found in the pediatric myocardium. CK-MB activity and mass could be measured in the cardiomyocytes. The adult cardiomyocytes were more difficult to maintain 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. Although rapid disorganization of the myofibrils occurs, the non-contractile cells can be identified as cardiomyocytes by morphological appearance, immunofluorescent staining, Northern blot analysis for HVMHC, and CK-MB activity.