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Creatine kinase isoenzymes in cardiomyocytes grown in serum-free defined medium
313INFLUENCE OF EXPLANTATION PROCEDURE ON THE PROPERTIES OF CULTURED NEONATAL RAT VENTRIDepartement de biophysique et d'anatomie CLE CELLS. O.F. Schanne, B. Fermini, J.Hugon. et biologic cellulaire, Faculte de mddecine, Universite de Sherbrooke, Sherbrooke, Canada, JlH 5N4 Exposure to trypsin was blamed for the changes in the ultrastructure found in cultured cardiac cells shortly after explantation. We undertook an electrophysiologic and ultrastructural study investigating the influence of trypsin and collagenase, with and without mechanical agitation on the electrogenesis and ultrastructure. Agitated collagenase treated cells (10.5 u/ml) showed heavy cell damage evidenced by des truction of intracellular organelles and the cells exhibited low resting potentials. When not agitated, after 2 days there was no morphological evidence for cell damage and the cells showed spontaneous activity and slow response action potentials. Trypsin ( 0.1%) treated and agitated cells showed chaotically oriented myofibrils 10 h after explnntation and upon stimulation, exhibited fast rising action potentials; 2 days later, the myofibrils resembled those of non explanted ventricle and there were spontaneous activity and slow response action potentials. We conclude: 1) the orientation of myofibrils does not reflect the state of electrical integrity of the cell; 2) when mechanically agitated, trypsin treatment preserves the cells better than collagenase; J) mechanical agitation acpears to be an important factor determining the state of cultured myocardial cells. Supported bv CRMC and QHF; B. Fermini holds a studentshin from FCAC.
314 ISOZYMES OF CREATINE KINASE IN MYOCARDIAL CELL CIILTURES. M.W. Seraydarian and J.J. Yang. School of Nursing, Center for The Health Sciences, University of California, Los Angeles, California, USA. Creatine kinase (CK) isozymes: MM,MB,BB and mitochondrial CK are identified in myocardial cell cultures derived from neonatal rat hearts. The relative activities of the four isozymes obtained from electrophoretograms demonstrate an increase in the percentage of mitochondrial CK isozyme with age in culture and with age of animals When cells are grown (between 1 and 7 days old) from which the cultures are derived. an increase of mitochondrial CK activity is obin the presence of 20 mM creatine, served at nil times in culture, e.g. 50% at 96h in culture, suggesting that creatine This isozyme is :night be a postnatal trigger for the mitochondrial CK synthesis. absent in non-muscle cell cultures derived from the same rat hearts and exogenous The creatine does not induce the mitochondrial CK synthesis in non-muscle cells. previously suggested essential role of mitochondrial CK in the regulation of myocardiIsozyme composition could be used al energy metabolism in thus further supported. (Supported by grants from as an index of homogeneinity of myocardial cell cultures. PHS NHLBI 821080 and the American Heart Association, with funds contributed in part bv the Greater Los Angeles Affiliate).
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CREATINE KLNASE ISOENZYMES IN CARDIOMYOCYTES CROWN IN SERUM-FREE DEFINED MEDIUM. Gania Kessler-Icekson*, Oded Sperling"" and J,ina Wasserman*. The Rogoff-Wellcome Medical Research Institute* and Clinical Chemistry Department**, Beilinson Medical Center, Petah-Tikva 49100, Israel. Primary cultures of neonate rat heart cells were grown on collagen coated plates in serum-free defined medium supplemented with insulin, transferrin, hydrocortison and fetuin. Differential plating along with the application of defined medium resulted in cultures consisting mostly of myocytes, as demonstrated by stainings for glycogen and myofibrils. Within 24 hours most cells resumed spontaneous beating and one day later the whole culture was beating synchronously. Total activity of cellular creatine kinase (CK), measured at different ages of culture, showed maximal values on days 3-6. The proportions of the three CK isoenzymes (MM,MB,BB) did not vary markedly with age of culture (up to 2 weeks) despite the observed changes in total activity. CK-MM constituted 70-90% of total enzyme activity, compatible with its proportion in the neonate rat heart. CK-MB did not exceed 30% of CK activity whereas CK-BB contributed less than 15% activity. In some cases the BB form was hardly detected. Lack of increase in CK-BB with age of culture indicated a limited growth of non-myocytes. The use of serum-free defined medium for culturine heart cells is highlv recommended since it limits non-myocytes growth and allows a long term maintenance of myocytes preserving their tissue specific features.
314 ISOZYMES OF CREATINE KINASE IN MYOCARDIAL CELL CIILTURES. M.W. Seraydarian and J.J. Yang. School of Nursing, Center for The Health Sciences, University of California, Los Angeles, California, USA. Creatine kinase (CK) isozymes: MM,MB,BB and mitochondrial CK are identified in myocardial cell cultures derived from neonatal rat hearts. The relative activities of the four isozymes obtained from electrophoretograms demonstrate an increase in the percentage of mitochondrial CK isozyme with age in culture and with age of animals When cells are grown (between 1 and 7 days old) from which the cultures are derived. an increase of mitochondrial CK activity is obin the presence of 20 mM creatine, served at nil times in culture, e.g. 50% at 96h in culture, suggesting that creatine This isozyme is :night be a postnatal trigger for the mitochondrial CK synthesis. absent in non-muscle cell cultures derived from the same rat hearts and exogenous The creatine does not induce the mitochondrial CK synthesis in non-muscle cells. previously suggested essential role of mitochondrial CK in the regulation of myocardiIsozyme composition could be used al energy metabolism in thus further supported. (Supported by grants from as an index of homogeneinity of myocardial cell cultures. PHS NHLBI 821080 and the American Heart Association, with funds contributed in part bv the Greater Los Angeles Affiliate).
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CREATINE KLNASE ISOENZYMES IN CARDIOMYOCYTES CROWN IN SERUM-FREE DEFINED MEDIUM. Gania Kessler-Icekson*, Oded Sperling"" and J,ina Wasserman*. The Rogoff-Wellcome Medical Research Institute* and Clinical Chemistry Department**, Beilinson Medical Center, Petah-Tikva 49100, Israel. Primary cultures of neonate rat heart cells were grown on collagen coated plates in serum-free defined medium supplemented with insulin, transferrin, hydrocortison and fetuin. Differential plating along with the application of defined medium resulted in cultures consisting mostly of myocytes, as demonstrated by stainings for glycogen and myofibrils. Within 24 hours most cells resumed spontaneous beating and one day later the whole culture was beating synchronously. Total activity of cellular creatine kinase (CK), measured at different ages of culture, showed maximal values on days 3-6. The proportions of the three CK isoenzymes (MM,MB,BB) did not vary markedly with age of culture (up to 2 weeks) despite the observed changes in total activity. CK-MM constituted 70-90% of total enzyme activity, compatible with its proportion in the neonate rat heart. CK-MB did not exceed 30% of CK activity whereas CK-BB contributed less than 15% activity. In some cases the BB form was hardly detected. Lack of increase in CK-BB with age of culture indicated a limited growth of non-myocytes. The use of serum-free defined medium for culturine heart cells is highlv recommended since it limits non-myocytes growth and allows a long term maintenance of myocytes preserving their tissue specific features.