Background information. Cultivation techniques promoting three-dimensional organization of mammalian cells areof increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power forregenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-basedgels have become attractive tools, because of the ease with which their mechanical properties can be controlled.In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culturesubstrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes,the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins:HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase).Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culturewith cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasmamembrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmentalstimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOSexpression and localization are affected by changes in ECM composition. Elaboration of the images revealed, onMatrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localizationcoefficients, which indicated the highest correlation with respect to the other substrates.Conclusions. Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell–ECM interactionsat electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocytephenotype.
HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes
AMELIO, DANIELA;CERRA, Maria Carmela;
2007-01-01
Abstract
Background information. Cultivation techniques promoting three-dimensional organization of mammalian cells areof increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power forregenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-basedgels have become attractive tools, because of the ease with which their mechanical properties can be controlled.In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culturesubstrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes,the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins:HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase).Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culturewith cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasmamembrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmentalstimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOSexpression and localization are affected by changes in ECM composition. Elaboration of the images revealed, onMatrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localizationcoefficients, which indicated the highest correlation with respect to the other substrates.Conclusions. Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell–ECM interactionsat electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocytephenotype.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
