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.
BACKGROUND
Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels 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 culture substrates, 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-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, 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 interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype.
Di Felice
Valentina
V
Human Anatomy Section E. Luna, Department of Experimental Medicine, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy. valentina.difelice@unipa.it
Cappello
Francesco
F
Montalbano
Antonella
A
Ardizzone
Nella Maria
NM
De Luca
Angela
A
Macaluso
Filippo
F
Amelio
Daniela
D
Cerra
Maria Carmela
MC
Zummo
Giovanni
G
D000818:Animals; D001672:Biocompatible Materials / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D005353:Fibronectins; D005455:Fluorescent Antibody Technique; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000648:ultrastructure*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats
D001672:Biocompatible Materials; D004338:Drug Combinations; D005353:Fibronectins; D018841:HSP90 Heat-Shock Proteins; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D052250:Nitric Oxide Synthase Type III