Membrane cholesterol depletion by methyl-beta-cyclodextrin enhances the expression of cardiac differentiation markers.
Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-beta-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/beta-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. beta-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac beta-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Carolina
C
Pontes Soares
Débora Morueco
DM
Portilho
Luzia
L
da Silva Sampaio
Marcelo
M
Einicker-Lamas
Marcelo Marcos
MM
Morales
Manoel Luis
ML
Costa
Cláudia
C
Dos Santos Mermelstein
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D024722:Cardiac Myosins / Q000378:metabolism; D051244:Caveolin 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D002784:Cholesterol / Q000378:metabolism*; D018031:Connexin 43 / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003893:Desmin / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D000943:Antigens, Differentiation; D015820:Cadherins; D051244:Caveolin 3; D018031:Connexin 43; D017077:Culture Media, Conditioned; D003893:Desmin; D014157:Transcription Factors; D014335:Tropomyosin; D051176:beta Catenin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D024722:Cardiac Myosins; D018995:Myosin Heavy Chains