Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.
OBJECTIVE
In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes.


RESULTS
Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation.


CONCLUSIONS
Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.
Parra
Valentina
V
Centro FONDAP Estudios Moleculares de la Célula, Universidad de Chile, Olivos 1007, Santiago 8380492, Chile.
Eisner
Veronica
V
Chiong
Mario
M
Criollo
Alfredo
A
Moraga
Francisco
F
Garcia
Alejandra
A
Härtel
Steffen
S
Jaimovich
Enrique
E
Zorzano
Antonio
A
Hidalgo
Cecilia
C
Lavandero
Sergio
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D004317:Doxorubicin / Q000494:pharmacology; D034281:Dynamins / Q000032:analysis; D020558:GTP Phosphohydrolases; D008565:Membrane Proteins / Q000032:analysis; D008929:Mitochondria, Heart / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D024101:Mitochondrial Proteins / Q000032:analysis; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002518:Ceramides; C503403:Fis1 protein, rat; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D004317:Doxorubicin; D020558:GTP Phosphohydrolases; C489710:Mfn2 protein, rat; C519851:Dnm1l protein, rat; D034281:Dynamins