Cardiovascular Research Advance Access [Accepted Manuscript] published online on October 4, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm029
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Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis
a Centro FONDAP Estudios Moleculares de la Célula
b Facultad de Ciencias Químicas y Farmacéuticas
c Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
d Institute for Research in Biomedicine (IRB) and Departament de Bioquímica y Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Spain
* Corresponding author: Centro FONDAP de Estudios Moleculares de la Célula, Universidad de Chile, Olivos 1007, Santiago-8380492, Chile. Telephone: +562-9782919. Fax: +562-7378920. E-mail: slavander{at}uchile.cl (S. Lavandero)
Aims: 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.
Methods: Neonatal rat cardiomyocytes were incubated with C2-ceramide or the inactive analog dihydro-C2-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 (
m) and cytochrome c distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry while cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2).
Results: C2-ceramide, but not dihydro-C2-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time dependent manner. C2-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 m and cytochrome c redistribution induced by C2-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation.
Conclusion: Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.
KEYWORDS Ceramide; Mitochondrial fission; Mitochondrial dynamics; Drp-1; Fis1; Mitofusin-2; Doxorubicin; Cell death; Apoptosis; Cardiomyocytes
Time for primary review: 19 days
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