Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis

Abstract

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 and 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-retated protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (Delta Psi(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 tactic 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-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 Delta Psi(m) and cyt 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.