Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

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-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. CONCLUSION Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

[1]  J. Martinou,et al.  Mitochondrial fission and apoptosis: an ongoing trial. , 2006, Biochimica et biophysica acta.

[2]  M. McNiven,et al.  The Mitochondrial Protein hFis1 Regulates Mitochondrial Fission in Mammalian Cells through an Interaction with the Dynamin-Like Protein DLP1 , 2003, Molecular and Cellular Biology.

[3]  S. Klassen,et al.  Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: A potential mechanism for loss of mitochondrial transmembrane potential and apoptosis , 2005, Molecular and Cellular Biochemistry.

[4]  M. Palacín,et al.  The Charcot-Marie-Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system. , 2005, Human molecular genetics.

[5]  V. Skulachev,et al.  Mitochondrial filaments and clusters as intracellular power-transmitting cables. , 2001, Trends in biochemical sciences.

[6]  J. Zierath,et al.  Mitofusin-2 Determines Mitochondrial Network Architecture and Mitochondrial Metabolism , 2003, The Journal of Biological Chemistry.

[7]  A. Criollo,et al.  Aldose Reductase Induced by Hyperosmotic Stress Mediates Cardiomyocyte Apoptosis , 2003, Journal of Biological Chemistry.

[8]  L. Galluzzi,et al.  Mechanisms of cytochrome c release from mitochondria , 2006, Cell Death and Differentiation.

[9]  Y. Yoon,et al.  Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A. Dobrzyń,et al.  Accumulation of specific ceramides in ischemic/reperfused rat heart; effect of ischemic preconditioning. , 2002, Journal of physiology and pharmacology : an official journal of the Polish Physiological Society.

[11]  Lawrence M. Lifshitz,et al.  Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. , 1998, Science.

[12]  T. Letellier,et al.  Mitochondrial bioenergetics and structural network organization , 2007, Journal of Cell Science.

[13]  Heping Cheng,et al.  Mitofusin-2 Is a Major Determinant of Oxidative Stress-mediated Heart Muscle Cell Apoptosis* , 2007, Journal of Biological Chemistry.

[14]  M. Prasad,et al.  Enhanced membrane protein kinase C activity in myocardial ischemia , 2004, Basic Research in Cardiology.

[15]  D. Wood,et al.  Involvement of caspase 3- and 8-like proteases in ceramide-induced apoptosis of cardiomyocytes. , 2000, Journal of cardiac failure.

[16]  R. Kennedy,et al.  Positive inotropic effect of ceramide in adult ventricular myocytes: mechanisms dissociated from its reduction in Ca2+ influx. , 2003, American journal of physiology. Heart and circulatory physiology.

[17]  M. Lagarde,et al.  Ceramide in the antiapoptotic effect of ischemic preconditioning. , 2004, American journal of physiology. Heart and circulatory physiology.

[18]  L. Galluzzi,et al.  Mitochondrial control of cell death induced by hyperosmotic stress , 2006, Apoptosis.

[19]  R. Gottlieb,et al.  Proapoptotic BCL-2 family members and mitochondrial dysfunction during ischemia/reperfusion injury, a study employing cardiac HL-1 cells and GFP biosensors. , 2006, Biochimica et biophysica acta.

[20]  R. Testi,et al.  Mitochondria as sensors of sphingolipids. , 2002, Biochimie.

[21]  P Athias,et al.  [Development of cardiac physiopathological models from cultured cardiomyocytes]. , 2006, Annales de cardiologie et d'angeiologie.

[22]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[23]  Pierre Athias,et al.  Développement de modèles physiopathologiques cardiaques à partir des cultures de cardiomyocytes , 2006 .

[24]  J. Martinou,et al.  Which came first, the cytochrome c release or the mitochondrial fission? , 2006, Cell Death and Differentiation.

[25]  A J Levi,et al.  Cultured adult cardiac myocytes: future applications, culture methods, morphological and electrophysiological properties. , 1998, Cardiovascular research.

[26]  R. Rizzuto,et al.  Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis. , 2004, Molecular cell.

[27]  G. Díaz-Araya,et al.  A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes , 2001, Cell and Tissue Research.

[28]  C. Leeuwenburgh,et al.  Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio. , 2002, Cancer research.

[29]  M. Verheij,et al.  Ceramide: second messenger or modulator of membrane structure and dynamics? , 2003, The Biochemical journal.

[30]  M. Lorusso,et al.  Ceramide interaction with the respiratory chain of heart mitochondria. , 2000, Biochemistry.

[31]  D. Zechner,et al.  Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. , 1996, The Journal of clinical investigation.

[32]  A. Bielawska,et al.  Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion. , 1997, The American journal of pathology.

[33]  R. Youle,et al.  Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. , 2004, Molecular biology of the cell.

[34]  R. Urrutia,et al.  Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p. , 1998, Journal of cell science.

[35]  J. Jaffrezou,et al.  Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes. , 1999, Cardiovascular research.

[36]  A. Godzik,et al.  Mitochondrial fission in apoptosis, neurodegeneration and aging. , 2003, Current opinion in cell biology.

[37]  D. Stojanovski,et al.  Levels of human Fis1 at the mitochondrial outer membrane regulate mitochondrial morphology , 2004, Journal of Cell Science.

[38]  Y. Kakinuma,et al.  Doxorubicin induces apoptosis by activation of caspase-3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. , 2006, Journal of pharmacological sciences.

[39]  D. Liem,et al.  Mitochondrial function: the heart of myocardial preservation. , 2003, The Journal of laboratory and clinical medicine.

[40]  C. Long,et al.  Sympathetic modulation of the cardiac myocyte phenotype: studies with a cell-culture model of myocardial hypertrophy. , 1992, Basic research in cardiology.

[41]  J. Eaton,et al.  Aging of Cardiac Myocytes in Culture: Oxidative Stress, Lipofuscin Accumulation, and Mitochondrial Turnover , 2004, Annals of the New York Academy of Sciences.

[42]  R. Kolesnick,et al.  Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations. , 2006, Mitochondrion.

[43]  J. Martinou,et al.  hFis1, a Novel Component of the Mammalian Mitochondrial Fission Machinery* , 2003, Journal of Biological Chemistry.

[44]  Young-Jae Nam,et al.  The Mitochondrial Death Pathway and Cardiac Myocyte Apoptosis , 2004, Circulation research.

[45]  A. M. van der Bliek,et al.  Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. , 2001, Molecular biology of the cell.

[46]  Tullio Pozzan,et al.  The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide‐induced apoptosis: significance for the molecular mechanism of Bcl‐2 action , 2001, The EMBO journal.

[47]  D. Arnoult.  Mitochondrial fragmentation in apoptosis. , 2007, Trends in cell biology.

[48]  Erik E. Griffin,et al.  Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development , 2003, The Journal of cell biology.

[49]  Martin Hermann,et al.  Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: possible physiological role? , 2006, Biochimica et biophysica acta.

[50]  S. Barrow,et al.  Effects of Secretagogues and Bile Acids on Mitochondrial Membrane Potential of Pancreatic Acinar Cells , 2004, Journal of Biological Chemistry.

[51]  D. Chan.  Mitochondria: Dynamic Organelles in Disease, Aging, and Development , 2006, Cell.

[52]  J. Martinou,et al.  Inhibiting the Mitochondrial Fission Machinery Does Not Prevent Bax/Bak-Dependent Apoptosis , 2006, Molecular and Cellular Biology.

[53]  E. Craig,et al.  Regulated Cycling of Mitochondrial Hsp70 at the Protein Import Channel , 2003, Science.

[54]  C. Leeuwenburgh,et al.  Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. , 2002, Biochimica et biophysica acta.

[55]  R. Urrutia,et al.  Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps 1 p , 1998 .