thapsigargin and Mitochondrial-Myopathies

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.

Topics: Actins; Ataxia; Benzimidazoles; Calcium; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line, Tumor; Cytoskeleton; DNA, Mitochondrial; Fluorescent Dyes; Fura-2; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Ionophores; Membrane Potentials; Microscopy, Fluorescence; Mitochondrial Myopathies; Muscle Weakness; Mutation; Osteosarcoma; Phalloidine; Retinitis Pigmentosa; Rhodamines; Thapsigargin

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.

Topics: Calcium; Calcium Signaling; Carbachol; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cytological Techniques; Cytosol; DNA; Electron Transport; Fluorescent Dyes; Homeostasis; Humans; Membrane Potentials; Microscopy, Electron; Mitochondria; Mitochondrial Myopathies; Neuroblastoma; Neurodegenerative Diseases; Thapsigargin; Tumor Cells, Cultured