Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria.
Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)-mediated cytosolic Ca2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. To measure the amount of Ca2+ taken up by the mitochondria, we used a novel approach that involves simultaneous fluorescence imaging of mitochondrial and cytosolic [Ca2+] in permeabilized H9c2 myotubes and RBL-2H3 mast cells. Communication between sarco-endoplasmic reticulum (SR/ER) and mitochondria is maintained in these permeabilized cells, as evidenced by the large RyR/IP3R-driven mitochondrial matrix [Ca2+] and NAD(P)H signals and also by preservation of the morphology of the SR/ER-mitochondrial junctions. Ca2+ was released from the SR/ER by addition of saturating caffeine or IP3 and subsequently thapsigargin (Tg), an inhibitor of SR/ER Ca2+ pumps. The amount of Ca2+ transmitted to the mitochondria was determined by measuring increases of global [Ca2+] in the incubation medium (cytosolic [Ca2+] ([Ca2+]c)). Mitochondrial Ca2+ uptake was calculated from the difference between [Ca2+]c responses recorded in the absence and presence of uncoupler or from [Ca2+]c elevations evoked by uncoupler or ionophore applied after complete Ca2+ mobilization from the SR/ER. [Ca2+]c increases were calibrated by adding Ca2+ pulses to the permeabilized cells. In H9c2 cells, caffeine induced partial mobilization of SR Ca2+ and mitochondria accumulated 26% of the released Ca2+. Sequential application of caffeine and Tg elicited complete discharge of SR Ca2+ without further increase in mitochondrial Ca2+ uptake. In RBL-2H3 mast cells, IP3 by itself elicited complete discharge of the ER Ca2+ store and the increase of the ionophore-releasable mitochondrial Ca2+ content reached 50% of the Ca2+ amount mobilized by IP3 + Tg. Thus, RyR/IP3R direct a substantial fraction of released Ca2+ to the mitochondria.
Pacher
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P
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Csordás
P
P
Schneider
T
T
Hajnóczky
G
G
D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008407:Mast Cells / Q000502:physiology / Q000648:ultrastructure; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D009994:Osmolar Concentration; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium