Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria

1. Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)-med iated cytosolic Ca2+ signals propagate to the mitochondria, initiating chai ns of events vital in the regulation of different cellular functions. Howev er, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. 2. To measure the amount of Ca2+ taken up by the mitochondria, we used a no vel approach that involves simultaneous fluorescence imaging of mitochondri al and cytosolic [Ca2+] in permeabilized H9c2 myotubes and RBL-2H3 mast cel ls. Communication between sarco-endoplasmic reticulum (SR/ER) and mitochond ria is maintained in these permeabilized cells, as evidenced by the large R yR/IP3R-driven mitochondrial matrix [Ca2+] and NAD(P)H signals and also by preservation of the morphology of the SR/ER-mitochondrial junctions. 3. Ca2+ was released from the SR/ER by addition of saturating caffeine or I P3 and subsequently thapsigargin (Tg), an inhibitor of SR/ER Ca2+ pumps. Th e amount of Ca2+ transmitted to the mitochondria was determined by measurin g increases of global [Ca2+] in the incubation medium (cytosolic [Ca2+] ([C a2+](3))). Mitochondrial Ca2+ uptake was calculated from the difference bet ween [Ca2+](0) responses recorded in the absence and presence of uncoupler or from [Ca2+](0) elevations evoked by uncoupler or ionophore applied after complete Ca2+ mobilization from the SR/ER. [Ca2+](0) increases were calibr ated by adding Ca2+ purses to the permeabilized cells. 4. In H9c2 cells, ca ffeine induced partial mobilization of SR Ca2+ and mitochondria accumulated 26% of the released Ca2+. Sequential application of caffeine and Tg elicit ed complete discharge of SR Ca2+ without further increase in mitochondrial Ca2+ uptake. In RBL-2H3 mast cells, TP, by itself elicited complete dischar ge of the ER Ca2+ store and the increase of the ionophore-releasable mitoch ondrial Ca2+ content reached 50% of the Ca2+ amount mobilized by IP3 + Tg. Thus, RyR/IP3R direct a substantial fraction of released Ca2+ to the mitoch ondria.