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. Increasing evidence suggests that mitochondria are important in intracellular Ca2+ signalling (for recent reviews see Babcock & Hille, 1998; Jouaville et al. 1998; Simpson & Russell, 1998; Duchen, 1999; Rizzuto et al. 1999; Hajnoczky et al. 2000; Huser et al. 2000). It has been shown that cytosolic calcium signals elicited by activation of IP3R or RyR are transmitted to the mitochondria (Rizzuto et al. 1993, 1998; Hajnoczky et al. 1995; Chacon et al. 1996; Drummond & Tuft, 1999). Mitochondrial Ca2+ content measured by different techniques has also been shown to be elevated in cells exposed to Ca2+ mobilizing agonists (e.g. Wendt-Gallitelli & Isenberg, 1989; Hoek et al. 1997). An important function of mitochondrial Ca2+ signals is the control of energy metabolism. Increases in mitochondrial [Ca2+] ([Ca2+]m), coupled to elevations in [Ca2+]c, participate in activation of the respiratory chain through stimulation of Ca2+-sensitive mitochondrial dehydrogenases, thereby ensuring adequate ATP synthesis (Denton & McCormack, 1980; Hansford, 1981; Duchen, 1992; Pralong et al. 1994; Hajnoczky et al. 1995; Robb-Gaspers et al. 1998; Jouaville et al. 1999). Propagation of calcium signals to the mitochondria is also important for apoptotic cell death (Stout et al. 1998; Szalai et al. 1999). Furthermore, mitochondrial Ca2+ transport appears to modulate the spatio-temporal pattern of [Ca2+]c responses evoked by IP3, suggesting that mitochondria are also involved in the shaping of [Ca2+]c signals (Jouaville et al. 1995; Budd & Nicholls, 1996; Babcock et al. 1997; Hoth et al. 1997; Ichas et al. 1997; Simpson et al. 1997; Landolfi et al. 1998; Boitier et al. 1999; Hajnoczky et al. 1999). However, how much calcium is utilized in mitochondrial signalling during intracellular Ca2+ mobilization has not yet been measured. Our aim was to determine the amount of Ca2+ transmitted from SR/ER to the mitochondria during RyR/IP3R-mediated [Ca2+]c signals. Since calibration of Ca2+ amounts is difficult in intact cells, we established a novel fluorescence Ca2+ imaging approach using permeabilized H9c2 myotubes and RBL-2H3 mast cells, which allowed calibration of Ca2+ release and uptake by intracellular organelles by adding known amounts of calcium. In particular, this method was suitable for estimating the relation between SR/ER release and mitochondrial Ca2+ uptake. We have already shown that the Ca2+ coupling between reticular and mitochondrial Ca2+ stores is well preserved in adherent permeabilized cells (Hajnoczky et al. 1999; Csordas et al. 1999). Furthermore, we found that the morphology of the SR/ER-mitochondrial junctions is retained in permeabilized H9c2 myotubes and RBL-2H3 mast cells. Using these experimental models we show that a large fraction (25-50%) of Ca2+ released through RyR and IP3R can be accumulated by the mitochondria.