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

Abstract

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.

Excitation-contraction coupling and mitochondrial energetics

“…Several studies indicate that by buffering [Ca 2+ ] c , rapid mitochondrial Ca 2+ uptake directly affects EC coupling [61,100,117,119,124,147,148,171]. When blocking mitochondrial Ca 2+ uptake, an increase of cytosolic Ca 2+ transients was observed ( Fig.…”

“…In the following years, a number of studies attempted to measure mitochondrial Ca 2+ uptake during EC coupling more directly, with quite controversial results [12,26,33,[59][60][61]68,[72][73][74]100,117,119,136,137,140,141,144,147,148,161,170,171,176,191,200,201,213] [26,117,119,148,176,191,200,201] or fluo-3 [33,144,170]) penetrate sarcolemmal and mitochondrial membranes and accumulate (to different degrees) in the mitochondrial matrix. To avoid contamination of the fluorescence signal by cytosolic Ca 2+ , the groups of Hansford, Silverman [59,60,74,136,137] and Bers [26,222] applied MnCl 2 in the extracellular solution.…”

“…7B) is in agreement with the K 0.5 of the mCU for Ca 2+ transport determined in isolated mitochondria (10-20 µM; [54,76,77]). In a parallel study from the lab of Hajnoczky, Pacher et al [147] calculated that ∼26% of the Ca 2+ released from the SR after caffeineexposure was taken up by mitochondria. This contrasts to the 1% of Ca 2+ removed by mitochondria after SR Ca 2+ release as calculated by Bassani et al [9].…”

“…Taken together, these results suggest that mitochondria may function as spatial buffers for Ca 2+ , regulating the propagation of Ca 2+ release events and thus, contractility of the cell. However, regarding the controversy on the amount of Ca 2+ taken up by mitochondria during EC coupling [9,117,147], the definite relevance of mitochondrial Ca 2+ uptake for cytosolic Ca 2+ homeostasis is still incompletely understood.…”

Ca 2+ marks: Miniature calcium signals in single mitochondria driven by ryanodine receptors

“…For measurements of [Ca 2ϩ ] m in permeabilized myotubes, the cells were loaded with 4 M rhod2͞AM in the presence of 0.003% (wt͞vol) pluronic acid at 37°C for 50 min. Dye-loaded cells were washed with Ca 2ϩ -free extracellular buffer composed of 120 mM NaCl, 20 mM Na⅐Hepes, 5 mM KCl, 1 mM KH 2 PO 4 , and 100 M EGTA͞Tris at pH 7.4 and then permeabilized with 15-20 g͞ml digitonin for 4 min in intracellular medium (ICM) composed of 120 mM KCl, 10 mM NaCl, 1 mM KH 2 PO 4 , 20 mM Tris⅐Hepes, and 2 mM MgATP at pH 7.2, supplemented with 1 g͞ml each of antipain, leupeptin, and pepstatin (21,22). For fluorescence imaging of [Ca 2ϩ ] c , labeling of cells with fura-C 18 (1 M) was carried out during permeabilization.…”