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Review
. 2006 Nov-Dec;40(5-6):553-60.
doi: 10.1016/j.ceca.2006.08.016. Epub 2006 Oct 30.

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

György Hajnóczky  1 , György Csordás, Sudipto Das, Cecilia Garcia-Perez, Masao Saotome, Soumya Sinha Roy, Muqing Yi
Affiliations
Review

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

György Hajnóczky et al. Cell Calcium. 2006 Nov-Dec.

Abstract

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.

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Figures

Figure 1
Figure 1. Mechanisms of the mitochondrial Ca2+ transport
Abbreviations used: CSMDH, Ca2+ sensitive mitochondrial dehydrogenase; PTP, permeability transition pore; UP, uniporter; VDAC, voltage dependent anion selective channel.
Figure 2
Figure 2. Ca2+-induced mitochondrial membrane permeabilization
Schemes illustrating possible mechanisms for the cellular Ca2+ overload (A)- and ER/SR Ca2+ mobilization (B,C) induced mitochondrial membrane permeabilization. Pro-survival mechanisms are depicted in green and pro-death mechanisms are shown in purple. Abbreviations used: AA, arachidonic acid; ROS, reactive oxygen species; cyto c, cytochrome c.
Figure 3
Figure 3. Approaches for preventing the mitochondrial Ca2+ uptake in intact cells
Figure 4
Figure 4. RuRed-and Ru360-dependent changes in calcium signalling in intact and permeabilized cells
Cells cultures were transfected with mitochondria targeted inverse pericam (24h) and subsequently, pretreated with RuRed (Fluka, ≥ 85% purity, red) or Ru360 (Calbiochem, cyan) for 12-18h in serum-free medium: RBL-2H3, RuRed 5μM (A); HepG2, RuRed 10μM (B); H9c2, RuRed or Ru360 10μM each. Subsequently, cells were loaded with fura2/AM and incubated in a Ca2+ free extracellular medium. For the RuRed/Ru360 treated cells the drug was also present during the fura2 loading and the recording. Fluorescence [Ca2+]c and [Ca2+]m imaging was carried out as described before [3, 20]. Traces represent the means for 3-7 experiments for each condition. (A) The RBL-2H3 cells were co-transfected with pericam and M1 muscarinic receptor. Cells were treated with carbachol, CCH of 100μM, thapsigargin, Tg of 2μM and CaCl2, Ca of 2mM as indicated by the arrows. (B) HepG2 cells were treated with ATP 100μM, Tg of 2μM and CaCl2 of 5mM. (C) H9c2 cells were treated with vasopressin, VP of 100nM, Tg of 2μM and CaCl2 of 10mM (i). Bar charts show the size of the VP- and Ca-induced [Ca2+]m and [Ca2+]m rise calculated in single cells (ii). The rates for the Ca-induced [Ca2+]c rise were 31.3±4.1 nM/s; 29.4±2.7 nM/s; 23.6±4 nM/s and for the [Ca2+]m rise were 2.4±0.4 %/s; 2.6±0.5 %/s; 2.6±0.3 %/s in control, RuRed-and Ru360-pretreated cells, respectively (n=34, 30 and 32 cells). (D) RuRed-and Ru360 induced suppression of the mitochondrial Ca2+ uptake in permeabilized H9c2 cells (i) Pericam-transfected adherent H9c2 cells were permeabilized with saponin and incubated in an intracellular medium containing fura2 5μM. [Ca2+]c and [Ca2+]m were followed in single cells with CCD imaging of fura2 and pericam fluorescence, respectively. To evoke a Ca2+ release from the ER IP3 7.5μM was added. RuRed of 5μM was added 3min before IP3 (red trace). Traces are means of three experiments. (ii,iii) Suspensions of H9c2 cells (1mg protein/ml) were incubated in an intracellular medium and were permeabilized by digitonin. [Ca2+]c was monitored in a fluorometer using fura2FF (0.5μM) added to the bathing medium. CaCl2 30μM and FCCP of 5μM was added as indicated by the arrows. RuRed (red) and Ru360 (blue) 1μM each was added 1min before Ca2+ addition. Representative traces for the fluorometer records (ii) and bar charts showing the RuRed/Ru360 sensitive fraction of the decay in [Ca2+]c (iii).
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