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

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.

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. 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. Approaches for preventing the mitochondrial Ca²⁺ uptake in intact cells

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).