A modified calcium retention capacity assay clarifies the roles of extra- and intracellular calcium pools in mitochondrial permeability transition pore opening

Abstract

Calcium homeostasis is essential for cell survival and is precisely controlled by several cellular actors such as the sarco/endoplasmic reticulum and mitochondria. Upon stress induction, Ca²⁺ released from sarco/endoplasmic reticulum stores and from extracellular Ca²⁺ pools accumulates in the cytosol and in the mitochondria. This induces Ca²⁺ overload and ultimately the opening of the mitochondrial permeability transition pore (mPTP), promoting cell death. Currently, it is unclear whether intracellular Ca²⁺ stores are sufficient to promote the mPTP opening. Ca²⁺ retention capacity (CRC) corresponds to the maximal Ca²⁺ uptake by the mitochondria before mPTP opening. In this study, using permeabilized cardiomyocytes isolated from adult mice, we modified the standard CRC assay by specifically inducing reticular Ca²⁺ release to investigate the respective contributions of reticular Ca²⁺ and extracellular Ca²⁺ to mPTP opening in normoxic conditions or after anoxia–reoxygenation. Our experiments revealed that Ca²⁺ released from the sarco/endoplasmic reticulum is not sufficient to trigger mPTP opening and corresponds to ∼50% of the total Ca²⁺ levels required to open the mPTP. We also studied mPTP opening after anoxia–reoxygenation in the presence or absence of extracellular Ca²⁺. In both conditions, Ca²⁺ leakage from internal stores could not trigger mPTP opening by itself but significantly decreased the CRC. Our findings highlight how a modified CRC assay enables the investigation of the role of reticular and extracellular Ca²⁺ pools in the regulation of the mPTP. We propose that this method may be useful for screening molecules of interest implicated in mPTP regulation.

Introduction

Ca²⁺ homeostasis supports the cell ability to integrate stimuli, trigger Ca²⁺ signals, and ultimately control molecular pathways essential for cellular physiology. This cascade of events is accurately controlled in space and time (reviewed in Ref. 1). Ca²⁺ channeling between the sarco/endoplasmic reticulum and the mitochondria occurs in microdomains known as mitochondria-associated membranes, which have been reported to control specific cell functions (reviewed in Ref. 2) such as mitochondrial bioenergetics, lipid metabolism, and cell fate. Disruption of mitochondria-associated membranes has been associated to different pathologies including hypoxia–reoxygenation (3–5). Ischemia or hypoxia induces a drop in ATP content, which in turn decreases ATP-dependent Ca²⁺ pumps in both sarcolemma (plasma membrane Ca²⁺ ATPase) and sarco/endoplasmic reticulum (SERCA),³ leading to an increase in cytosolic (6) and consequently mitochondrial (7) Ca²⁺ concentration. This Ca²⁺ overload and enhancing factors like reactive oxygen species (8), partial mitochondrial membrane depolarization (9), pH restoration at reperfusion (10, 11), gangliosides (12), P_i (13), and outer mitochondrial membrane components (14) induce mitochondrial swelling and the opening of mPTP and ultimately promote cell death (1, 15, 16).

Although mitochondrial Ca²⁺ overload has been well-identified as being a main contributor to the latter phenomena, the origin of this Ca²⁺ is still unclear. Little is known about whether the internal Ca²⁺ stores alone are sufficient to promote mPTP opening in cardiomyocytes or whether extracellular Ca²⁺ entry is also required. Growing evidences have involved sarcolemmal channels and transporters such as the connexin and pannexin hemichannels (17) or the Na⁺/Ca²⁺ exchanger NCX (18). Their inhibition reduced Ca²⁺ overload and decreased myocardial injury, suggesting the involvement of extracellular Ca²⁺. However, none of these studies have evaluated the respective participation of this extracellular Ca²⁺ and the internal Ca²⁺ stores to the mitochondria Ca²⁺ overload process. Answering this question may help rationalizing therapeutic strategies relying on Ca²⁺ fluxes modulation.

This maximum ability of Ca²⁺ uptake by mitochondria before mPTP opening and mitochondrial swelling is defined as Ca²⁺ retention capacity (CRC) (reviewed in Ref. 19). This feature of mitochondria may vary according to the cell type, as well as the physiological (e.g. oxidative stress and pH) or pathological cellular state. CRC measurement is experimentally performed on isolated mitochondrial preparations or on cellular models and is conventionally realized with the addition of exogenous Ca²⁺ enabling the simple quantification of the Ca²⁺ amount required to open mPTP. Therefore, the role of reticular Ca²⁺ stores (the major Ca²⁺ store besides the mitochondria) in the mitochondrial Ca²⁺ overload was not studied.

Although several factors are implicated in mPTP opening, numerous studies have shown evidence of the crucial implication of Ca²⁺ overload in mPTP opening especially during ischemia reperfusion (20, 21). In this work we focused particularly on Ca²⁺ contribution. However, we ensure that the other factors contributing to mPTP opening are still present in our experimental condition by using a cellular environment. We investigated concomitantly the role of reticular and extracellular Ca²⁺ in the regulation of mPTP on a model of isolated cardiomyocytes. We modified the standard CRC method by combining the effect of extracellular Ca²⁺ (depicted by the addition of Ca²⁺ pulses) and reticular Ca²⁺ mobilization by stimulating ryanodine receptors using caffeine or ryanodine. We validated our experimental model by showing that, as expected, both pharmacological treatment of cardiomyocytes with cyclosporine A (CsA), an inhibitor of cyclophilin D (CypD), or the genetic ablation of CypD in cyclophilin D knockout (CypD-KO) cardiomyocytes required a greater amount of Ca²⁺ in the mitochondria to open mPTP. In addition, we demonstrated that in both normoxia and anoxia–reoxygenation (AR) conditions, reticular Ca²⁺ stores alone were not sufficient to trigger mPTP opening and that extracellular Ca²⁺ import (Ca²⁺ pulses addition) was required to open mPTP. Finally, we proposed an estimation of these Ca²⁺ sources contributions to mPTP opening.

Results

Validation of the experimental model

As shown in Fig. 1 (A and B), Ca²⁺ transfers were properly recorded with both probes in isolated cardiomyocyte preparation. Ca²⁺ Green-5N probe reported Ca²⁺ uptake by the mitochondria after each Ca²⁺ pulse, followed by a spontaneous cytoplasmic release indicating mPTP opening (Fig. 1A). Conversely, Ca²⁺ uptake by mitochondria was reported by the increase in Rhod2-AM fluorescence after each Ca²⁺ pulse (Fig. 1B), proving that exogenous Ca²⁺ added during the assay was indeed taken up by the mitochondria. When cardiomyocytes were subjected to ∼900 s of incubation before Ca²⁺ pulses (Fig. 1C), CRC was still similar to basal condition (Fig. 1A) (i.e. 10 Ca²⁺ pulses were needed to open mPTP). In addition, cardiac mitochondria in situ and permeabilized cellular integrity was preserved during at least 2000 s of incubation in the presence of Ca²⁺ Green-5N probe (Fig. 1D). Fluorescence was stable throughout the experiment reflecting the absence of intracellular Ca²⁺ leak and the absence of fluorescence bleaching. We also used Rhod2-AM staining to confirm that the mitochondrial network was not affected in permeabilized cardiomyocytes (40 μm digitonin) (Fig. 1E) and that Rhod2-AM colocalized with MitoTracker Green, proving a strict mitochondrial localization of Rhod2-AM staining in our experimental conditions (Fig. 1F).

Figure 1.

CRC in adult cardiomyocytes. A and B, CRC was performed in the presence of Ca²⁺ Green-5N probe to detect extramitochondrial Ca²⁺ (A) or in the presence of Rhod2-AM probe to detect intramitochondrial free Ca²⁺ (B). C, cardiomyocytes were incubated for 900 s in CRC medium before CRC measurement. D, cardiomyocytes were incubated in CRC medium and recorded for 2000 s in the presence only of Ca²⁺ Green-5N to assess cellular integrity and stability. For all experiments, fluorescence (F) is expressed in A.U. Ca²⁺ was added every 120 s by increments of 20 nmol/injection. E, confocal microscopy images were acquired after 30 min incubation of cardiomyocytes with 5 μm Rhod2-AM in the presence or absence of 40 μm digitonin. F, confocal microscopy images were acquired after 30 min of incubation of cardiomyocytes with 5 μm Rhod2-AM and 0.2 μm MitoTracker Green. 1024 × 1024 pixels images were acquired with an average of four scanning lines. Displayed graphs and images are representative of three independent experiments.

Another control experiment for our method validation was performed on isolated mitochondria (250 μg of protein). Standard CRC was performed in basal conditions (Fig. 2A), and in the presence of 5 mm caffeine (Fig. 2B) or 10 nm ryanodine (Fig. 2C). CRC values were similar in all experiments (Fig. 2D), indicating that, at the used concentrations, these ryanodine receptors agonists had no direct mitochondrial targets.

Figure 2.

Effect of caffeine and ryanodine on isolated mitochondria. A–C, the effect of caffeine and ryanodine was tested on isolated mitochondria. CRC was performed on isolated cardiac mitochondria using Ca²⁺ Green-5N probe in basal condition (A) or in the presence of 5 mm caffeine (B) or 10 nm ryanodine (C). D, CRC values were quantified for all conditions. Fluorescence (F) is expressed in A.U. Ca²⁺ was added every 120 s by increments of 20 nmol/injection. Displayed graphs are representative of three independent experiments. The CRC values in D are presented as means ± S.D. nmol Ca²⁺/mg protein of three independent experiments (Kruskal–Wallis; H = 0.6382, p = 0.7786).

Effects of the reticular Ca²⁺ release on mPTP opening

To study the effect of reticular Ca²⁺ on mPTP opening, CRC was evaluated either by adding exogenous Ca²⁺ pulses alone (Fig. 3A, black panel) or by using 5 mm caffeine or 10 nm ryanodine followed by exogenous Ca²⁺ pulse addition (Fig. 3A, gray and blue panels, respectively). In basal conditions (Fig. 3, B and C, black plots), 10 pulses (20 nmol Ca²⁺/pulse) of exogenous Ca²⁺ were needed to open mPTP. The addition of caffeine (Fig. 3B, gray plot) or ryanodine (Fig. 3C, blue plot) did not induce fluorescence elevation, suggesting that mitochondria take up the amount of Ca²⁺ released by caffeine or ryanodine stimulation (Fig. 3, B and C, insets). This was confirmed when cardiomyocytes preincubated with 1 μm FCCP were stimulated with 5 mm caffeine: an increase in extramitochondrial fluorescence was observed (Fig. 3D), representing the caffeine-induced reticular Ca²⁺ release that cannot be taken up by the mitochondria. The same observation was made after stimulation with 5 mm caffeine in the presence of 1 μm RU360, a specific mitochondrial Ca²⁺ intake inhibitor (Fig. 3E).

Figure 3.

Reticular Ca²⁺ contribution to mPTP opening. A, CRC measurement was performed using Ca²⁺ Green-5N probe and conditions. B and C, Ca²⁺ pulses added in basal condition (black plots) were compared with Ca²⁺ pulses added after stimulation with 5 mm caffeine (B, gray plot) or 10 nm ryanodine (C, blue plot) in adult cardiomyocytes. D and E, as a control, extramitochondrial Ca²⁺ fluorescence was measured in cardiomyocytes preincubated with 1 μm FCCP (D) or in the presence of 1 μm RU360 (E) after reticular Ca²⁺ release stimulation with 5 mm caffeine. Mitochondrial free Ca²⁺ was also stained using Rhod2-AM after 5 mm caffeine or 10 nm ryanodine stimulation (F, gray plot and blue plot, respectively). At 900 s, a series of 20 nmol of Ca²⁺ pulses were necessary to induce mPTP opening. In B and C, insets represent Ca²⁺ Green fluorescence variation from 0 to 500 s and reflect extramitochondrial Ca²⁺ after ryanodine receptor stimulation. In F, the inset represents Rhod2-AM fluorescence variation from 0 to 500 s and reflects mitochondrial free Ca²⁺ uptake after ryanodine receptor stimulation. G, Ca²⁺ amount added to open mPTP in basal condition or after reticular Ca²⁺ stimulation was quantified and presented as dot plots. Fluorescence (F) is expressed in A.U. Ca²⁺ was added every 120 s by increments of 20 nmol/injection. The displayed graphs are representative of three or four independent experiments. The values in G are presented as means ± S.D. nmol Ca²⁺/mg protein of four independent experiments (Kruskal–Wallis; H = 7.69, p = 0.0081), followed by Dunn's post-test. a, p < 0.05 versus basal.

No massive Ca²⁺ depletion from mitochondria was observed after a 900-s caffeine treatment, indicating that the amount of Ca²⁺ released from reticulum stores was not sufficient to open mPTP. Five additional pulses of 20 nmol of Ca²⁺ were required to induce mPTP opening (Fig. 3, B and C). The same experiments performed in Rhod2-AM–loaded cardiomyocytes showed an increase in fluorescence after stimulation with 5 mm caffeine (Fig. 3F, gray plot and inset) or 10 nm ryanodine (Fig. 3F, blue plot and inset). Overall, these experiments show that the caffeine-induced Ca²⁺ is quickly and efficiently taken up by mitochondria with a limited Ca²⁺ leak toward cytosol.

Maximal release of reticular Ca²⁺ was triggered by three repetitive stimulations with 5 mm caffeine. However, the amount of Ca²⁺ released was still insufficient, and additional pulses of 20 nmol Ca²⁺ were necessary to open mPTP (data not shown).

After stimulation with 5 mm caffeine or 10 nm ryanodine, the amount of Ca²⁺ necessary to open mPTP significantly decreased to 127 ± 13 nmol/mg protein (p = 0.0286) and 137 ± 16 nmol/mg protein (p = 0.0375), respectively, when compared with basal condition value (267 ± 22 nmol/mg protein) (Fig. 3G). In our conditions, reticular Ca²⁺ released after stimulation with 5 mm caffeine or 10 nm ryanodine represents ∼50% (48 ± 7% and 52 ± 6%, respectively) of the Ca²⁺ amount necessary for mPTP opening in the basal condition (Fig. 3G). This suggests that the reticular Ca²⁺ stores of cardiomyocytes cannot induce mPTP opening by itself.

Involvement of extracellular Ca²⁺ in mPTP opening

To confirm our previous results, we assessed whether preloading cardiac mitochondria with 50% of the Ca²⁺ amount necessary to open mPTP could allow the reticular Ca²⁺ content to drive mPTP opening (Fig. 4A). In three independent experiments, the addition of 5 mm caffeine (Fig. 4B) or 10 nm ryanodine (Fig. 4C) induced a strong and sustained increase in fluorescence intensity indicating a massive mitochondrial Ca²⁺ leak. The reticular Ca²⁺ release induced by either caffeine or ryanodine was sufficient to trigger mPTP opening when cardiomyocytes were loaded with 50% of the Ca²⁺ amount necessary to open mPTP. Both caffeine and ryanodine induced a massive reticular Ca²⁺ release after mitochondria Ca²⁺ preloading, suggesting an absence of or a limited induction of the Ca²⁺-induced Ca²⁺-release (CICR) mechanism. This was confirmed by the addition of one 20-nmol Ca²⁺ pulse after the massive fluorescence increase induced by caffeine and ryanodine. This Ca²⁺ pulse was not taken up by the mitochondria, ensuring that the mPTP was indeed opened. As a control of the potential effect of the experiment duration on mPTP opening, we preloaded cardiomyocytes with the same Ca²⁺ amount and recorded fluorescence without the addition of caffeine or ryanodine. In this condition, mPTP did not open, even after 2000 s of incubation time (Fig. 4D), demonstrating that mPTP opening observed in Fig. 4 (B and C) was triggered by caffeine- or ryanodine-induced Ca²⁺ release. In the presence of 110 ± 10-nmol Ca²⁺ load, no additional external Ca²⁺ was needed to open mPTP after internal Ca²⁺ stimulation with caffeine or ryanodine (Fig. 4E). These results are consistent with the previous observation from Fig. 3 and confirm our estimation that caffeine/ryanodine-induced reticular Ca²⁺ release represented ∼50% of the Ca²⁺ amount necessary to open mPTP.

Figure 4.

mPTP opening triggered by a combination of external and reticular calcium. A, measurements conditions. CRC was measured after the addition of a Ca²⁺ load with 50% of the Ca²⁺ amount necessary to open mPTP. B and C, at 900 s, ryanodine receptors were stimulated with 5 mm caffeine (B) or 10 nm ryanodine (C). In both situations, mPTP was opened. An additional pulse of 20 nmol of Ca²⁺ was added but was not taken up by the mitochondria, confirming mPTP opening. D, as a control, cardiomyocytes were recorded during 2000 s after the addition of Ca²⁺ load without caffeine or ryanodine stimulation. E, the amount of external Ca²⁺ added to open mPTP was quantified in basal condition and in the presence of Ca²⁺ load (110 ± 10 nmol) followed by caffeine or ryanodine stimulation. All experiments were performed in the presence of Ca²⁺ Green-5N probe. Fluorescence (F) is expressed in A.U. and represents extramitochondrial Ca²⁺. Ca²⁺ was added every 120 s by increments of 20 nmol/injection. Displayed graphs are representative of three independent experiments. The values in E are presented as means ± S.D. nmol Ca²⁺/mg protein of three independent experiments (Kruskal–Wallis; H = 7.624, p = 0.0357), followed by Dunn's post-test. a, p < 0.05 versus basal.

One of the most studied cardioprotective strategy within the last decade has relied on either the pharmacological inhibition or the genetic ablation of cyclophilin D, which, under Ca²⁺ stimulation, is known to enhance mPTP opening. Assuming that the reticular Ca²⁺ content is stable, we wondered whether CypD inhibition/suppression would also shift the amount of extracellular Ca²⁺ required to open mPTP in our experimental model. We first determined the amount of Ca²⁺ necessary to open mPTP after CypD inhibition/suppression and then loaded cardiomyocytes with 50% of this amount prior to inducing reticular Ca²⁺ release with 10 nm ryanodine (Fig. 5A). When CsA was added to the preparation, an additional 63.3 ± 14.5 nmol Ca²⁺/mg protein were necessary to induce mPTP opening after ryanodine stimulation (Fig. 5B, blue plot). If rotenone, a complex I inhibitor, was added to CsA, the amount of Ca²⁺ required to open mPTP was raised to 133.7 ± 27.0 nmol Ca²⁺/mg protein (Fig. 5C, blue plot). Similar results were observed in CypD-KO adult cardiomyocytes (Fig. 5D, blue plot), in which the addition of 177.7 ± 15.3 nmol Ca²⁺/mg protein was necessary to induce mPTP opening. Compared with the control condition tested in Fig. 4C (where no additional Ca²⁺ was needed to open mPTP after stimulation of reticular Ca²⁺ release), the inhibition/suppression of CypD in our model significantly increase mitochondria capacity to uptake Ca²⁺ (Fig. 5E).

Figure 5.

Effects of pharmacological and genetic protection on mPTP opening. A, Ca²⁺ amount necessary to induce mPTP opening was determined without reticular Ca²⁺ stimulation (black) or after stimulation with 10 nm ryanodine (blue). B–D, measurements were performed in the presence of 1 μm CsA (B) or 1 μm CsA + 1 μm rotenone (C) in WT adult cardiomyocytes or in CypD-KO adult cardiomyocytes (D). CRC was measured after the addition of 50% of the amount of Ca²⁺ necessary to open the mPTP. This amount was calculated based on basal CRC (black plots) with each CypD inhibition or suppression. At 900 s, ryanodine receptors were stimulated with 10 nm ryanodine. 900 s later, fluorescence level did not increase, indicating no mPTP opening. Further pulses of 20 nmol of Ca²⁺ were added to induce mPTP opening (B–D, blue plots). E, amount of Ca²⁺ added to open mPTP after ryanodine stimulation: in control (without CypD inhibition/suppression (i.e. Fig. 4C) or when CypD is inhibited/suppressed. The data are presented as means ± S.D. nmol Ca²⁺/mg protein of three independent experiments: one-way ANOVA (F(3, 8) = 62.41; p < 0.0001), followed by Tukey post-test. a, p < 0.05 versus control; b, p < 0.005 versus CsA. All experiments were performed in the presence of Ca²⁺ Green-5N probe. Fluorescence (F) is expressed in A.U. and represents extramitochondrial Ca²⁺. Ca²⁺ was added every 120 s by increments of 20 nmol/injection. Displayed graphs are representative of three independent experiments.

Effect of reticular Ca²⁺ mobilization on mPTP opening after AR

Altogether, our results emphasized the major contribution of both internal Ca²⁺ stores and extracellular Ca²⁺ to mitochondrial/cellular fate in normoxic condition. We next assessed whether AR would modify this dynamic.

Cardiomyocytes were subjected to 30 min of anoxia followed by a 15-min reoxygenation period (in the presence or absence of 1 mm extracellular Ca²⁺) prior to CRC measurement. As a control (sham groups), cardiomyocytes were incubated for 45 min in normoxic condition also in the presence or absence of 1 mm extracellular Ca²⁺ (Fig. 6A).

Figure 6.

CRC value variations after AR in the absence or presence of extracellular Ca²⁺. A, design of the AR protocol and CRC measurement methods are presented. Adult cardiomyocytes were stored for 45 min in normoxic conditions (sham groups) or subjected to 30 min of anoxia followed by 15 min of reoxygenation (AR groups). Both sham and AR were performed in the presence or in the absence of 1 mm extracellular Ca²⁺. Two methods were used to measure CRC: in sham groups and AR1 groups, only Ca²⁺ pulses were added (20 nmol/pulse) until mPTP opens. In AR2 groups, caffeine (5 mm) was added first to induce reticular Ca²⁺ release, and then Ca²⁺ pulses were added (20 nmol/pulse) until mPTP opens. B, values of Ca²⁺ amount added to open mPTP are presented as means ± S.D. nmol Ca²⁺/mg protein of four or five independent experiments. Two-way ANOVA was conducted on the influence of the presence of 1 mm extracellular Ca²⁺ on mPTP opening in the different above protocols. Both AR (F(2, 22) = 81.27; p < 0.0001) and 1 mm Ca²⁺ addition (F(1, 22) = 48.2; p < 0.0001) had a significant effect on CRC. Tukey post-test showed significant differences: a, p < 0.0001 versus respective sham; b, p < 0.0001 with extracellular Ca²⁺ versus without extracellular Ca²⁺; c, p < 0.005 AR1 versus AR2. The interaction was, however, not significant (F(2, 22) = 1. 228; p = 0.3121). Ctrl, control.

AR protocol was determined based on our previous work and on a recent study from Panel et al. (22), to guarantee a sufficient amount of viable cardiomyocytes after AR and to achieve a valuable measurement of mPTP opening. The amount of Ca²⁺ needed to open mPTP was determined (as in Fig. 3) either using only Ca²⁺ pulses addition (sham groups and AR1 groups) or using 5 mm caffeine (to induce reticular Ca²⁺ release) followed by Ca²⁺ pulse addition (AR2 groups) (Fig. 6A).

As expected, the amount of Ca²⁺ added to open mPTP decreased after AR protocol. AR1 with or without 1 mm Ca²⁺ compared with respective sham (Fig. 6B): 108 ± 19 versus 210 ± 35 nmol Ca²⁺/mg protein (p < 0.0001; with 1 mm extracellular Ca²⁺) and 193 ± 22 versus 290 ± 40 nmol Ca²⁺/mg protein (p < 0.0001; without 1 mm extracellular Ca²⁺). In AR2, our results showed that even after AR protocol, either with or without 1 mm extracellular Ca²⁺, reticular Ca²⁺ release after caffeine stimulation was not sufficient to open mPTP. Additional Ca²⁺ pulses were added to trigger mPTP opening.

Moreover, in the presence of 1 mm extracellular Ca²⁺, CRC values significantly decreased when compared with the respective condition in the absence of 1 mm extracellular Ca²⁺ (sham, AR1, and AR2 with 1 mm extracellular Ca²⁺ versus sham, AR1, and AR2 without 1 mm extracellular Ca²⁺, respectively; p < 0.0001). Strikingly, in none of these conditions were intracellular Ca²⁺ stores sufficient to trigger mPTP opening, even though the presence of extracellular Ca²⁺ during AR facilitates mPTP opening. In all these conditions, the addition of exogenous Ca²⁺ pulses was needed to induce mPTP opening. Interestingly, other biophysical features can be evaluated using our measurement method, which is described below.

The mobilized internal Ca²⁺ stores

These stores can be estimated by measuring the difference between CRC value in sham without extracellular Ca²⁺ and the CRC value in AR2 without extracellular Ca²⁺ (Table 1). The mobilized internal Ca²⁺ stores in our model would be ∼187 ± 38 nmol Ca²⁺/mg protein.

Table 1.

Estimation of the mobilized Ca²⁺ cross-talk during AR

The values represent means ± S.D. of four or five independent experiments.

Mobilized internal Ca2+ stores	Remaining internal Ca2+ stores	Proportion of internal stores Ca2+ leak during AR	External Ca2+ amount that entered the cell during AR
(Sham without Ca2+) − (AR2 without Ca2+)	(AR1 without Ca2+) − (AR2 without Ca2+)	100 − [(2) × 100/(1)]	(AR2 without Ca2+) − (AR2 without Ca2+)
187 ± 38 nmol/mg protein	80 ± 39 nmol/mg protein	57 ± 23%	48 ± 14 nmol/mg protein
(1)	(2)	—	—

The remaining internal Ca²⁺ stores after AR

The measured CRC values without 1 mm extracellular Ca²⁺ were significantly different in AR1 compared with AR2 (p < 0.005). Therefore, the remaining internal Ca²⁺ stores after AR can be estimated by subtracting CRC value in AR1 from CRC value in AR2 without 1 mm extracellular Ca²⁺ and were equivalent to 80 ± 39 nmol Ca²⁺/mg protein (Table 1).

The proportion of internal Ca²⁺ stores leak during AR

Knowing both the total internal Ca²⁺ amount (187 ± 38 nmol Ca²⁺/mg protein) and the remaining internal Ca²⁺ after AR (80 ± 39 nmol Ca²⁺/mg protein), we can estimate the internal Ca²⁺ that leaked during AR to 57 ± 23% (Table 1).

The extracellular Ca²⁺ amount that entered the cell during AR

The difference between the CRC values of AR2 in the absence and the presence of 1 mm extracellular Ca²⁺ represents an estimation of the amount of extracellular Ca²⁺ that entered the cells during AR and was equal to 48 ± 14 nmol Ca²⁺/mg protein (Table 1). The 48 ± 14 nmol Ca²⁺/mg protein represented approximately half of the internal Ca²⁺ stores that leaked to the mitochondria (187 − 80 = 107 nmol Ca²⁺/mg protein). Overall, we can conclude that in our experimental conditions, two-thirds of the Ca²⁺ loaded to the mitochondria during AR may come from internal Ca²⁺ stores (mainly from the reticulum), whereas approximately one-third seems to be of extracellular origin.

Discussion

Mitochondria are involved in multiple cell processes such as the control of cytoplasmic Ca²⁺ level because of their Ca²⁺ uptake capacity. This participation is particularly important when the cellular environment is subjected to major stress such as ischemia–reperfusion during which the absence of oxygen induces the drop of ATP synthesis and consequently the inhibition of SERCA activity contributing thus to several physiological mishandling including a massive intracellular Ca²⁺ overload (23). In such a situation, mPTP transient activity could act as a safety valve, which enables Ca²⁺ leak with an increase rate of efflux. Ultimately, this mechanism triggers mitochondrial swelling, which has been associated with cell death. This finely tuned regulation, balanced by Ca²⁺ and mediated by CypD, has been extensively studied (24–26). However, in most of the studies, no distinctions were made regarding the origin of Ca²⁺ (internal Ca²⁺ stores and/or extracellular Ca²⁺) inducing the opening of mPTP, more likely because there was no reliable method to investigate it.

The proposed method in this study is derived from classical CRC assay and proposes a simple mean to quantitatively estimate the respective contribution of internal Ca²⁺ stores and extracellular Ca²⁺ toward mPTP opening. CRC can be achieved by the addition of exogenous Ca²⁺ pulses to isolated mitochondria (27) or permeabilized cells (28). However, this classical CRC method does not allow the study of reticular Ca²⁺ input. In parallel to CRC experiment, most of the standard protocols use Ca²⁺ ionophores and Ca²⁺ addition to induce mPTP opening. These Ca²⁺ ionophores can induce artifacts in cellular models including in isolated adult cardiomyocytes (22) and can also induce a release of internal Ca²⁺ stores in a nonspecific manner, as compared with caffeine or ryanodine treatment, which specifically induces reticular Ca²⁺ release.

Interestingly, our presented method relies on the analysis of mitochondria–reticulum interaction in situ. We used freshly isolated adult murine cardiomyocytes as a cellular model. Cardiomyocytes plasma membranes were permeabilized with 40 μm digitonin. This chemical agent and the used concentration are both respectful of the intracellular membrane structure (29–31). The intracellular microdomains and organelle interactions are thus preserved in this model, allowing the study of mitochondria–reticulum interaction within an integrated physiological-like cellular environment (31, 32). This was also shown by confocal microscopy images showing no impact of 40 μm digitonin on the mitochondrial network in our model (Fig. 1E). We also confirmed the reliability of our cellular model and our experimental conditions, as well as the absence of Ca²⁺ Green-5N fluorescence bleaching throughout the duration of our experiments.

In our permeabilized model, the low ATP level ensures a natural inhibition of SERCA activity and therefore excludes reticular Ca²⁺ uptake (33). In addition, with this method, one can activate the reticular Ca²⁺ release via ryanodine receptors with specific agonists such as caffeine or ryanodine (34, 35) to estimate its involvement in mPTP opening. In isolated adult cardiomyocytes, we estimated that reticular Ca²⁺ represents ∼50% of the Ca²⁺ amount necessary to open mPTP in basal conditions. This proportion may represent an indicator of physiological Ca²⁺ steady state between the mitochondria and the reticulum. A second biophysical parameter that can be deduced with this method is the proportion of Ca²⁺ content that leaks from the internal stores during AR: ∼57% during a 15-min reoxygenation. Finally, the third biophysical parameter that can be estimated is the proportion of extracellular Ca²⁺ entering the cells during AR, which represents ∼50% of the internal Ca²⁺ leaking during AR protocol. Considering these two last parameters, we can suggest that ∼15–20% of the total Ca²⁺ content inducing mPTP opening comes from the extracellular Ca²⁺ pulses added during the CRC experiments measured after AR protocol. To our knowledge, this is the first demonstration that internal Ca²⁺ leaks during AR require additional factors like external Ca²⁺ to open mPTP.

Another advantage offered by this method is the quantification estimate of the relationship between extracellular Ca²⁺ content and steady-state mitochondrial Ca²⁺ content. It has always been assumed that Ca²⁺ homeostasis translates extracellular Ca²⁺ changes to shifts in Ca²⁺ concentration in the cytosol and organelles. Although the quantification of this interdependence could be realized in live cells with fluorescent probes, the calibration of these probes in mitochondria is much harder than in the cytosol. With our method, we found that the CRC value in cardiomyocytes incubated for 45 min (sham groups) in the absence of extracellular Ca²⁺ was increased by 80 nmol Ca²⁺/mg protein compared with an incubation in the presence of 1 mm extracellular Ca²⁺. This emphasizes the fact that extracellular Ca²⁺ contribute to the mitochondrial Ca²⁺ homeostasis. Future experiments will be performed as a dose-effect response to fully quantify and understand this interdependence.

One limitation of our method could be that a proportion of the Ca²⁺ released by caffeine leaks in the medium instead of entering the mitochondria. However, this would mean that upon caffeine stimulation, the fluorescence of Ca²⁺ Green-5N probe should rise. The dissociation constant of Ca²⁺ Green-5N probe is ∼14 μm. In our experimental conditions, the caffeine-mediated Ca²⁺ released is ∼70 μm (187 nmol Ca²⁺/mg protein with 750 μg of protein in 2 ml of CRC medium). It is thus very unlikely that the probe could not detect a reticular Ca²⁺ leak, if there is any. Along the same line, it should be noted that the insets in Fig. 3 (B and C) showed no significant Ca²⁺ variation in the cytosol, suggesting that it was rapidly taken up by the mitochondria, whereas a fluorescence increase was observed in the mitochondria as shown by a Rhod2-AM probe, which is sensitive to free Ca²⁺ variation (insets Fig. 3F). This was also confirmed by the absence of reticular Ca²⁺ uptake in the presence of the mitochondrial uncoupler FCCP (Fig. 3D) and mitochondrial Ca²⁺ uptake inhibitor, RU360 (Fig. 3E). These results highlight the efficiency of mitochondria–reticulum connection to channel Ca²⁺ between both organelles in isolated adult cardiomyocytes.

Another limitation is that in our experimental model, CICR is very limited because of cellular permeabilization and very low ATP amount. However, in the situation of ischemia–reperfusion injury, the absence of ATP production makes CICR unlikely to occur, suggesting the absence of CICR contribution to mPTP opening during ischemia–reperfusion (36) similarly to our experimental model.

Finally, pH restoration during reperfusion, one of the factors that could participate in enhancing mPTP opening (11, 37), is probably underestimated in our experimental model because of the use of buffered medium in AR protocol. Further experiments are needed to assess any synergetic activation of mPTP with this factor.

In this work, we showed that the mitochondrial Ca²⁺ level required to open mPTP exceeded the reticular Ca²⁺ content in both sham and AR experimental conditions either when the protocol was performed in the presence or in the absence of 1 mm extracellular Ca²⁺. In all these conditions, additional Ca²⁺ pulses were needed to induce mPTP opening. This work emphasizes the prior knowledge that both extracellular Ca²⁺ and internal Ca²⁺ stores are important triggers of mPTP opening, by discerning, estimating, and contextualizing their respective contribution. Our results highlight the involvement of extracellular Ca²⁺ to mitochondrial Ca²⁺ homeostasis but also suggest that the mPTP opening process requires additional mechanisms such as protein and membrane oxidation by reactive oxygen species (38) or pH restoration (10, 11).

This original and simple approach could be applied to a large panel of cell types and cellular models to detect in situ reticulum–mitochondria Ca²⁺ transfer dysfunction and would open new perspectives for the study and screening of pharmacological molecules for mitochondrial and reticular targets. This method could thus bring new and valuable insights into the physiopathological investigation of numerous diseases.

Experimental procedures

Animals

We obtained 8–12-week-old male C57BL/6J mice from Charles River Laboratories (L'arbresle, France). CypD-KO mice under C57Bl/6/SV129 background were issued from Korsmeyer's laboratory (Dana Farber Cancer Institute, Boston, MA). CypD-KO male mice (8–12 weeks old, 20–30 g) were obtained by homozygous intercross in our laboratory.

The present study was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 85-23, revised 1996), and all experiments were approved by the University of Lyon Ethics Committee (UCBL1 approval BH2007-07).

Cardiomyocytes isolation

Adult mouse cardiomyocytes were freshly isolated using two different enzymatic digestions as previously described (39, 40). Briefly, hearts from cervically dislocated male mice were quickly removed and retrogradely perfused with Krebs–Henseleit buffer. Ventricular cardiomyocytes were isolated using enzymatic digestion with 0.167 mg·ml⁻¹ Liberase (Roche) and 0.14 mg·ml⁻¹ trypsin 2.5% (Invitrogen), or with 2.4 mg·ml⁻¹ collagenase type II (Gibco). Cellular protein concentration was measured using the Bradford method. Isolated cardiomyocytes viability was between 85 and 95%. The cells were used within 5 h after isolation.

Mitochondria isolation

Isolated mitochondria were used as part of the validation of the study model. After being removed, the hearts were quickly placed in an ice-cold isolation buffer (70 mm sucrose, 210 mm mannitol, and 10 mm EGTA, 50 mm Tris-HCl, pH 7.4). Myocardial tissue was finely minced and then homogenized in the same buffer. Mitochondria were isolated in accordance with our previous studies (39, 41). Briefly, the homogenate was centrifuged at 1300 × g for 3 min, and the supernatant was centrifuged at 10,000 × g for 10 min. The mitochondrial pellet was then suspended in a cold buffer containing: 70 mm sucrose and 210 mm mannitol in 50 mm Tris-HCl, pH 7.4, and centrifuged at 10,000 × g for 10 min. Mitochondrial protein concentration was measured using the Bradford method. Mitochondria were used within 3 h after isolation.

Ca²⁺ retention capacity assay

This study was mainly based on the measurement of CRC, an in vitro surrogate for the susceptibility of mPTP opening following a Ca²⁺ overload. Briefly, the sample was placed in the CRC medium (150 mm sucrose, 50 mm KCl, 2 mm KH₂PO₄, 20 mm Tris-HCl, and 5 mm succinate-Tris) and under continuous stirring in the spectrofluorometer. After 120 s of stabilization time, 20 nmol of CaCl₂ (pulses) were added every 120 s. Modification of extramitochondrial Ca²⁺ concentration was continuously recorded in the presence of a Ca²⁺ fluorescent probe. After sufficient Ca²⁺ loading, an increase in fluorescence intensity represents an elevation of extramitochondrial Ca²⁺ concentration, indicating a massive release of Ca²⁺ by mitochondria caused by mPTP opening. The amount of Ca²⁺ necessary to trigger a massive Ca²⁺ release, expressed as the CRC value, was used as an indicator of mPTP susceptibility to Ca²⁺ overload.

CRC assay can be performed on isolated mitochondria or permeabilized cells and uses only exogenous Ca²⁺ addition to achieve mitochondrial Ca²⁺ overload. The purpose of the present study was to evaluate mitochondria–reticulum Ca²⁺ cross-talk.

Consequently, (a) CRC was specifically performed on a model of permeabilized cardiomyocytes to benefit from the entire intracellular environment. (b) Intracellular Ca²⁺ store release was triggered using caffeine or ryanodine addition. Another specification of the permeabilized cell context is the very low ATP amount. ATP is diluted in the assay medium and is not sufficient to induce SERCA activity (33), which guarantees no refilling of the sarco/endoplasmic stores and prevent CICR. (c) CRC assay was then performed as previously described. Ca²⁺ fluorescence was measured using a spectrofluorometer (F-2500, Hitachi, High-tech) with two different Ca²⁺ probes: Ca²⁺ Green-5N and Rhod2-AM (Invitrogen). (d) Ca²⁺ Green-5N probe (excitation, 506 nm; emission, 530 nm) detects extramitochondrial calcium. Cardiomyocytes (750 μg of protein) were added in 2 ml of CRC medium. This medium was supplemented with 0.4 μm Ca²⁺ Green-5N and 40 μm digitonin for cardiomyocytes permeabilization (42). (e) Rhod2-AM probe (excitation, 551 nm; emission, 582 nm) detects free intramitochondrial Ca²⁺ and attests that Ca²⁺ was actually taken up by the mitochondria. Cardiomyocytes (750 μg of protein) were incubated with 5 μm of Rhod2-AM for 30 min at room temperature and then washed for 30 min with a buffer containing 50 mm Tris-HCl, 70 mm saccharose, and 210 mm of mannitol, pH 7.4, to remove remaining Rhod2-AM, as well as Ca²⁺ traces.

Confocal microscopy

Cardiomyocytes (750 μg of protein) were incubated with 5 μm of Rhod2-AM and 0.2 μm MitoTracker Green under the same conditions described above. Images were taken on a confocal microscope Nikon A1r using an oil-immersion 40× objective (N.A. 1.3). MitoTracker Green and Rhod2-AM were excited with 488 and 560-nm wavelength laser lines, respectively. The emitted light was filtered by 525 ± 25 and 595 ± 25 bandpass filters, respectively. 1024 × 1024 pixels images were acquired with an average of four scanning lines.

Anoxia–reoxygenation protocol

The effect of depleting reticulum Ca²⁺ stores on mPTP opening (as described above) was evaluated after AR in the presence of Ca²⁺ Green-5N. Cardiomyocytes (750 μg of protein) were subjected to 30 min of hypoxia followed by 15 min of reoxygenation in the presence or absence of 1 mm extracellular Ca²⁺ (nonpermeabilized cardiomyocytes). Anoxia was achieved using CRC medium (with or without 1 mm Ca²⁺) degassed for 5 min with nitrogen and supplemented with 0.5 mm dithionite. At the end of anoxia, the cells were washed and centrifuged at 20 × g for 3 min and resuspended with 2 ml of fresh oxygenated CRC medium. Sham groups (cardiomyocytes placed for 45 min in normoxic conditions) were subjected to centrifugation/resuspension and used as controls for AR groups. At the end of reoxygenation time, CRC was measured following two different procedures: either using only Ca²⁺ pulses addition (Sham groups and AR1 groups) or after the addition of 5 mm caffeine first (to release reticular Ca²⁺) followed by the addition of Ca²⁺ pulses to complete the measurement (AR2 groups).

Data processing and presentation

The animals were randomly distributed between groups. The data were analyzed using GraphPad Prism 6 (GraphPad software, San Diego, CA). Displayed graphs are representative of three independent experiments and presented as fluorescence arbitrary unit (A.U.) as a function of time in seconds.

Histograms and scatter plots are represented as means ± S.D. nmol Ca²⁺/mg protein of three to five distinct experiments. Interactions and comparisons between groups were made using one-way ANOVA or two-way ANOVA followed by Tukey multicomparison post-test. Kruskal–Wallis followed by Dunn's multicomparison post-test were used as nonparametric tests in absence of Gaussian normality. The values for p < 0.05 were considered significant.

Author contributions

R. H., M. A., R. A.-M., C. V., and A. G. conceptualization; R. H., M. A., P. C., R. A.-M., C. V., and A. G. data curation; R. H., N. T., and A. G. formal analysis; R. H., G. B., and A. G. validation; R. H., M. A., P. C., G. B., and A. G. investigation; R. H., M. A., P. C., R. A.-M., N. T., G. B., and A. G. methodology; R. H., P. C., G. B., and A. G. writing-original draft; R. H., M. A., P. C., G. B., M. O., and A. G. writing-review and editing; C. V. and A. G. resources; G. B., M. O., and A. G. funding acquisition; M. O. and A. G. project administration; A. G. supervision.