Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress

Yamanaka, Ryu; Tabata, Sho; Shindo, Yutaka; Hotta, Kohji; Suzuki, Koji; Soga, Tomoyoshi; Oka, Kotaro

doi:10.1038/srep30027

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Published: 26 July 2016

Mitochondrial Mg²⁺ homeostasis decides cellular energy metabolism and vulnerability to stress

Ryu Yamanaka¹,
Sho Tabata²,
Yutaka Shindo¹,
Kohji Hotta¹,
Koji Suzuki³,
Tomoyoshi Soga² &
…
Kotaro Oka¹

Scientific Reports volume 6, Article number: 30027 (2016) Cite this article

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Abstract

Cellular energy production processes are composed of many Mg²⁺ dependent enzymatic reactions. In fact, dysregulation of Mg²⁺ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg²⁺ stores. Several biological stimuli alter mitochondrial Mg²⁺ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg²⁺ alteration affect cellular energy metabolism remains unclear. Mg²⁺ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg²⁺ uptake system. Here, we comprehensively analyzed intracellular Mg²⁺ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg²⁺ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg²⁺ via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg²⁺ level in response to physiological stimuli.

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Introduction

Adenosine triphosphate (ATP) is the universal energy currency of cells. ATP binds to magnesium ion (Mg²⁺) to compose biologically functional form and most of intracellular ATP and Mg²⁺ assumed to form Mg-ATP complexes. Because both ATP and Mg²⁺ are mutually and strongly buffered in cytosol, it had been believed that Mg²⁺ deeply contributes on energy metabolism. Actually, studies performed in vitro has revealed that the variety of enzymatic activities are dependent on [Mg²⁺]¹ and some of the Mg²⁺-dependent enzymes are operated in mitochondria to sustain the cellular function and viability. Some of enzymatic activities of tricarboxylic acid cycle (TCA cycle)^2,3 are regulated by [Mg²⁺] and therefore, computer simulation by a realistic metabolic model of TCA cycle also showed mitochondrial Mg²⁺ level is most important regulating factor⁴. Mg²⁺ homeostasis is crucial for maintenance of electron transport chain⁵. Moreover, the mitochondrial ATP-Mg/P_i carrier exports mitochondrial ATP into cytosol^6,7. Therefore, Mg²⁺ has been implicated as an important regulator of metabolic status in mitochondria^8,9,10.

Mg²⁺ is an important cation for maintain cellular functions and, therefore, suggested the relation of Mg²⁺ to various diseases such as cancer, obesity, type 2 diabetes and neurological diseases^11,12,13,14. Furthermore, intracellular Mg²⁺ plays roles as a second messenger in the immune system^15,16,17,18 and it has been recognized as a multi-target metabolic regulator^8,19. Therefore, regulation of intracellular Mg²⁺ is critical for maintenance of cellular functions and tissue integrity. To reveal the regulatory mechanism of intracellular Mg²⁺, we have developed Mg²⁺ sensitive fluorescence probes and imaging techniques^{20,21,22,23,24}. These intracellular Mg²⁺ imaging works revealed Mg²⁺ mobilization in pathological and physiological conditions^25,26,27,28 and mitochondria are intracellular Mg²⁺ stores²⁹. Mitochondria redistribute cytosolic and mitochondrial Mg²⁺ sufficient to change the cytosolic Mg²⁺ concentration ([Mg²⁺]_cyto) in response to several physiological stimuli^25,26. Recent studies using novel Mg²⁺ fluorescent probe successfully visualized that mitochondrial Mg²⁺ concentration ([Mg²⁺]_mito) dynamically changes^22,30. However, it is not clear, in cells, how the changes of [Mg²⁺]_mito comprehensively affect the cellular energy metabolism in detail.

Although regulation of [Mg²⁺]_mito has not been elucidated in detail, mitochondrial Mg²⁺ channel MRS2 is known to be a molecular machinery associated with Mg²⁺ influx into mitochondria^31,32,33,34. The rats with functional inactivation of mutated MRS2 have major mitochondrial deficits with a reduction in ATP and increased numbers of mitochondria in oligodendrocytes³⁵. Mg²⁺ uptake into mitochondria via MRS2 is essential for the maintenance of respiratory chain and cell viability⁵.

In this study, we investigate how dysregulation of mitochondrial Mg²⁺ homeostasis affects cellular energy maintenance and viability using single-cell fluorescence imaging and metabolomics analysis in Mrs2 knockdown (KD) cells. Mrs2 KD induces disruption of mitochondrial Mg²⁺ homeostasis, which results in supression of mitochondrial ATP production and increased cellular stress susceptibility. These findings suggest that mitochondrial Mg²⁺ plays important roles to maintain energy supply in cells and its dysregulation causes cellular malfunction and multiple diseases.

Results

RNAi-mediated Mrs2 KD in HeLa cells

We investigated the importance of mitochondrial Mg²⁺ homeostasis by an RNAi-mediated Mrs2 KD in HeLa cells. The best miRNA to knockdown MRS2 expression was selected by comparing loss of Mrs2 mRNA expression in HeLa cells with quantitative real time RT-PCR after 3 days of transfection of miR expression vector (Fig. S1). The miR expression vector #1 was optimal for Mrs2 KD in HeLa cells and it was used for Mrs2 KD.

Effects of Mrs2 KD on Intracellular Mg²⁺ Homeostasis

MRS2 is primary Mg²⁺ uptake machinery in mitochondria^31,32,34. To assess the effects of Mrs2 KD on [Mg²⁺]_mito homeostasis, [Mg²⁺]_mito was compared by using a ratiometric Mg²⁺ indicator Mag-Fura-2. The cell membrane permeabilization protocol was used for the quantification of the [Mg²⁺]_mito³⁶. Briefly, after loading Mag-Fura-2, cytosolic Mag-Fura-2 was washed out by cell membrane permeabilization with a detergent digitonin. While Mag-Fura-2 is normally used for the measurement of [Mg²⁺]_cyto (Fig. 1a–c), the co-localization of Mag-Fura-2 and a mitochondrial marker MitoFluor Red signals was observed after Mag-Fura-2 wash out from cytosol by digitonin treatment (Fig. 1d–f), indicating that this cell membrane permeabilization protocol by using Mag-Fura-2 enables mitochondrial Mg²⁺ measurement. [Mg²⁺]_mito in Mrs2 KD cells was lower than that in normal cells (Fig. 1g). Mrs2 KD cells were identified by expression of emGFP also coded in miRNA expression vector. [Mg²⁺]_cyto was also estimated with Mag-Fura-2 in normal usage (Fig. 1h–j). The comparative analysis revealed the Mrs2 KD induced an increase in [Mg²⁺]_cyto (Fig. 1k). These results indicate that Mrs2 KD in HeLa cells induces a decrease in [Mg²⁺]_mito and an increase [Mg²⁺]_cyto.

Effects of Mrs2 KD on Mitochondrial Mg²⁺ Regulatory Systems

The alternation in intracellular distribution of Mg²⁺ level in Mrs2 KD cells indicates that MRS2 maintain mitochondrial Mg²⁺ concentration. To investigate the roles of MRS2 on stimulation-induced intracellular Mg²⁺ redistribution, we visualized cytosolic and mitochondrial Mg²⁺ dynamics during stimulation with a mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP; 5 μM) in HeLa cells loaded with highly sensitive cytosolic Mg²⁺ indicator KMG-104 (Fig. 2b,c) or mitochondrial Mg²⁺ indicator KMG-301 (Fig. 2e,f). To distinguish Mrs2 KD cells from control cells, Mrs2 miR expression vector coding tagBFP was used. As both control and tagBFP-labeled Mrs2 KD cells existed in an observed area of single experiments, the traces of [Mg²⁺]_cyto and [Mg²⁺]_mito in both control and Mrs2 KD cells for comparative analysis were simultaneously obtained in the same batch experiments (Fig. 2a–f). As shown in previous studies reporting FCCP-induced Mg²⁺ release from mitochondria into the cytosol^22,25,26,29, the application of FCCP induced [Mg²⁺]_cyto increase (Fig. 2g). Measurement of [Mg²⁺]_cyto dynamics using KMG-104 revealed that the averaged amplitude of FCCP-induced [Mg²⁺]_cyto increase in 2–4 min (initial phase of [Mg²⁺]_cyto increase) in Mrs2 KD cells was smaller than that in control cells (Fig. 2i, left), supporting the idea that the amount of Mg²⁺ stored in mitochondria before FCCP treatment is lower in Mrs2 KD cells. The [Mg²⁺]_cyto increase was sustained at least for 8 min in Mrs2 KD cells, whereas [Mg²⁺]_cyto in control cells returned back to the same level of [Mg²⁺]_cyto in Mrs2 KD cells in 6–8 min (Fig. 2i, right). In [Mg²⁺]_mito imaging using KMG-301 (Fig. 2h), in 2–3 min (initial phase of [Mg²⁺]_mito decrease), the averaged amplitudes of FCCP-induced [Mg²⁺]_mito decrease in both cells were not different (Fig. 2j, left). During 6–7 min, the averaged amplitudes of FCCP-induced [Mg²⁺]_mito decrease in Mrs2 KD cells was larger than that in control cells (Fig. 2j, right). These results also indicate that, after the initial phase of FCCP-induced mitochondrial Mg²⁺ release, mitochondria reuptake the released cytosolic Mg²⁺ through MRS2 and mitochondria in Mrs2 KD cells failed it. Taken together, MRS2 plays a role as the mitochondrial Mg²⁺ uptake systems in mammalian cells and Mrs2 KD causes the deletion of mitochondrial Mg²⁺ uptake in both steady state and Mg²⁺ mobilization.

Suppression of TCA Cycle Induced by Disruption of Mitochondrial Mg²⁺ Homeostasis

To directly assess the effect of Mrs2 KD-induced homeostatic malfunction of mitochondrial Mg²⁺ on global metabolism and, in particular, mitochondrial energy generation, the metabolomics of Mrs2 KD and control cells were investigated using capillary electrophoresis mass spectrometry (CE-MS) technique. CE-MS comprehensively quantify metabolites in biological samples³⁷. We examined the differences in metabolite levels between control and Mrs2 KD cells. We quantitatively identified a total of 133 metabolites (control and Mrs2 KD samples, n = 6 respectively) and 24 metabolites were found to significantly differ between control and Mrs2 KD samples, which are overviewed in Fig. 3a (pathway information was obtained by reference to KEGG [http://www.genome.jp/kegg/pathway.html] and a previous report³⁸). Especially, most metabolites of TCA cycle such as malate, citrate, cis-aconitate and succinate, were reduced in Mrs2 KD cells (Fig. 3b). These results suggest that dysregulation of mitochondrial Mg²⁺ homeostasis causes suppression of TCA cycle turnover in mitochondria. Despite suppression of TCA cycle activity, NADH/NAD⁺ was not affected in Mrs2 KD cells (Fig. 3b). No effect on product of TCA cycle suggests that the direct impact of Mrs2 KD is downstream of TCA cycle in mitochondrial energy production.

Mrs2 KD Disrupt the Mitochondrial Membrane Potential

Using electron released from NADH and FADH₂ produced in TCA cycle, proton gradient is generated across mitochondrial inner membrane via electron-transport chain, which result in generation of mitochondrial membrane potential (ΔΨ). To assess the ΔΨ, we quantified the ratio (red to green) of average mitochondrial fluorescence intensity of 5, 5′, 6, 6′-terachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) per cells (Fig. 4a). In Mrs2 KD HeLa cells, the red-to-green ratio of JC-1 fluorescence, which indicates the mitochondrial inner membrane potential, was lower than control cells (Fig. 4b). This result suggests the mitochondrial Mg²⁺ uptake through MRS2 is crucial for maintaining the ΔΨ.

Mrs2 KD Disrupt the Energy Metabolism in Mitochondria

In mitochondria, ATP is made from ADP and phosphate by ATP synthase using an electrochemical gradient of protons across the mitochondrial inner membrane. Therefore, inside-negative ΔΨ across the mitochondrial inner membrane is crucial for maintaining the physiological function of the oxidative phosphorylation to generate ATP. To access the intracellular energy status, we performed live imaging of cells expressing ATeam, which is genetically-encoded fluorescent resonance energy transfer (FRET)-based ATP indicator³⁹. In cytosol and nucleus, the ATeam ratio, which indicates ATP level, were lower in Mrs2 KD cells than that in control cells (Fig. 4c,e). In contrast to the decrease in extra-mitochondrial ATP levels, in Mrs2 KD cells, ATP level increased in mitochondria (Fig. 4d) despite decrease of TCA cycle turnover (Fig. 3) and ΔΨ_m (Fig. 4a,b), suggesting that dysregulation of mitochondrial Mg²⁺ causes the imbalances of ATP exports from mitochondria.

Dysregulation of Mitochondrial Mg²⁺ Affect the Mitochondrial Morphology

Mitochondria are highly dynamic organelle^12,40,41 and their morphological changes regulate cellular metabolic processes and vice versa¹². We assessed whether the malfunction of mitochondrial Mg²⁺ regulatory system affects mitochondrial morphology. To obtain the exact mitochondrial morphology, mitochondrion selective probe insensitive to ΔΨ, Mito Tracker Green FM, was loaded into both control and Mrs2 KD cells (Fig. 5b,c). To distinguish Mrs2 KD cells from control cells, Mrs2 miR expression vector coding tagBFP was used (Fig. 5a,c). Whereas the normal morphology of mitochondria in cells was tubular¹⁰ (Fig. 5d), the increased accumulation of rounding mitochondria was observed in Mrs2 KD cells (Fig. 5e). In stress condition, the abnormal accumulation of large swollen mitochondria is reported in previous studies^42,43. To quantitatively analyze the morphological difference of mitochondria between in control and in Mrs2 KD cells, a morphological feature was evaluated by computer-assisted image processing. The acquired images (Fig. 5f) were processed to generate a mitochondria-specific binary image (Fig. 5g) allowing the quantification of mitochondrial shape (Fig. 5h) as previously described⁴⁴. From quantitative morphological analysis of mitochondria, the aspect ratio (the ratio between the major and minor axes of the ellipse equivalent to the mitochondrial object) was calculated for each mitochondrion. The mitochondria in Mrs2 KD cells had lower aspect ratio than that in control cells (Fig. 5i), indicating that Mrs2 KD induced mitochondrial rounding.

Cell Vulnerability to Cellular Stress enhanced by Mitochondrial Mg²⁺ dysregulation

We revealed the dysregulation of mitochondrial Mg²⁺ results in the reduction of extra-mitochondrial ATP concentration. The negative effects of low ATP levels on cellular vulnerability are suggested⁴⁵. To investigate the physiological effects of inaccessibility to Mg²⁺ induced energy imbalance on cellular vulnerability to stress, the viabilities were compared under cellular stress conditions between control and Mrs2 KD cells by MTT assay. In Mrs2 KD cells, cell viabilities were lower than in control cells in the condition of 20 ng/mL TNF-α and 1 μg/mL cycloheximide (CHX) (Fig. 6a) and also 1 mM H₂O₂ conditions (Fig. 6b), respectively. These results indicate that dysregulation of mitochondrial Mg²⁺ homeostasis causes increased susceptibility under cellular stress conditions. Metabolome analysis revealed that, under H₂O₂ condition, ATP level in Mrs2 KD cells was lower than that in control cells (Fig. 6c), suggesting Mrs2 KD-induced lower ATP level cause cellular vulnerability against oxidative stress. Next, to investigate the effects of mitochondrial Mg²⁺ dysregulation on cellular metabolome under stress condition, the overall impact of Mrs2 KD was compared using principal component analysis (PCA) for all detected 104 metabolites among 4 conditioned samples; control and Mrs2 KD cells with/without H₂O₂. PCA revealed that the clusters of control and Mrs2 KD cells under H₂O₂ condition were more separated than in normal condition, indicating that Mrs2 KD-induced defection of mitochondrial ATP synthesis had larger impacts under stress conditions (Fig. 6d).

Discussion

We demonstrated that deficit of MRS2, which is a mitochondrial primary Mg²⁺ regulatory system, causes disruption of mitochondrial energy metabolism and cellular sensitization against cellular stress. First, we confirmed that Mrs2 KD causes malfunction of mitochondrial Mg²⁺ uptake (Fig. 2) and reduction of Mg²⁺ stored in mitochondria (Fig. 1). Second, we revealed that Mrs2 KD induces decreases in substrates of TCA cycle (Fig. 3), ΔΨ and extra-mitochondrial ATP levels (Fig. 4) in contrast increased mitochondrial ATP level (Fig. 4). In addition, we observed that mitochondria in Mrs2 KD cells have abnormal morphology (Fig. 5). Lastly, we showed that the effect of Mrs2 KD was noticeable under stress conditions, which sensitized cells to cellular stress (Fig. 6). These results indicate that mitochondrial Mg²⁺ regulate the cellular energy status and it changes mitochondrial morphology and affects cell vulnerability against biological stress.

Physiological Significance of mitochondrial Mg²⁺ homeostasis

Although partial information has been accumulated about Mg²⁺-dependent regulation of energy metabolism, the comprehensive effects of mitochondrial Mg²⁺ on metabolic status in living cells have not been elucidated. As far as we know, this is the first work to demonstrate a series of relationships between Mg²⁺ regulatory system and cellular energy metabolism. In Mrs2 KD cells, in contrast to decreased substrates of TCA cycle (Fig. 3), collapse of ΔΨ_m and decreased extra-mitochondrial ATP levels (Fig. 4), mitochondrial ATP level are increased (Fig. 4). These results indicate the mitochondrial dis-accessibility to Mg²⁺ suppress ATP efflux from mitochondria, which is possibly mediated by ATP-Mg/P_i carrier⁶. Mitochondrial ATP accumulation inhibits many enzymatic processes in TCA cycle⁴⁶ and electron transport chain activities⁵ in a negative feedback manner. Suppression of TCA cycle and electron transport chain activities would result in reduced ATP production in mitochondria. Consequently, mitochondrial Mg²⁺ regulates coupled reactions in mitochondrial energy metabolism, i.e. TCA cycle, electron transport chain and ADP/ATP translocation. In addition, morphological changes of mitochondria were also observed in Mrs2 KD cells (Fig. 5). Abnormal large and round mitochondria are also observed under pharmaceutically ATP synthesis-inhibited condition⁴⁷. These are consistent with the idea that mitochondrial morphology is controlled by energy metabolism⁴⁸. Abnormal mitochondrial morphology is associated with cancer¹¹ obesity, type 2 diabetes¹² and neurodegenerative disorders^12,13,14. It may be explained by the idea that metabolic impairment induces cellular vulnerability⁴⁵. Actually, sensitization against cellular stress by mitochondrial Mg²⁺ dysregulation was observed in Mrs2 KD cells (Fig. 6). In contrast, in a cellular model experiments of Parkinson’s disease, increase in [Mg²⁺]_cyto, which probably links to [Mg²⁺]_mito increase, protects cells from neurodegeneration by maintaining cellular ATP concentration and suppressing ROS production²⁸. In summary, mitochondrial Mg²⁺ regulate the cellular metabolic process via shift of mitochondrial energy metabolism and it changes mitochondrial morphology and affects the cell viability through changing stress susceptibility.

In normal cells, mitochondrial Mg²⁺ would play a role as a regulator of metabolic state under physiological condition. A wide variety of hormonal regulations of intracellular Mg²⁺ homeostasis has been reported⁴⁹. In human, circadian rhythm for the serum Mg²⁺ level with the peak around noon are reported⁵⁰, which is corresponding to circadian Mg²⁺ excretory rhythm with the peak at night⁵¹. In facts, a recent study revealed circadian rhythms in the intracellular [Mg²⁺] regulate cellular metabolism⁵². Because Mg²⁺ transport through MRS2 depends on extra-mitochondrial Mg²⁺ concentration⁵³, mitochondrial Mg²⁺ homeostasis is also probably governed by circadian regulation. In addition, we recently demonstrated mitochondrial Mg²⁺ regulation mediated by second messenger pathways²⁶. These studies support that cells dynamically regulate [Mg²⁺]_mito in response to various signals and circadian rhythm under physiological condition. A respiration-dependent mitochondrial Mg²⁺ regulation⁵⁴ is consistent with the coupling of [Mg²⁺]_mito regulation with mitochondrial activities. Consequently, cells would modulate energy production and metabolic states via regulation of mitochondrial Mg²⁺ in response to environmental information.

Methods

HeLa cells culture

HeLa cells were seeded in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 50 U/mL penicillin and 50 mg/mL streptomycin (Life technologies, Carlsbad, CA, USA) and cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were plated on glass-bottomed dishes (Iwaki, Tokyo, Japan), 100 mm cell culture dishes (Thermo Fisher Scientific, Waltham, MA, USA), or 48 well dishes (Thermo Fisher Scientific) for fluorescence imaging, metabolomics analysis or MTT assay, respectively. The medium was changed in every other day.

Knockdown of Mrs2 in HeLa cells

For knockdown of Mrs2, BLOCK-iT^TM Pol II miR RNAi Expression Vector Kits (Life technologies) was used. BLOCK-iT RNAi Designer from Life Technologies was used to design four single-stranded DNA oligonucleotides encoding the target pre-miRNA. These vectors are specifically designed to allow expression of miRNA sequences and contain specific miR flanking sequences that allow proper processing of the miRNA. Sequences of pre-miRNA insert in miRNA vectors for knockdown were designed as follow:

5′-GTAGCTACTGATTCCAAGAGTAGTTTTGGCCACTGACTGACTACTCTTGATCAGTAGCTA-3′ (Mrs2 KD #1),

5′-GAATGCTAGCTACTGATTCCAAGTTTTGGCCACTGACTGACTTGGAATCTAGCTAGCATT-3′ (Mrs2 KD #2),

5′-GTGAAGAATGCTAGCTACTGATGTTTTGGCCACTGACTGACATCAGTAGAGCATTCTTCA-3′ (Mrs2 KD #3), and

5′-GTTTGAAGAATGCTAGCTACTGGTTTTGGCCACTGACTGACCAGTAGCTCATTCTTCAAA-3′ (Mrs2 KD #4), respectively.

To identify the cells transfected with microRNA expression vector for Mrs2 knockdown, the sequence coding EmGFP or tagBFP were incorporated into the vectors. The miR RNAi vectors were transfected into cells 3 days prior to the experiments using Lipofectamine LTX (Life technologies) in the experiments except preparation for metabolome analysis.

Real time RT-PCR

To determine Mrs2 mRNA levels in miR transfected cells, total RNA from HeLa cells were isolated and purified by using the RNeasy mini kit (QIAGEN, Tokyo, Japan). Total RNA was reverse-transcripted using SuperScript VILO (Life technologies) and the generated cDNA was used as template for quantitative real time PCR amplification with SYBR GreenER^TM (Life technologies). The Mrs2 mRNA levels were normalized to GAPDH signals as an internal standard. Quantitative real time PCR were performed using the following primers: Mrs2 forward 5′-CAGTTGTCTGGAGAGGGTCAA-3′, Mrs2 reverse 5′-AAGGATCAGTGGCTGCAAAA-3′, GAPDH forward 5′-CACCCACTCCTCCACCTTTG-3′ and GAPDH reverse 5′-CATGAGGTCCACCACCCTGT-3′.

Fluorescence imaging of the cytosolic or mitochondrial Mg²⁺ levels

For the quantification of cytosolic Mg²⁺ level in cells, HeLa cells were loaded with Mag-Fura-2-AM (Life technologies). For dye loading, HeLa cells were incubated in medium with 1 μM and 0.02% F-127 for 45 min at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were gently washed twice with 1.0 mL of Hanks’ balanced salt solution (HBSS) at pH7.4 (adjusted with NaOH) that consisted of (in mM) 137 NaCl, 5.4 KCl, 1.3 CaCl₂, 0.5 MgCl₂, 0.4 MgSO₄, 0.3 Na₂HPO₄, 0.4 KH₂PO₄, 4.2 NaHCO₃, 5.6 D-glucose, 5.0 HEPES. Then, further incubation was carried out for 15 min to allow for complete de-esterification of AM esters. For the measurement of the emitted fluorescence values at the excitation of 340 nm and 380 nm, a fluorescence microscope ECLIPSE TE300 (Nikon, Tokyo, Japan) equipped with a ×20 objective (S Fluor, Nikon) was used. A Xe lamp (150 W) with a monochromator unit was used for 340 nm and 380 nm excitation and fluorescence was measured with a CCD camera (HiSCA, Hamamatsu Photonics, Shizuoka, Japan). The ratios of the fluorescence with excitation at 340 nm to that at 380 nm were calculated as an indicator of the cytosolic Mg²⁺ levels. For the detection of Mrs2 KD cells, emGFP was illuminated with the excitation at 488 nm. Region of interest (ROI) was located on the respective cell areas. In each ROI, spatial averaged ratios of the emitted fluorescence with excitation at 340 nm to that at 380 nm was calculated as an indicator of the Mg²⁺ levels.

For the quantification of mitochondrial Mg²⁺ level in cells using ratiometric Mg²⁺ indicator Mag-Fura-2, the cell permeabilization method using digitonin was performed as described previously³⁶. Briefly, cells were stained with Mag-Fura-2 as described above. Cells loaded with Mag-Fura-2 were permeabilized with 20 μg/mL digitonin in intracellular-like medium (ICM; 120 mM KCl, 10 mM NaCl, 1 mM KH₂PO₄, 20 mM HEPES-Tris at pH7.2 and 2 mM MgATP) buffer for 3 min, followed by washout of the released cytosolic Mag-Fura-2 with ICM. For the confirmation of mitochondrial localization and leakage from cytosol of Mag-Fura-2, fluorescence imaging was conducted with a confocal laser scanning microscope system (FluoViewFV1000; Olympus, Tokyo, Japan) mounted on an inverted microscope (IX81; Olympus) equipped with a ×60 oil objective. For the measurements Mag-Fura-2 signals, the cells were illuminated with the excitation wavelength at 405 nm from diode laser and the signals from Mag-Fura-2 and Mito Fluor Red were separated using a 560 nm dichroic mirror. The fluorescence images were obtained by detecting the signals at 500–550 nm for Mag-Fura-2 and at 570–670 nm for Mito Fluor Red, respectively. For the measurement of the emitted fluorescence values at the excitation of 340 nm and 380 nm, a fluorescence microscope ECLIPSE TE300 equipped with a ×20 objective (S Fluor) was used. The procedure of Mag-Fura-2 detection was mentioned above. A ROI is located on the all spotted area with strong intensities in each cell (Fig. 1D).

Real time imaging of cytosolic Mg²⁺ dynamics

For real time imaging of cytosolic Mg²⁺ dynamics, highly selective Mg²⁺ fluorescent dye KMG-104AM was used²⁴. For loading KMG-104, cells were incubated with 5 μM KMG-104AM and 0.02% F-127 in pH adjusted HBSS for 30 min at 37 °C. Then, the cells were washed twice with HBSS and incubated in HBSS for 15 min at 37 °C in a humidified atmosphere containing 5% CO₂ to allow for complete hydrolysis of the acetoxymethyl ester form. Fluorescence imaging was conducted with a confocal laser scanning microscope system, FV1000, equipped with a ×40 oil objective. For the measurements of KMG-104 signals, the cells loaded with KMG-104 were illuminated with the excitation wavelength at 488 nm from argon (Ar) laser. The fluorescence was obtained by detecting the signals at 500–600 nm. For the detection of tagBFP-labeled Mrs2 KD cells, the cells were illuminated with the excitation wavelength at 405 nm from diode laser and its fluorescence was obtained by detecting the signals at 425–475 nm.

Real time imaging of mitochondrial Mg²⁺ dynamics

For real time imaging of mitochondrial Mg²⁺ dynamics, HeLa cells were stained with 20 μM highly selective mitochondrial Mg²⁺ fluorescent dye KMG-301AM in pH adjusted HBSS for 10 min on ice, so that hydrolysis of the acetoxymethyl ester by esterase present in the cytosol would be avoided²². Then, the cells were washed twice with HBSS and incubated in HBSS for 15 min at 37 °C in a humidified atmosphere containing 5% CO₂ to allow for complete hydrolysis of the acetoxymethyl ester form in mitochondria. The detailed property and usage of KMG-301AM was described in our previous report²². Fluorescence imaging was conducted with a confocal laser scanning microscope, FV1000, equipped with a ×40 oil objective. The cells loaded with KMG-301 were illuminated with the excitation wavelength at 559 nm from diode laser and its fluorescence was obtained by detecting signals at 570–670 nm. For the detection of tagBFP-labeled Mrs2 KD cells, the cells were illuminated with the excitation wavelength at 405 nm from diode laser and its fluorescence was obtained by detecting the signals at 425–475 nm.

Fluorescence imaging of the intracellular ATP levels

For the quantification of intracellular ATP levels in living cells, ATeam1.03³⁹ was transfected into HeLa cells using Lipofectamine LTX. The transfection was conducted the day before observation. Fluorescent imaging was conducted with a confocal laser scanning microscope system equipped with a ×20 air objective. For the measurements of ATeam signals, the cells were illuminated with the excitation wavelength at 440 nm from diode laser and the signals from mseCFP and cp173-mVenus were separated using a 510 nm dichroic mirror and obtained at 480–510 nm for mseCFP and at 535–565 nm for cp173-Venus, respectively. The ratios of cp173-mVenus to mseCFP signals were calculated as an indicator of the cytosolic ATP levels.

Fluorescence imaging of the mitochondrial membrane potentials

For the quantification of mitochondrial membrane potential in living cells, HeLa cells were loaded with mitochondrial membrane potential sensitive dye 5, 5′, 6, 6′-terachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Life technologies). For dye loading, HeLa cells were incubated in pH adjusted HBSS with 10 μg/mL JC-1 for 15 min at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were gently washed twice with 1.0 mL of HBSS. Fluorescent imaging was conducted with a confocal laser scanning microscope system, FV1000, equipped with a ×20 air objective. For the measurements of JC-1 signals, the cells were illuminated with the excitation wavelength at 488 nm from Ar laser and the signals from JC-1 were separated using a 560 nm dichroic mirror and obtained by detecting the signals at 520–560 nm for green channel and at 575–620 nm for red channel, respectively. The ratio of signals from red channels to that from green channels was calculated as an indicator of the mitochondrial membrane potential. For the detection of tagBFP-labeled Mrs2 KD cells, the cells were illuminated with the excitation wavelength at 405 nm from diode laser and its fluorescence images were obtained by detecting the signals at 425–475 nm.

Measurement of mitochondrial morphology

For assessment of mitochondrial morphology in living cells, mitochondria were stained with Mito Tracker Green FM and their morphology were quantified by handmade digital image processing software using MATLAB (MathWorks, Cambridge, UK). The algorithm for quantification of morphological feature is previously described⁴⁴. Briefly, mitochondrial binary images were obtained from Mito Tracker Green FM-stained images and aspect ratio of each mitochondrion was calculated as the ratio between the major and minor axes of the ellipse equivalent to the mitochondrial object.

Measurement of cell viability

For quantification of vulnerability against cellular stress, cell viabilities under the stress inducer H₂O₂- or TNFα/CHX-treated condition were measured using MTT assay. Control and Mrs2 KD cells were treated with H₂O₂ (1 mM) or TNFα (20 ng/mL) plus cycloheximide (CHX; 1 μg/mL) for 24 h. Then, the cells were incubated in the medium containing 0.5 mg/mL of MTT for 2 h at 37 °C in a humidified atmosphere containing 5% CO₂. Then, the medium was removed and 100 μL of dimethylsulfoxide (DMSO, nacalai tesque, Kyoto, Japan) was added in each well to dissolve the precipitation. The absorbance at 570 nm under stress condition (ABS_stress) and normal condition (ABS_control) were measured using a microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific). Cell viability was defined as the ABS_stress/ABS_control.

Image analysis and statistics

The fluorescence was calculated as the mean intensity over a ROI on the cell body of each cell using the software package, FluoView (Olympus), Aquacosmos (Hamamatsu Photonics) or handmade software by MATLAB.

Metabolome analysis

The cells were transfected with a plasmid for Mrs2 knockdown by electroporation using Neon (Life Technologies). The cells were plated on a 100 mm dish and grown for 3 days at 37 °C in a humidified atmosphere containing 5% CO₂. Culture medium was changed every day. Procedure for sample preparation of metabolome analysis was previously described³⁷. Briefly, after washing cells twice with ice-cold 5% mannitol, metabolites were extracted by 1 mL ice-cold methanol containing internal standards (25 μM each of methionine sulfone (MetSul; Wako, Osaka, Japan), 2-(N-morpholino)ethanesulfonic acid (MES; wako), D-Camphor-10-sulfonic acid (CSA; Wako). 400 μL of collected extracts were transferred into another tube, mixed with 400 μL chloroform and 200 μL Milli-Q water and centrifuged at 10,000 × g for 3 min at 4 °C. A 400 μL aliquot of the aqueous layer was centrifugally filtered through a 5 kDa cutoff membrane (UltrafreeMC-PLHCC for Metabolome Analysis; Human Metabolome Technologies, Yamagata, Japan) to remove proteins from samples, followed by the centrifugal-concentration at 42 °C. CE-MS experiments were performed using Agilent CE Capillary Electrophoresis System.

Additional Information

How to cite this article: Yamanaka, R. et al. Mitochondrial Mg²⁺ homeostasis decides cellular energy metabolism and vulnerability to stress. Sci. Rep. 6, 30027; doi: 10.1038/srep30027 (2016).

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Acknowledgements

The authors are grateful to Dr. H. Imamura for the generous gift of intracellular ATP indicator ATeam 1.03. This work was supported by Grant-in-Aid for Scientific Research, KAKENHI (24240045, 16H01751) and Ministry of Education, Culture, Sport, Science and Technology, Japan (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities in 2014–2018 (S1411003).

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Center for Biosciences and Informatics, School of Fundamental Science and Technology Graduate School of Science and Technology, Keio University, Yokohama, 223-8522, Kanagawa, Japan
Ryu Yamanaka, Yutaka Shindo, Kohji Hotta & Kotaro Oka
Institute for Advanced Biosciences, Keio University, Kakuganji, Tsuruoka, 997-0052, Yamagata, Japan
Sho Tabata & Tomoyoshi Soga
Center for Science and Technology for Designing Functions, School of Integrated Design Engineering, Graduate School of Science and Technology, Keio University, Yokohama, 223-8522, Kanagawa, Japan
Koji Suzuki

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Ryu Yamanaka
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Sho Tabata
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Yutaka Shindo
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Kohji Hotta
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Koji Suzuki
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Tomoyoshi Soga
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Kotaro Oka
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Contributions

R.Y., Y.S., K.H. and K.O. designed the research. R.Y. and S.T. performed the experiments and analyzed the data. K.S. designed and synthesized the compounds. R.Y., Y.S. and K.O. designed and wrote the paper. T.S. and K.O. supervised the project. All authors participated in the discussion of the data and in production of the final version of the manuscripts.

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Yamanaka, R., Tabata, S., Shindo, Y. et al. Mitochondrial Mg²⁺ homeostasis decides cellular energy metabolism and vulnerability to stress. Sci Rep 6, 30027 (2016). https://doi.org/10.1038/srep30027

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Received: 04 May 2016
Accepted: 28 June 2016
Published: 26 July 2016
DOI: https://doi.org/10.1038/srep30027

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