Alteration in mitochondrial thiol enhances calcium ion dependentmembrane permeability transition and dysfunction in vitro:a cross-talk between mtThiol, Ca2+, and ROS

Brijesh Kumar Singh • Madhulika Tripathi •

Pramod Kumar Pandey • Poonam Kakkar

Received: 24 February 2011 / Accepted: 28 May 2011 / Published online: 12 July 2011

� Springer Science+Business Media, LLC. 2011

Abstract Mitochondrial permeability transition (MPT)

and dysfunctions play a pivotal role in many patho-physi-

ological and toxicological conditions. The interplay of

mitochondrial thiol (mtThiol), MPT, Ca2? homeostasis,

and resulting dysfunctions still remains controversial

despite studies by several research groups. Present study

was undertaken to ascertain the correlation between Ca2?

homeostasis, mtThiol alteration and reactive oxygen spe-

cies (ROS) in causing MPT leading to mitochondrial dys-

function. mtThiol depletion significantly enhanced Ca2?

dependent MPT (swelling) and depolarization of mito-

chondria resulting in release of pro-apoptotic proteins like

Cyt c, AIF, and EndoG. mtThiol alteration and Ca2?

overload caused reduced mitochondrial electron flow,

oxidation of pyridine nucleotides (NAD(P)H) and signifi-

cantly enhanced ROS generation (DHE and DCFH-DA

fluorescence). Studies with MPT inhibitor (Cyclosporin A),

Ca2? uniport blocker (ruthenium red) and Ca2? chelator

(BAPTA) indicated that mitochondrial dysfunction was

more pronounced under dual stress of altered mtThiol and

Ca2? overload in comparison with single stress of

excessive Ca2?. Transmission electron microscopy con-

firmed the changes in mitochondrial integrity under stress.

Our findings suggest that the Ca2? overload itself is not

solely responsible for structural and functional impairment

of mitochondria. A multi-factorial cross-talk between

mtThiol, Ca2? and ROS is responsible for mitochondrial

dysfunction. Furthermore, minor depletion of mtThiol was

found to be an important factor along with Ca2? overload

in triggering MPT in isolated mitochondria, tilting the

balance towards disturbed functionality.

Keywords Calcium overload � Membrane permeability

transition � Mitochondrial thiol � Reactive oxygen species

Introduction

Mitochondria are one of the most important organelle, as

they are involved in ATP synthesis which is a pre-requisite

for basal cellular activities, and also regulate apoptotic and

necrotic cell death. Although mitochondria stores Ca2? (to

maintain Ca2? homeostasis) [1, 2], but under conditions of

Ca2? overload accompanied by other factors, most notably

oxidative stress, high phosphate concentrations or low

adenine nucleotide concentrations, mitochondria undergo

rapid membrane permeability transition (MPT) [3–5].

Precisely, pores in the inner membrane open (known as the

MPT pore constituted by voltage dependent anion channel

(VDAC) in outer membrane and adenine nucleotide

translocator (ANT) in inner membrane), causing the

membrane to become non-specifically permeable to any

molecule less than 1.5 kDa including protons, thus making

mitochondria uncoupled, leading to loss in pH gradient or

membrane potential (DWm) [6]. These functionally

impaired mitochondria are not only incapable of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11010-011-0908-0) contains supplementarymaterial, which is available to authorized users.

B. K. Singh � M. Tripathi � P. Kakkar (&)

Herbal Research Section, Indian Institute of Toxicology

Research (CSIR), Formerly-Industrial Toxicology Research

Centre, Mahatma Gandhi Marg, P. O. Box-80, Lucknow 226001,

Uttar Pradesh, India

e-mail: poonam_kakkar@yahoo.com

B. K. Singh � P. K. Pandey

Department of Biotechnology, J.C. Bose Institute of Life

Sciences, Bundelkhand University, Jhansi 284128,

Uttar Pradesh, India

123

Mol Cell Biochem (2011) 357:373–385

DOI 10.1007/s11010-011-0908-0

maintaining their volume and ATP synthesis but also

actively degrade ATP followed by early apoptosis, cellular

necrosis, and ultimately tissue damage [6]. Along with

DWm, the role of mitochondrial swelling (increase in

mitochondrial volume) also needs to be elucidated. A

growing body of evidences suggests that mitochondrial

swelling is only an indication of cell injury and not rep-

resent final stage of mitochondrial dysfunction but plays a

crucial role in cytotoxicity [5]. DWm also plays an

important role in controlling movement of biologically

important molecules across the membrane and is often

associated with apoptosis involving reactive oxygen spe-

cies (ROS) [5, 6].

Free radicals are generated during normal cellular

metabolism and these ROS cause damage to normal cells.

Hence, a balance between pro-oxidants/antioxidants is

critical for normal cellular functions. State of oxidative

stress is found critical in various patho-physiologies [7]. A

battery of antioxidants play an indispensable role in

maintaining the intracellular redox environment; especially

free thiols (majorly reduced glutathione, GSH, as non-

enzymatic antioxidant) being one of them. GSH, an

endogenous antioxidant which protects cells from free

radicals, is a major determinant of this intracellular redox

potential. Although several other reducing couples such

as NADP?/NADPH and thioredoxin (TrxSS/Trx(SH)2)

helps in maintaining the same, GSH concentration is

100–10,000-fold greater than other couples, therefore is a

determining factor of the redox potential [8]. GSH is dis-

tributed mainly between cytosolic and mitochondrial

compartments at similar concentrations, i.e., 5–10 mM

(10–15% is found in the mitochondria versus 80–85% in

the cytosol) [9, 10]. The normal redox potential GSH/

GSSG is important to maintain normal physiology of many

proteins and enzymes involved in cell survival/death.

Mitochondria transiently stores Ca2?, and their ability to

rapidly take up Ca2? for later release makes them very

good cytosolic buffers. Ca2? is accumulated in the matrix

through the Ca2? uniporter on the inner mitochondrial

membrane [11]. Cellular Ca2? homeostasis as well as GSH

level is extremely important in regulation of human dis-

eases including diabetes, cancer, neuro-degenerative, car-

dio-vascular, and liver diseases [12]. Role of GSH in liver

diseases and hepatotoxicity is very well reviewed by Yuan

and Kaplowitz [8]. Most of liver diseases occur during

compromised hepatic GSH pool, through impairment of

synthesis or transport and/or over-consumption. Thus,

mtThiol alteration and Ca2? overload together may prove

to have more deleterious effects on human health.

Mitochondrial GSH uptake is critical for protection

against stress and the function of mtThiol in hepatic

mitochondrial redox maintenance is equally important.

Although efforts have been made to unravel the importance

of mitochondrial redox system in maintaining mitochon-

drial structural and functional integrity during Ca2? over-

load but the literature is insufficient to conclude any

significant correlation. In this study, authors have tried to

explore the role of mtThiol in Ca2? dependent MPT in

isolated mitochondria. Authors have successfully estab-

lished from the study that alteration in mtThiol amplifies

oxidative stress in Ca2? overloaded mitochondria. Also,

MPT is not solely dependent on Ca2? or mtThiol alteration

as independent phenomenon, but a minor alteration in

mtThiol under conditions of Ca2? overload enhances MPT

significantly. Thus, the study suggests a cross-talk between

mitochondrial Ca2? homeostasis, mtThiol and ROS.

Materials and methods

Materials

Anti-cytochrome c (Cyt c), apoptosis inducing factor

(AIF), and endonuclease G (EndoG), antibodies were

procured from Santa Cruz Biotechnology, Inc. Anti-VDAC

and b-actin antibodies were purchased from Sigma-

Aldrich, St. Louis, MO, USA and anti-cytochrome c oxi-

dase subunit-IV (Cyto Ox-IV) antibody from Abcam,

Cambridge, UK. Unless indicated, all other chemicals were

obtained from Sigma Chemicals Co. (St. Louis, MO,

USA). Solutions were prepared in de-ionized ultra-pure

water (Direct Q5, Millipore, Bangalore, India).

Animals

Mitochondria were isolated from male Sprague-Dawley

rats weighing 180 ± 20 g, which were procured from

Indian Institute of Toxicology Research (IITR) animal

colony. Rats were acclimatized for 1 week in IITR animal

house before euthanize. Animals were fed on standard

pellet diet (Ashirwad Pellet Diet, Mumbai, Maharashtra,

India) and water ad libitum. Rats were kept under standard

conditions of humidity (60–70%), temperature (25 ± 2�C),

and a controlled 12 h light/dark cycle. All the guidelines of

Institutional Animal Ethics Committee (ITRC/IAEC/20/

2006) were followed while handling the animals.

Methods

Isolation of rat liver mitochondria and protein estimation

Standardized mitochondria isolation protocol of Frezza

et al. [13] was followed with some modifications [14, 15].

1 g liver tissue from cervically dislocated rat was homog-

enized in 10 ml ice cold MSH buffer (10 mM HEPES, pH

7.5, 200 mM mannitol, 70 mM sucrose, 1.0 mM EGTA,

374 Mol Cell Biochem (2011) 357:373–385

123

and 2.0 mg/ml fatty acid free serum albumin). Resulting

10% (w/v) liver homogenate was centrifuged (Sigma-

3K18, Germany) at 5009g for 10 min. The supernatant

was again centrifuged at 15,0009g for 10 min at 4�C to

obtain crude mitochondrial pellet. The pellet was then

washed three times with ice cold homogenization buffer to

get contamination free mitochondria [16]. Final mito-

chondrial pellet was re-suspended in swelling buffer

(0.15 M KCl, 0.02 M Tris, pH 7.4; for mitochondrial

swelling) or suspension buffer (5 mM MOPES, containing

0.075 M sucrose, 0.225 M mannitol, and 1 mM EDTA, pH

7.4; for mitochondrial electron flow and flow cytometry) to

produce a suspension containing 30–40 mg mitochondrial

protein/ml [17]. 2 mg mitochondrial protein/ml was used to

observe mitochondrial swelling and 40 mg mitochondrial

protein/ml (unless stated otherwise) was used for other

enzymatic assays and flow cytometric preparations. After

giving treatments, samples were sonicated (3 cycles of 20 s

each with 5 s time interval) (Labsonic� M, Sartorius,

Germany) and centrifuged at 15,0009g for 10 min at 4�C.

The mitochondrial lysate thus obtained was then subjected

to further biochemical assays.

Transmission electron microscopy (TEM)

Electron microscopy was performed by the Department of

Pathology, Sanjay Gandhi Post Graduate Institute of

Medical Sciences, Lucknow, India. Samples were fixed in

2.5% glutaraldehyde for 2 h, followed by 100 mM caco-

dylate buffer, pH 7.4 for 1.5 h. Samples were then finally

fixed in 1% osmium tetroxide in 50 mM cacodylate buffer,

pH 7.4. Serial dehydration was performed in acetone after

fixation followed by infiltration with toluene and resin.

Ultra-thin sections were cut with an ultra-microtome (LKB

Ultrotome Nova, Leica, Broma, Sweden) then placed on

grid and stained with uranyl acetate and lead citrate. Grids

were analyzed on transmission electron microscope (Phi-

lips EM10 TEM, Germany) and negatives were developed

manually and digitized by scanning. Total mitochondrial

population was categorized into three groups according to

their morphology as follows: (i) intact/condensed mito-

chondria with well defined cristae (ii) swollen mitochon-

dria, and (iii) compromised mitochondria, i.e., vacuolated

matrix, degenerated cristae, and loss of mitochondrial

morphology. Mitochondria were counted in a blind fash-

ion. Morphological distribution was analyzed and plot-

ted against total number of mitochondria analyzed

(percentage).

Immunoblot analysis

Cyt c (as mitochondrial marker) and b-actin (as cytosolic

marker) were used to check purity of mitochondrial

fraction using immunoblotting. Mitochondria specific

proteins (Cyt c, EndoG, AIF, and VDAC) released from

mitochondria were also detected in mitochondrial super-

natant. Cyto Ox-IV was used as mitochondrial internal

loading control. Samples were electro-phoretically sepa-

rated on SDS-PAGE and electro-blotted on PVDF mem-

brane (Amersham Biosciences UK Limited, Bucks, NA).

Membrane was incubated for 2 h with primary antibody

and further incubated for 1 h with horse-radish peroxidase-

conjugated secondary antibody. Protein bands were visu-

alized using ImmobilonTM Western Chemiluminescent

HRP substrate kit (Millipore Corporation, MA, USA) and

quantification of bands was done using ImageJ (version

1.41o, NIH, USA).

Treatments (mtThiol alteration, oxidative stress generation

and Ca2? overload in isolated mitochondria)

N-Ethylmaleimide (NEM, an alkylating agent, which binds

with critical thiols; like GSH, and membrane proteins) is

widely used to alter thiol levels [18–20]. Selection of NEM

concentration was dependent on the lowest (0.1 mM) and

highest (1 mM) thiol alteration, which are important to

study any effect on mitochondria under dual stress. In our

study, selection of Ca2? concentration was based on

reported free Ca2? level in extracellular and cytosolic fluid

which is millimolar to sub micromolar range [21] that

caused significant swelling of mitochondria. t-BHP

(250 lM) caused *50% decrease in MTT activity and was

used as positive control for oxidative stress [22]. BAPTA

(intra-cellular Ca2? chelator; 10 lM), Ruthenium Red

(RuRed; an inhibitor of the mitochondrial Ca2? uniporter;

30 lM) and Cyclosporin A (CyA; an MPT inhibitor;

1 lM) were also used as negative controls for studies

involving Ca2? and MPT [15].

Mitochondrial thiol determination

mtThiol was measured according to Halvey et al. [23] and

Oyama et al. [24] using Cell Tracker Green 50-chlorom-

ethylflouroscein diacetate (CMF-DA; Molecular Probes,

Eugene, Oregon, USA). Samples were incubated for

30 min with the probe (1 lM) at 37�C in dark. Signals

were obtained using 530 nm band pass filter (FL-1 chan-

nel) for chloromethylfluorescien (CMF) on flow cytometer

(BD-LSR, Becton–Dickinson). Each determination is

based on mean fluorescence intensity of 10,000 events.

Determination of reduced mitochondrial pyridine

nucleotide [NAD(P)H] levels

Use of mitochondrial pyridine nucleotide fluorescence has

been very well correlated with mitochondrial function by

Mol Cell Biochem (2011) 357:373–385 375

123

Mayevsky and Barbiro-Michaely [25]. UV excited blue

autofluorescence was used to measure reduced pyridine

nucleotides, designated as NAD(P)H to represent contri-

butions by both NADH and NADPH [26]. Mitochondrial

samples were analyzed on flow cytometer after giving

treatments and signals were obtained at 455-nm band pass

filter (FL-5 channel).

ROS estimation in mitochondria

Level of ROS inside mitochondria was measured according

to Sharikabad et al. [27] by flow cytometry as the fluo-

rescence of dichlorofluorescein (DCF) and ethidium

(ETH). These are the oxidation products of 20,70,-dichlor-

ohydrofluorescien diacetate (DCFH-DA) and dihydroethi-

dium (DHE) with sensitivity for secondary ROS/RNS and

superoxide, respectively [24, 28, 29]. Samples were incu-

bated for 30 min with DCFH-DA (5 lg/ml) and DHE

(2.5 lg/ml) at 37�C in dark and analyzed on flow cytom-

eter. Signals were obtained using a 585-nm bandpass filter

(FL-2 channel) for ETH and a 530-nm band pass filter (FL-

1 channel) for DCF.

Determination of mitochondrial electron flow (MTT

reduction assay)

Study was carried out according to Cohen and Kesler [30]

with some modifications [31]. The samples were quenched

with DMSO and absorbance was measured at 592 nm.

ELISA plate reader (Synergy HT, Biotek, USA) was used to

read mitochondrial samples in 48-well plate.

Assessment of mitochondrial membrane potential (DWm)

DWm was assessed with JC-1 (5,50,6,60-tetrachloro-

1,10,3,30-tetraethylbenzemidazol carbocyanine iodide) dye

[15, 28]. It is a lipophilic cationic dye which is highly

specific for mitochondrial membrane. Samples were incu-

bated in dark for 30 min at room temperature with 2.5 lg/

ml of JC-1 dye. Signals were obtained using FL-1 (for

green monomer, depolarized) and FL-2 (for red J-aggre-

gate, polarized) channel settings on flow cytometer.

Determination of membrane permeability transition

(i.e., protein released from mitochondria)

Samples (20 mg mitochondrial protein) were incubated

with treatments for 10 min and centrifuged at 15,0009g for

10 min at 4�C. Mitochondrial supernatant was collected

and pellet was dissolved in 500 ll deionized water fol-

lowed by sonication (3 cycles of 20 s each with 5 s time

interval) to obtain mitochondrial lysate. These samples

were subsequently used to quantitate (Lowry protein esti-

mation) released proteins from mitochondria to the corre-

sponding supernatant.

Determination of mitochondrial swelling

Mitochondrial swelling was carried out as described earlier

[32]. Mitochondrial swelling was assessed by measuring

90� light scattering at 520 nm using Ultrospec 3100pro

(Amersham Biosciences, Sweden) UV/Visible spectro-

photometer at 25�C. 2 mg mitochondrial protein/ml was

used in the 2 ml swelling buffer. The swelling was recor-

ded as decrease in the absorbance at every 30 s time

interval for a period of 10 min.

Determination of calcium ion flux across mitochondrial

membrane

Mitochondrial Ca2? flux was analyzed using Fluo-4

DirectTM Calcium Assay Kit (Molecular Probes, Eugene,

Oregon, USA). Experiment was performed according to the

manufacturer’s protocol. Mitochondria were pre-treated

with BAPTA, RuRed or CyA for 10 min or used directly

with NEM or Ca2? treatment. Fluorescence was assessed

using spectrofluorometer (Varioskan Flash 4.00.53 equip-

ped with SkanIt Software 2.4.3.37 Research Edition;

Thermo Fisher Scientific, Finland) using Ex/Em-495/

516 nm wavelength.

Statistical analysis of data

The data are reported as mean ± SD. The probability values

(P) were derived using ANOVA followed by Student’s t test

(two-tailed distribution with unequal variance) (SPSS 14.0

for Windows, USA), wherein the differences were consid-

ered to be significant at *P \ 0.05, **P \ 0.01, and

***P \ 0.001, when compared to control.

Results

Assessment of quality of mitochondrial fraction

Quality and integrity of rat liver mitochondria (RLM) after

isolation was assessed using transmission electron

microscopy (Fig. 1a, b), where mitochondria retained their

normal morphology, i.e., well defined cristae structure and

intact outer membrane. Immunoblot analysis (Fig. 1c),

indicated that inter-membrane space protein cytochrome

c (Cyt c) was retained in mitochondria confirming intact-

ness of outer mitochondrial membrane. Loose mitochon-

drial fraction was separated and discarded.

376 Mol Cell Biochem (2011) 357:373–385

123

Ca2? enhances mitochondrial thiol (mtThiol) depletion

and oxidative stress

Different concentrations of NEM (0.1 and 1 mM) were

used to alter mtThiol. mtThiol was also determined under

Ca2? overloaded (100 lM; CaCl2) and oxidatively stressed

(t-BHP, 250 lM) conditions (Table 1A). At 0.1 mM con-

centration of NEM, only 25.11% (P \ 0.05) decrease in

mtThiol was observed which decreased further up to

80.73% (P \ 0.01) at 1 mM concentration (data not

shown). In the presence of Ca2? (100 lM), mtThiol was

depleted significantly (39.64%; P \ 0.05). However, under

combined stress of Ca2? (100 lM CaCl2) and NEM

(0.1 mM), 81.39% (P \ 0.01) mtThiol was depleted. It was

observed that alteration in mtThiol increased significantly

when Ca2? loaded mitochondria were subjected to NEM

treatment.

mtThiol alteration causes oxidation of pyridine

nucleotides (NADH/NADPH) under Ca2? overload

Apart from mtThiol, NADH, and NADPH are major

sources of cellular reducing equivalents. To determine

whether mtThiol alteration in Ca2? overloaded

Fig. 1 Purity of mitochondrial fraction. Purity of mitochondrial

fraction was determined using transmission electron microscopy and

immunoblotting. a, b Electron micrograph of ultra-thin section of

control (untreated) rat liver mitochondria (RLM) is shown. Mito-

chondria show an interconnected network structure with numerous

regularly arranged cristae, i.e., intact/condensed mitochondria

(a 910,500 and b 925,000). Immunoblot is shown in c. Marker of

mitochondrial fraction was Cyt c, and for cytosolic fraction, b-actin.

Both the proteins were analyzed in three fractions, (a) cytosolic

fraction, (b) loose mitochondrial fraction (which was discarded during

the preparation of pure mitochondria), and (c) mitochondrial fraction,

i.e., suspension of structurally intact mitochondria. Densitometry of

the bands were done using ImageJ (V. 1.41o, NIH, USA) software

and graph (100% scale) was plotted against percent densitometric

units

Table 1 Effect of mtThiol alteration on membrane permeability transition and mitochondrial activity

Treatments A. GSH content

(CMF-MFI)

B. Electron flow

(% MTT activity)

C. Mitochondrial Membrane Permeability Transition

Protein (mg/ml) in

mitochondrial supernatant

Protein (mg/ml) in

mitochondrial lysate

Control 356.83 ± 18.38 100 ± 2.46 0.18 ± 0.04 9.4 ± 0.17

Ca2? (100 lM) 214.16 ± 4.94* 70 ± 2.31* 0.68 ± 0.06** 8.42 ± 0.30**

t-BHP (250 lM) 132.71 ± 7.07* 54 ± 1.57** 0.39 ± 0.02 9.09 ± 0.33*

NEM (0.1 mM) 267.61 ± 4.43* 93 ± 2.63 0.44 ± 0.01* 8.25 ± 0.23**

Ca2? ? NEM 66.42 ± 2.82** 24 ± 0.95*** 0.95 ± 0.04*** 7.81 ± 0.18**

A. mtThiol in rat liver mitochondria. mtThiol in isolated rat liver mitochondria was estimated using CMFDA fluoroprobe on flow cytometer.

Data are represented as mean fluorescence intensity (MFI) of the fluorescent CMF-adduct. B. Mitochondrial electron flow. Mitochondrial

electron flow was measured by reduction of MTT (yellow colored dye) to formazan (violet colored crystals) at 3 min intervals. C. Mitochondrial

membrane permeability transition. Quantification of released proteins from mitochondria due to MPT in mitochondrial supernatant and mito-

chondrial lysate by Lowry protein estimation and represented as mg/ml proteins

Statistical significance was considered as *P \ 0.05; **P \ 0.01; ***P \ 0.001, when compared to control

Mol Cell Biochem (2011) 357:373–385 377

123

mitochondria causes any change in reducing equivalents,

NAD(P)H was measured (Fig. 2). Ca2? overload (100 lM

CaCl2) or mtThiol alteration (0.1 mM NEM) did not

caused significant decrease in NAD(P)H level (6.36 and

3.34%, respectively), whereas under oxidatively stressed

mitochondria, 18.04% reduction in NAD(P)H level was

observed. However, under dual stress of Ca2? and 0.1 mM

NEM, a sharp decrease in NAD(P)H (i.e., 48.36%;

P \ 0.05) was observed.

mtThiol alteration amplifies oxidative stress in Ca2?

overloaded mitochondria

To elucidate the role of mtThiol alteration and ROS pro-

duction, in the presence of Ca2? overload, highly sensitive

fluoroprobes, DHE, and DCFH-DA were used to assess the

generation of O��2 (superoxide anion radical) (Fig. 3a) and

secondary ROS/RNS (Fig. 3b), respectively. Under Ca2?

overloaded condition (100 lM CaCl2) (Fig. 3c), superox-

ide generation as indicated by ethidium (ETH) intensity,

was increased by 100% (P \ 0.01) whereas secondary

ROS/RNS (i.e., DCF intensity) increased by 85%

(P \ 0.05) suggesting that under Ca2? overload, genera-

tion of superoxide was more pronounced than secondary

ROS/RNS. NEM at 0.1 mM did not cause significant

increase in ETH and DCF intensities, while in oxidatively

stressed (t-BHP; 250 lM) mitochondria, 111% (P \ 0.01)

and 107% (P \ 0.01) increase was observed, respectively.

Ca2? overloaded mitochondria when subjected to mtThiol

alteration, showed increase in superoxide generation by

138% (P \ 0.001), however, secondary ROS/RNS gener-

ation increased by 180% (P \ 0.001), indicating that

secondary ROS/RNS increased substantially in response to

minor changes in mtThiol level in Ca2? overloaded mito-

chondria. Our studies also showed (Supplementary mate-

rial) that the mtThiol alteration makes Ca2? overloaded

mitochondria susceptible to oxidative membrane damage

and compromise of antioxidant defenses such as SOD,

GPx, and GR.

mtThiol alteration adversely affects mitochondrial

electron flow during Ca2? overload

Table 1B shows that mitochondria subjected to Ca2?

overload (100 lM CaCl2) caused 30% (P \ 0.05) decrease

in mitochondrial electron flow, while 0.1 mM NEM caused

no significant decrease in electron flow. As the concen-

tration of NEM was enhanced (1.0 mM), electron flow in

mitochondria decreased up to 53% (P \ 0.01) in 3 min

(data not shown), suggesting that significant mtThiol

alteration is required to affect mitochondrial electron flow.

Combined treatment of CaCl2 and NEM (0.1 mM) caused

significant decrease in mitochondrial electron flow, i.e.,

76% (P \ 0.001). Therefore, mtThiol along with Ca2?

plays a key role in decreasing mitochondrial electron flow.

mtThiol alteration increases number of depolarized

mitochondria under Ca2? overload

Depolarization of mitochondria is one of the key indicators

of its dysfunction. We observed (Fig. 4) that Ca2? overload

(100 lM CaCl2) and oxidative stress (250 lM t-BHP)

caused 20 and 10% increase in mitochondrial depolariza-

tion as compared to control. While under low mtThiol

Fig. 2 Determination of pyridine nucleotides (NAD(P)H). UV

excited blue auto-fluorescence was used to measure reduced pyridine

nucleotides by flow cytometry. Data are demonstrated as histogram

plot, and the distribution of low and high NAD(P)H content in

isolated mitochondria is shown under the markers, right marker (M1)

indicates mitochondrial population with high NAD(P)H content and

left marker (M2) indicates mitochondrial population with low

NAD(P)H content. The effect of mtThiol alteration on Ca2?

overloaded mitochondria was statistically significant (P \ 0.05) when

compared to mtThiol depleted mitochondria

378 Mol Cell Biochem (2011) 357:373–385

123

depletion (0.1 mM NEM), no significant depolarization in

mitochondria was observed. NEM and CaCl2 when given

in combination, a significant increase of 37% (P \ 0.05)

was found in number of depolarized mitochondria.

Therefore, low level of mtThiol alteration under Ca2?

overloaded condition can collapse DWm to a significant

extent.

mtThiol alteration causes significant Ca2? dependent

MPT and swelling in mitochondria

MPT is reported as a Ca2? dependent process linked with

the opening of non-specific channels in the inner mem-

brane which permits non-specific movement of molecules

B1.5 kDa. The present set of experiment was performed to

understand Ca2? dependent MPT under altered mtThiol

conditions. Proteins released from the mitochondria due to

MPT were quantified in the mitochondrial supernatant and

its respective lysate (Table 1C). During Ca2? overload

(CaCl2; 100 lM), 2.7-fold increase in protein release was

observed in supernatant which was significant (P \ 0.01).

At the same time when t-BHP (250 lM) and NEM

(0.1 mM) were used in isolation, 1.2- and 1.4-fold increase

in released proteins in mitochondrial supernatant were

observed with corresponding decrease in mitochondrial

lyate (Table 1C). Under combined stress of NEM and

Ca2?, release of proteins was found to be 5.3-fold

(P \ 0.001) in supernatant and decrease in lysate was 20%.

Apoptotic proteins found in mitochondria such as AIF,

EndoG, Cyt c and the anion channel protein VDAC are

some specific proteins which decide the fate of cell under

stress. Therefore, the release of these proteins was also

detected in both the fractions, i.e., mitochondrial superna-

tant and lysate. Figure 5a and b, indicate that under Ca2?

overload, release of Cyt c was not significant as compared

to control, whereas under mtThiol alteration 2.0-fold

increase was observed, which enhanced to 3.0-fold

(P \ 0.01) under combined stress of Ca2? overload and

mtThiol alteration. On the other hand, significant

enhancement of 3.3-fold (P \ 0.01) was observed in AIF

release under Ca2? overload which further enhanced to

5.7-fold (P \ 0.001) under combined stress of Ca2?

overload and mtThiol alteration. Release of EndoG and

VDAC as compared to control was only 0.9- and 0.4-fold

during Ca2? overload which significantly enhanced to 4.3-

fold (P \ 0.01) and 4.8-fold (P \ 0.001) during dual stress

of Ca2? overload and mtThiol alteration, respectively.

Our data indicates that, MPT changed significantly due

to the effect of mtThiol alteration and Ca2? overload,

hence it was logical to study the mitochondrial swelling

pattern. Thus, experiments were performed to compare

swelling traces under given treatments to confirm the ear-

lier results (Fig. 6a–d). Isolated rat liver mitochondria

when incubated with NEM (0.1 and 1 mM) showed large

amplitude swelling in the initial phase (Fig. 6a, b). During

mtThiol alteration, initial swelling started even at 0.1 mM

NEM and shifted towards shorter lag phase as the con-

centration of NEM was increased up to 1 mM. In case of

Ca2? overload 38% swelling was observed in isolated

mitochondria. In the presence of t-BHP (oxidative stress),

only 18% swelling was observed. Since Ca2? regulates

MPT, a minute change in mtThiol alteration (0.1 mM

NEM) together with Ca2? could alter MPT to 58%

(P \ 0.01) (Fig. 6c, d) which was very much comparable

to high mtThiol depleted condition, i.e., 1.0 mM NEM

(63%) without Ca2? overload. As Ca2? has its own regu-

latory mechanism for mitochondrial MPT, it was observed

Fig. 3 Mitochondrial oxidative load (Total ROS). Total ROS gen-

eration in isolated rat liver mitochondria was estimated using DHE

and DCFH-DA dye on flow cytometer with sensitivity for superoxide

and secondary ROS/RNS respectively. Data are demonstrated in two

forms: (i) overlay of histogram plots, a and b, and (ii) bar graph, c,

plotted against different concentrations of treatments versus mean

fluorescence intensity (MFI). In histograms, right shift in histogram

shows increase in ROS. Overlay of histogram plots represent isolated

mitochondrial samples under Ca2? overload (CaCl2, 100 lM),

oxidative stress (t-BHP, 250 lM), mtThiol alteration (NEM,

0.1 mM) and combination of CaCl2 and NEM. Bar graph shows

perceptible increase in total ROS content

Mol Cell Biochem (2011) 357:373–385 379

123

that in the presence of Ca2?, NEM induced mitochondrial

swelling is more pronounced as compared to mitochondria

stressed with NEM and t-BHP alone.

Large amplitude swelling of isolated mitochondria was

also confirmed by TEM (Fig. 6e) wherein vacuolation, loss

of cristae structure was apparent in the presence of Ca2?

overload and mtThiol alteration (photograph not shown).

Our data shows that, swelling produced by Ca2? overload

or mtThiol alteration in isolation was not sufficient to

release Cyt c or VDAC, but the dual stress, mtThiol

alteration along with Ca2? overload caused a significant

release of VDAC, Cyt c, AIF and EndoG. Thus, mtThiol

alteration under Ca2? overloaded condition causes per-

meabilization of the mitochondrial membranes, thereby,

significantly increasing mitochondrial volume, i.e.,

swelling.

Confirmation of Ca2? dependent MPT using inhibitors

and chelator

BAPTA (Ca2? ion chelator), RuRed (Ca2? uniport

blocker), and CyA (MPT blocker) were used as negative

control for Ca2? retention, Ca2? entry in the mitochondria

and Ca2? dependent MPT change, respectively. Inorganic

Fig. 4 Mitochondrial

membrane potential (DWm).

JC-1 dye, highly specific for

mitochondrial membrane, was

used for the detection of DWm

on flow cytometer. Data are

demonstrated as dot plot, and

the distribution of depolarized

and polarized mitochondria is

shown under lower and upperhalf, respectively. 100 lM

CaCl2, 250 lM t-BHP, 0.1 mM

NEM, and combinations of

CaCl2 and NEM were used to

evaluate DWm. The effect of

mtThiol alteration on Ca2?

overloaded mitochondria was

statistically significant

(P \ 0.01) when compared to

mtThiol depleted mitochondria

Fig. 5 Mitochondrial membrane permeability transition (MPT) pore

opening. MPT pore opening under Ca2? overload and mtThiol

alteration was assessed (a, b). Release of mitochondria specific

proteins (AIF, EndoG, VDAC, and Cyt c) was detected in supernatant

as well as mitochondrial fraction by immunoblot. Cyto Ox-IV was

used as mitochondrial loading control. ‘S’ represents mitochondrial

supernatant and ‘M’ represents mitochondrial lysate. Samples are;

control, Ca2? (100 lM), t-BHP (250 lM), NEM (0.1 mM) and

Ca2??NEM treated mitochondria

380 Mol Cell Biochem (2011) 357:373–385

123

phosphate (Pi; KH2PO4; 2 mM) was used as positive

control of MPT. Figure 7a shows that Ca2? influx

increased under Ca2? and Ca2? with NEM treatment in

mitochondria, whereas (Fig. 7b) negative controls (BAP-

TA, RuRed or CyA) inhibited swelling while Pi caused

large amplitude swelling in mitochondria. In the presence

of these negative controls, Ca2? influx and MPT was pre-

vented so that no swelling was observed when compared to

Ca2? or Pi (Fig. 7c, d).

Under dual stress of Ca2? and NEM, no Ca2? influx was

observed in the presence of BAPTA and RuRed whereas in

the presence of CyA Ca2? influx occurred (Fig. 7e).

However, significant swelling was observed in BAPTA and

RuRed treated mitochondria during the dual stress, whereas

in CyA, no swelling was observed (Fig. 7f). In case of CyA

pre-treated mitochondria, Ca2? dependent permeability

changes may have been inhibited due to Cyclophilin D and

ANT interaction in the matrix and inner membrane of

mitochondria, respectively. Thereby, it can be stated, that

in the presence of chelator and inhibitors of Ca2? uniporter,

swelling was observed due to the mtThiol alteration and is

not a Ca2? dependent phenomenon. This experiment

strongly suggests that MPT is not solely a Ca2? dependent

or mtThiol alteration induced phenomenon but depends on

the synergistic effect of both.

Discussion

ROS and oxidative stress have now been associated with

induction of the MPT in mitochondria which are estab-

lished key regulators in propagating apoptotic signaling

[33–38]. Increasing evidences have implicated mtThiol/

GSH pool and redox status of the cell as critical compo-

nents of the regulatory mechanisms for oxidative stress and

apoptotic signaling [39]. Efforts to establish a correlation

between ROS, Ca2?, and thiol/GSH have been made using

different cell types and models but the results are con-

flicting and direct evidence is lacking [40–44]. In this

study, authors have tried to elucidate and characterize the

mechanism of Ca2? dependent mitochondrial MPT mod-

ulation during mtThiol alteration using direct model sys-

tem, i.e., isolated mitochondria, to establish the link.

Mitochondrion has the capacity to accumulate Ca2? in the

Fig. 6 Mitochondrial swelling.

Increase in mitochondrial

volume (swelling) was

determined

spectrophotometrically and

absorbance was recorded at

520 nm as decrease in the

optical density at 30 s time

intervals for a period of 10 min.

Linear graph (a, c) represents

traces of swelling (slope) of

isolated rat liver mitochondria

and bar graph (b, d) shows the

phase distribution of swelling,

i.e., 0–1, 1–5, 5–10, and

0–10 min. 0–1 and 1–5 min are

early phases of swelling while

5–10 min is late phase of

swelling. Third phase is to show

total mitochondrial swelling

achieved under given treatment

of 10 min (0–10 min). a,

b Mitochondrial swelling under

single stress condition. c, d The

Ca2? dependent mitochondrial

swelling under mtThiol depleted

condition. e Ultra-structural

investigation of isolated

mitochondria by TEM.

Morphometric analysis of

electron micrographs of ultra-

thin sections is expressed as a

percentage from the total

number of analyzed

mitochondria after treatment

Mol Cell Biochem (2011) 357:373–385 381

123

presence of adenine nucleotides. However, if adenine

nucleotides are depleted, in the presence of pro-oxidants,

Ca2? uptake may trigger opening of a large conductance

trail in the mitochondrial inner membrane usually referred

to as the MPT pore. The major factors that promote MPT

pore opening are high intra-mitochondrial Ca2?, oxidative

stress and high Pi [4]. We proposed that pore opening is

also governed by the slight change in mtThiol in Ca2?

dependent manner. The spontaneous discharge of Ca2?

from mitochondria is associated with increased non-spe-

cific permeability of the mitochondrial inner membrane

(MPT), swelling of the mitochondria, loss of matrix ade-

nine nucleotides, followed by collapse of DWm. Moderate

Ca2? uptake stimulates pyruvate dehydrogenase and

Kreb’s cycle enzymes, resulting in increased NADH redox

potential and enhanced ATP synthesis [45]. On the con-

trary, higher amounts of mitochondrial Ca2? levels result

in a progressive decrease in electron transfer, because both

Ca2? maintenance and ATP production are dependent on

the DWm [4]. This study suggests that Ca2? along with

mtThiol alteration causes significant mitochondrial swell-

ing, loss of activity followed by depolarization and

uncoupling. It has also been elucidated that the ROS gen-

erated in Ca2? treated mitochondria selectively oxidizes

critical thiols, pyridine nucleotides and causes decrease in

mitochondrial activity along with membrane lipid peroxi-

dation (Supplementary material). Consequently, this leads

to change in structural integrity, as confirmed by TEM.

Redox state of the pyridine nucleotides (NAD/NADH or

NADP/NADPH) is also a major factor that regulates MPT.

NADH acts synergistically with ADP to inhibit the MPT

pores whereas either compound alone gives a mixed type

of inhibition, indicating that ADP and NADH may act at

the same site [46]. Therefore, it has been suggested that

unlike ADP and Pi, the oxidation state of the mitochondrial

pyridine nucleotides may be the controlling factor for MPT

pore and needs more attention. Our data suggests that, in

the state of mtThiol alteration, decrease in pyridine

nucleotide favors Ca2? dependent MPT as well as increase

in ROS production and finally depolarization of

Fig. 7 mtThiol alteration and

Ca2? flux during MPT

(Mitochondrial swelling).

Various inhibitors and chelator

were used to confirm the Ca2?

dependency of mtThiol to cause

MPT. BAPTA (10 lM), an

intracellular Ca2? chelator,

Ruthenium Red (RuRed;

30 lM), an inhibitor of

mitochondrial Ca2? uniporter

and Cyclosporin A (CyA;

1 lM), an inhibitor of

permeability transition process

in the presence of Ca2?, when

used in micromolar

concentration inhibited MPT.

KH2PO4 (2 mM) was used as

positive control of

mitochondrial swelling.

Mitochondrial Ca2? flux was

analyzed using Fluo-4 DirectTM

Calcium Assay Kit. The graphs

are plotted against AFU

(arbitrary fluorescent units)

versus time (10 min)

382 Mol Cell Biochem (2011) 357:373–385

123

mitochondria. Pyridine nucleotides are important co-fac-

tors in mitochondria, synthesized in the sub-organelle

because the intact inner mitochondrial membrane is

impermeable to these polar molecules. Thus, level of pyr-

idine nucleotide in mitochondria under oxidant stress could

be a determinant of the extent of oxidative damage.

In order to maintain mitochondrial redox environment, it

is also necessary to keep ROS in check. The known sites of

ROS production in the electron transport chain are com-

plexes I and III [47]. ROS generated are detoxified by the

antioxidants to maintain a state of equilibrium. However,

under lowered antioxidant defense or overproduction of

ROS, the equilibrium leans towards an increase of ROS

resulting in oxidative stress and cellular damage [6].

Decrease in mtThiol and increase in ROS generation alters

MPT and mitochondrial redox status. Our results advocate

that under mtThiol alteration in Ca2? overloaded mito-

chondria, secondary ROS/RNS generation is more than

superoxide formation suggesting that mtThiol alteration

impaired antioxidant defense in mitochondria (Supple-

mentary material), so that unquenched superoxide leads to

generation of other reactive species. These ROS, besides

causing peroxidation of the phospholipid bilayer, resulting

in severe damage to membrane integrity, induce MPT with

release of Cyt c, EndoG, and AIF, thereby, initializing the

apoptotic cascade as consolidated from our data. mtThiol

depleting agents can also induce rapid peroxidation of

cellular lipids as confirmed by our experiments.

Conclusion

The previous work from our laboratory [32, 37, 38, 48]

showed that in isolated mitochondria MPT is significantly

induced during oxidative stress and can be modulated in

the presence of different antioxidants/oxidants. In contin-

uation to our earlier findings, the present study established

that minor mtThiol alteration in Ca2? overloaded mito-

chondria induced MPT as well as reduced mitochondrial

activity significantly. This is assisted by alteration in redox

homeostasis, compromised antioxidant defense and

enhanced ROS generation leading to ultimate collapse of

DWm. Overall (Fig. 8) it appears to be a multi-factorial

cross-talk between Ca2?, mtThiol, and ROS, centered on

the mitochondria and suggests that alteration in mtThiol

along with Ca2? overload (dual stress) cause enhanced

Fig. 8 Schematic representation of proposed mitochondrial cross-

talk between mtThiol, Ca2? and ROS. As evident from the data and

literature we propose a multi-factorial cross-talk between Ca2?,

mtThiol, and ROS, in which a number of simultaneous events occur.

Two independent process take place initially which synchronize

afterwards. Initially when mitochondria are overloaded with Ca2?, it

influences MPT followed by alteration in electron flow, ROS

generation, disruption of DWm, and finally mitochondrial collapse.

On the other hand, mtThiol alteration makes mitochondrial mem-

branes vulnerable to per-oxidative damage, redox imbalance followed

by ROS generation, disruption of DWm, and mitochondrial collapse.

In between these two independent processes, loss of reduced pyridine

nucleotides (NAD(P)H), compromised antioxidant defense, increased

ROS generation makes a bridge, so that, one effect (mtThiol

alteration) compliments the other (Ca2? overload), i.e., afterwards,

when ROS generation exceeds a threshold, it dismantles mitochon-

drial redox homeostasis followed by loss of antioxidant defense which

further leads to membrane oxidative damage leading to altered MPT

(release of pro-apoptotic proteins like AIF, EndoG, Cyt c, etc., giving

apoptotic signals to cell), mitochondrial swelling, loss of mitochon-

drial activity, mitochondrial depolarization, and finally mitochondrial

collapse. In view of this, the proposed mechanistic approach may help

to understand the effect of mtThiol on Ca2? dependent MPT and

mitochondrial dysfunction

Mol Cell Biochem (2011) 357:373–385 383

123

membrane permeability transition and mitochondrial

dysfunction.

Acknowledgments The author’s are grateful to Director, IITR for

his interest in this work. Author’s are also thankful to Dr. Yogeshwar

Shukla for support in flow cytometric analysis. Author’s are also

grateful to Dr. Manjula Murari, Department of Pathology, Sanjay

Gandhi Post Graduate Institute of Medical Sciences for support in

performing TEM. Mr. Brijesh Kumar Singh is indebted to Council of

Scientific and Industrial Research (CSIR) and Ms. Madhulika Tripathi

is grateful to Indian Council of Medical Research (ICMR) for grant of

Senior Research Fellowship. Authors also thank IITR Publication

Review Committee for allocation of manuscript number 2766.

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