The

role

of

mitochondria

in

protection

of

the

heart

by

preconditioning

A

prolonged

period

of

ischaemia

followed

by

reperfusion

irreversibly

damages

the

heart

Such

reperfusion

injury

-LRB-

RI

-RRB-

involves

opening

of

the

mitochondrial

permeability

transition

pore

-LRB-

MPTP

-RRB-

under

the

conditions

of

calcium

overload

and

oxidative

stress

that

accompany

reperfusion

Protection

from

MPTP

opening

and

hence

RI

can

be

mediated

by

ischaemic

preconditioning

-LRB-

IP

-RRB-

where

the

prolonged

ischaemic

period

is

preceded

by

one

or

more

brief

-LRB-

min

-RRB-

cycles

of

ischaemia

and

reperfusion

Following

a

brief

overview

of

the

molecular

characterisation

and

regulation

of

the

MPTP

the

proposed

mechanisms

by

which

IP

reduces

pore

opening

are

reviewed

including

the

potential

roles

for

reactive

oxygen

species

-LRB-

ROS

-RRB-

protein

kinase

cascades

and

mitochondrial

potassium

channels

It

is

proposed

that

IP-mediated

inhibition

of

MPTP

opening

at

reperfusion

does

not

involve

direct

phosphorylation

of

mitochondrial

proteins

but

rather

reflects

diminished

oxidative

stress

during

prolonged

ischaemia

and

reperfusion

This

causes

less

oxidation

of

critical

thiol

groups

on

the

MPTP

that

are

known

to

sensitise

pore

opening

to

calcium

The

mechanisms

by

which

ROS

levels

are

decreased

in

the

IP

hearts

during

prolonged

ischaemia

and

reperfusion

are

not

known

but

appear

to

require

activation

of

protein

kinase

Cε

either

by

receptor-mediated

events

or

through

transient

increases

in

ROS

during

the

IP

protocol

Other

signalling

pathways

may

show

cross-talk

with

this

primary

mechanism

but

we

suggest

that

a

role

for

mitochondrial

potassium

channels

is

unlikely

The

evidence

for

their

activity

in

isolated

mitochondria

and

cardiac

myocytes

is

reviewed

and

the

lack

of

specificity

of

the

pharmacological

agents

used

to

implicate

them

in

IP

is

noted

Some

K

channel

openers

uncouple

mitochondria

and

others

inhibit

respiratory

chain

complexes

and

their

ability

to

produce

ROS

and

precondition

hearts

is

mimicked

by

bona

fide

uncouplers

and

respiratory

chain

inhibitors

IP

may

also

provide

continuing

protection

during

reperfusion

by

preventing

a

cascade

of

MPTP-induced

ROS

production

followed

by

further

MPTP

opening

This

phase

of

protection

may

involve

survival

kinase

pathways

such

as

Akt

and

glycogen

synthase

kinase

-LRB-

-RRB-

either

increasing

ROS

removal

or

reducing

mitochondrial

ROS

production

Introduction

The

heart

is

one

of

the

most

energy

demanding

tissues

in

the

body

and

is

totally

dependent

upon

oxidative

phosphorylation

to

supply

the

large

amount

of

ATP

required

for

beat-by-beat

contraction

and

relaxation

If

the

blood

flow

to

the

heart

is

impaired

-LRB-

ischaemia

-RRB-

as

occurs

when

a

blood

clot

occludes

a

coronary

artery

-LRB-

coronary

thrombosis

-RRB-

or

where

cardiac

surgery

requires

the

heart

to

be

disconnected

from

the

blood

supply

the

source

of

oxygen

is

removed

leading

to

the

cessation

of

oxidative

phosphorylation

This

causes

tissue

ATP

and

creatine

phosphate

concentrations

to

decrease

with

a

concomitant

rise

in

ADP

AMP

and

Pi

concentrations

Although

glycolysis

is

activated

it

is

unable

to

meet

the

demand

of

the

beating

heart

for

ATP

Consequently

the

heart

rapidly

ceases

to

beat

as

the

contractile

machinery

is

inhibited

by

elevated

Pi

and

ADP

combined

with

the

decreasing

pH

that

accompanies

the

accumulation

of

glycolytic

lactic

acid

-LSB-

-RSB-

The

heart

can

usually

survive

a

short

period

of

ischaemia

and

then

recover

upon

reperfusion

Although

the

performance

of

the

heart

may

be

impaired

initially

-LRB-

stunning

-RRB-

given

time

recovery

is

complete

However

if

the

period

of

ischaemia

is

too

long

the

tissue

becomes

irreversibly

damaged

Hence

if

the

heart

is

to

be

salvaged

it

is

important

to

restore

the

blood

flow

as

soon

as

possible

Yet

paradoxically

such

reperfusion

can

exacerbate

the

damage

occurring

during

the

ischaemic

period

This

is

known

as

reperfusion

injury

and

is

accompanied

by

enzyme

release

and

morphological

changes

characteristic

of

necrosis

-LSB-

-RSB-

The

extent

of

damage

can

be

visualised

as

an

area

of

necrotic

tissue

known

as

the

infarct

whose

area

can

be

determined

to

provide

a

quantitative

measure

of

injury

Quantification

of

damage

may

also

be

provided

by

measuring

the

release

of

intracellular

proteins

such

as

lactate

dehydrogenase

or

troponin

I

-LSB-

-RSB-

In

addition

to

the

necrotic

cell

death

that

represents

the

major

damage

to

the

reperfused

heart

there

is

also

evidence

that

some

myocytes

around

the

periphery

of

the

infarct

die

by

apoptosis

-LSB-

-RSB-

Understanding

the

causes

of

reperfusion

injury

and

devising

ways

of

preventing

it

is

of

major

clinical

importance

in

cardiac

surgery

and

the

treatment

of

coronary

thrombosis

There

is

increasing

evidence

that

mitochondrial

dysfunction

plays

a

central

role

in

mediating

both

the

necrotic

and

apoptotic

components

of

reperfusion

injury

and

that

one

of

the

most

effective

ways

of

protecting

hearts

from

such

injury

known

as

ischaemic

preconditioning

-LRB-

IP

-RRB-

acts

to

attenuate

this

-LSB-

-RSB-

This

review

will

address

the

causes

of

reperfusion

injury

emphasising

the

role

of

the

mitochondrial

permeability

transition

pore

-LRB-

MPTP

-RRB-

and

prevention

of

its

opening

by

IP

The

proposed

signalling

mechanisms

through

which

IP

may

exert

its

effects

will

be

discussed

including

the

proposed

role

of

mitochondrial

potassium

channels

which

has

been

a

major

but

controversial

area

of

research

-LRB-

see

-LSB-

-RSB-

However

first

it

is

necessary

to

explain

the

phenomenon

of

preconditioning

The

phenomenon

of

ischaemic

preconditioning

Ischaemic

preconditioning

-LRB-

IP

-RRB-

involves

exposing

the

heart

to

brief

periods

of

ischaemia

-LRB-

typically

min

-RRB-

interspersed

with

periods

of

normal

perfusion

prior

to

the

prolonged

ischaemia

initiated

within

an

hour

of

the

preconditioning

protocol

This

protocol

was

first

shown

to

offer

strong

protection

against

reperfusion

injury

in

the

dog

heart

by

Murry

et

al.

in

-LSB-

-RSB-

and

has

since

been

confirmed

in

all

species

investigated

including

humans

-LSB-

-RSB-

However

it

should

be

noted

that

significant

reduction

in

infarct

size

only

occurs

when

the

optimal

duration

for

the

test

period

of

ischemia

is

chosen

and

is

lost

if

the

ischaemic

period

is

too

long

Preconditioned

hearts

exhibit

a

smaller

infarct

size

and

intracellular

enzyme

release

-LRB-

indicators

of

necrotic

cell

death

-RRB-

and

fewer

arrhythmias

-LSB-

-RSB-

whilst

contractile

function

is

preserved

-LSB-

-RSB-

Typically

brief

-LRB-

min

-RRB-

cycles

of

ischaemia

and

reperfusion

are

used

in

IP

there

being

little

advantage

of

additional

cycles

-LSB-

-RSB-

If

ischaemia

is

initiated

more

than

h

after

the

preconditioning

protocol

protection

is

lost

but

re-emerges

again

after

about

h

and

lasts

for

up

to

days

This

is

termed

the

second

window

of

preconditioning

-LSB-

-RSB-

A

wide

variety

of

drugs

that

activate

different

signalling

pathways

thought

to

be

involved

in

mediating

IP

-LRB-

see

Section

-RRB-

can

also

induce

protection

and

thus

preconditioning

is

often

used

as

a

generic

term

to

encompass

any

protocol

applied

before

prolonged

ischaemia

that

protects

the

heart

during

reperfusion

Not

to

be

confused

with

such

preconditioning

is

a

more

recently

described

phenomenon

known

as

post-conditioning

Here

hearts

are

given

several

very

brief

ischaemic

interludes

-LRB-

s

-RRB-

during

the

early

phase

of

reperfusion

which

also

improves

the

recovery

of

the

heart

and

reduces

infarct

size

-LSB-

-RSB-

This

protocol

has

considerable

clinical

potential

since

intermittent

reperfusion

can

be

induced

during

angioplasty

prior

to

permanent

restoration

of

blood

flow

It

is

beyond

the

scope

of

this

review

to

consider

the

similarities

and

differences

between

preconditioning

and

postconditioning

in

any

detail

However

many

of

the

mechanisms

and

signalling

pathways

mediating

protection

are

thought

to

be

the

same

-LSB-

-RSB-

and

where

effects

on

mitochondria

have

been

demonstrated

in

postconditioning

they

will

be

noted

Causes

of

reperfusion

injury

Increases

in

cellular

-LSB-

-RSB-

and

reactive

oxygen

species

-LRB-

ROS

-RRB-

initiated

in

ischaemia

and

then

amplified

upon

reperfusion

are

thought

to

be

the

main

causes

of

reperfusion

injury

Mitochondria

are

involved

both

in

the

production

of

ROS

and

as

targets

for

the

damaging

action

of

both

ROS

and

calcium

-LSB-

-RSB-

Damage

occurring

in

ischaemia

-LRB-

Reviewed

in

-LSB-

-RSB-

-RRB-

During

ischaemia

the

increased

rate

of

glycolysis

causes

lactic

acid

to

accumulate

and

the

intracellular

pH

-LRB-

pHi

-RRB-

to

drop

rapidly

This

activates

the

H

antiporter

as

the

cell

endeavours

to

restore

pHi

However

the

rapidly

declining

ATP

concentrations

cause

inhibition

of

the

Na/K

ATPase

and

lead

to

a

rise

in

intracellular

-LSB-

-RSB-

This

in

turn

reduces

the

ability

of

the

cell

to

restore

its

pHi

and

increases

intracellular

-LSB-

-RSB-

since

the

antiporter

that

usually

pumps

out

of

the

cell

is

inhibited

or

reversed

The

conversion

of

ATP

to

ADP

and

AMP

is

rapid

and

reversible

AMP

is

slowly

converted

into

adenosine

and

then

inosine

and

xanthine

through

a

purine

degradation

pathway

These

nucleosides

leak

out

of

the

cell

-LRB-

and

may

have

vasodilator

effects

through

purinergic

receptors

-RRB-

and

lead

to

a

gradual

depletion

of

adenine

nucleotide

which

may

contribute

to

the

reduced

cardiac

performance

-LRB-

stunning

-RRB-

seen

on

reperfusion

Another

feature

of

ischaemia

that

is

thought

to

contribute

to

the

damage

seen

during

reperfusion

is

the

production

of

reactive

oxygen

species

-LRB-

ROS

-RRB-

There

is

a

small

increase

in

ROS

production

immediately

the

heart

becomes

ischaemic

which

remains

stable

for

min

but

then

increases

dramatically

-LSB-

-RSB-

This

large

increase

is

thought

to

play

a

major

role

in

damaging

the

heart

during

ischaemia

and

sensitising

it

to

reperfusion

as

described

below

However

the

source

of

the

ROS

is

unclear

and

might

involve

complex

and

complex

of

the

respiratory

chain

or

perhaps

more

likely

xanthine

oxidase

acting

on

xanthine

formed

from

the

degradation

of

adenosine

as

noted

above

-LSB-

-RSB-

The

depletion

of

ATP

combined

with

elevated

-LSB-

-RSB-

and

ROS

leads

to

a

gradual

decline

in

cellular

integrity

as

degradative

enzyzmes

are

activated

and

ATP-dependent

repair

processes

are

unable

to

operate

-LSB-

-RSB-

If

the

tissue

remains

ischaemic

for

only

short

periods

and

the

mitochondria

remain

sufficiently

intact

to

generate

the

ATP

tissue

damage

is

slight

and

can

be

repaired

by

ATP-dependent

processes

upon

reoxygenation

However

a

critical

point

is

reached

when

recovery

is

not

possible

and

reperfusion

actually

causes

further

damage

to

the

heart

causing

extensive

necrosis

with

associated

enzyme

release

-LSB-

-RSB-

Damage

occurring

during

reperfusion

Reperfusion

is

associated

with

a

burst

of

reactive

oxygen

species

-LRB-

ROS

-RRB-

production

-LSB-

-RSB-

but

here

too

the

source

of

the

ROS

is

debated

Although

some

may

be

produced

by

xanthine

oxidase

and

NADPH

oxidase

it

is

probable

that

most

is

formed

by

complex

and

complex

of

the

respiratory

chain

-LSB-

-RSB-

When

the

respiratory

chain

is

inhibited

by

lack

of

oxygen

and

then

re-exposed

to

oxygen

ubiquinone

can

become

partially

reduced

to

ubisemiquinone

This

can

then

react

with

the

oxygen

to

produce

superoxide

that

is

reduced

to

hydrogen

peroxide

by

superoxide

dismutase

Hydrogen

peroxide

is

removed

by

glutathione

peroxidase

or

catalase

but

if

ferrous

ions

-LRB-

or

other

transition

metals

such

as

copper

-RRB-

are

present

it

will

form

the

highly

reactive

hydroxyl

radical

through

the

Fenton

reaction

-LSB-

-RSB-

Mitochondrial

proteins

are

especially

susceptible

to

ROS

induced

damage

and

this

is

reflected

in

the

impaired

respiratory

chain

activity

of

mitochondria

isolated

from

ischaemic

hearts

-LRB-

see

-LSB-

-RSB-

-RRB-

Thus

ROS

have

direct

effects

on

several

respiratory

chain

components

most

especially

on

complex

but

also

on

complex

and

other

iron

sulphur

proteins

such

as

aconitase

ROS

can

also

cause

thiol

oxidation

and

inhibition

of

the

ATPase

and

adenine

nucleotide

translocase

In

addition

ROS

cause

oxidation

of

glutathione

that

may

then

form

mixed

disulphides

with

proteins

Such

protein

modification

is

thought

to

have

inhibitory

effects

on

ion

pumps

and

therefore

exacerbate

the

effects

of

ATP

deprivation

on

ionic

homeostasis

-LSB-

-RSB-

ROS

also

cause

peroxidation

of

the

unsaturated

fatty

acid

components

of

the

phospholipids

and

especially

cardiolipin

of

the

inner

mitochondrial

membrane

and

this

leads

to

further

inhibition

of

respiratory

chain

activity

-LSB-

-RSB-

Furthermore

lipid

peroxidation

causes

the

release

of

reactive

aldehydes

such

as

that

can

modify

membrane

proteins

-LSB-

-RSB-

Overall

it

is

thought

that

the

combined

effects

of

ROS

and

elevated

-LSB-

-RSB-

play

a

critical

role

in

the

transition

from

reversible

to

irreversible

reperfusion

injury

and

that

mitochondria

are

the

major

target

of

these

agents

In

particular

they

lead

to

the

opening

of

the

mitochondrial

permeability

transition

pore

that

is

now

widely

accepted

to

play

a

critical

role

in

reperfusion

injury

-LSB-

-RSB-

The

mitochondrial

permeability

transition

pore

-LRB-

MPTP

-RRB-

Properties

of

the

MPTP

Under

normal

physiological

conditions

the

mitochondrial

inner

membrane

is

impermeable

to

all

but

a

few

selected

metabolites

and

ions

this

is

essential

to

maintain

the

membrane

potential

and

pH

gradient

that

together

drive

ATP

synthesis

through

oxidative

phosphorylation

However

under

conditions

of

high

matrix

calcium

especially

when

this

is

accompanied

by

oxidative

stress

high

phosphate

and

low

adenine

nucleotide

concentrations

a

non-specific

pore

opens

in

the

inner

mitochondrial

membrane

known

as

the

mitochondrial

permeability

transition

pore

-LRB-

MPTP

-RRB-

The

properties

and

regulation

of

the

MPTP

are

reviewed

extensively

elsewhere

-LSB-

-RSB-

Once

open

this

pore

allows

free

passage

of

any

molecule

of

kDa

and

so

disrupts

the

permeability

barrier

of

the

inner

membrane

This

has

two

major

consequences

First

it

allows

unrestricted

proton

movement

across

the

inner

membrane

causing

oxidative

phosphorylation

to

be

uncoupled

Not

only

does

this

prevent

ATP

synthesis

but

it

also

enables

the

proton-translocating

ATPase

to

reverse

direction

and

so

actively

hydrolyse

ATP

rather

than

synthesise

it

Under

such

conditions

intracellular

ATP

concentrations

rapidly

decline

leading

to

the

disruption

of

ionic

and

metabolic

homeostasis

and

activation

of

degradative

enzymes

such

as

phospholipases

nucleases

and

proteases

-LSB-

-RSB-

Unless

pore

closure

occurs

these

changes

will

cause

irreversible

damage

to

the

cell

resulting

in

necrotic

death

It

should

be

noted

that

opening

of

just

a

single

pore

in

one

mitochondrion

is

likely

to

cause

its

immediate

depolarisation

This

will

then

activate

further

pore

opening

in

the

same

mitochondrion

since

MPTP

opening

of

calcium

loaded

mitochondria

is

activated

by

depolarisation

-LSB-

-RSB-

Thus

mitochondria

are

either

fully

open

or

closed

and

it

is

the

fully

open

state

that

leads

to

the

second

consequence

of

MPTP

opening

the

mitochondria

swell

extensively

-LSB-

-RSB-

This

occurs

as

all

small

molecular

weight

solutes

equilibrate

across

the

inner

membrane

leaving

the

high

concentration

of

matrix

proteins

to

exert

a

colloidal

osmotic

pressure

that

leads

to

the

uptake

of

water

and

matrix

swelling

Although

unfolding

of

the

cristae

allows

the

matrix

to

expand

without

rupture

of

the

inner

membrane

the

outer

membrane

will

break

and

lead

to

the

release

of

proteins

in

the

intermembrane

space

such

as

cytochrome

c

and

other

factors

that

play

a

critical

role

in

apoptotic

cell

death

-LSB-

-RSB-

The

molecular

identity

of

the

MPTP

The

molecular

identity

of

the

mitochondrial

permeability

transition

pore

remains

uncertain

-LSB-

-RSB-

but

it

is

generally

accepted

that

an

inner

membrane

component

undergoes

a

calcium-triggered

change

in

conformation

that

is

facilitated

by

cyclophilin

D

-LRB-

CyP-D

-RRB-

a

peptidyl-prolyl

cis-trans

isomerase

-LSB-

-RSB-

The

role

of

CyP-D

was

first

suggested

by

the

discovery

that

cyclosporin

A

-LRB-

CsA

-RRB-

acts

as

a

potent

inhibitor

of

pore

opening

-LSB-

-RSB-

Further

studies

revealed

that

the

potency

of

different

CsA

analogues

to

inhibit

pore

opening

correlates

with

their

ability

to

inhibit

the

peptidyl-prolyl

cis-trans

isomerase

activity

within

the

matrix

-LSB-

-RSB-

that

was

subsequently

identified

as

CyP-D

-LSB-

-RSB-

Extensive

work

from

many

laboratories

confirmed

the

critical

role

of

CyP-D

and

this

was

finally

put

beyond

doubt

by

the

demonstration

that

MPTP

opening

in

liver

mitochondria

from

CyP-D

knockout

mice

is

much

less

sensitive

to

calcium

than

normal

mitochondria

and

is

no

longer

inhibited

by

CsA

-LSB-

-RSB-

The

identity

of

the

membrane

component

of

the

MPTP

is

less

certain

However

the

most

widely

accepted

view

is

that

the

adenine

nucleotide

translocase

-LRB-

ANT

-RRB-

normally

fulfils

this

role

and

extensive

circumstantial

data

supports

this

view

-LRB-

see

-LSB-

-RSB-

-RRB-

Thus

opening

of

the

MPTP

is

inhibited

by

adenine

nucleotides

with

a

similar

concentration

dependence

and

specificity

as

they

exhibit

when

acting

as

substrates

for

the

ANT

and

this

inhibition

is

overcome

by

the

specific

inhibitor

of

the

ANT

carboxyatractyloside

-LRB-

CAT

-RRB-

that

traps

the

ANT

in

its

c

conformation

By

contrast

another

inhibitor

of

the

ANT

bongkrekic

acid

that

causes

the

carrier

to

take

up

the

alternative

m

conformation

inhibits

pore

opening

-LSB-

-RSB-

The

ANT

can

also

account

for

the

sensitisation

of

the

MPTP

to

calcium

by

oxidative

stress

and

the

vicinal

thiol

reagent

phenylarsine

oxide

-LRB-

PAO

-RRB-

-LSB-

-RSB-

Thus

cysteine

residues

and

of

rat

can

be

cross-linked

by

oxidative

stress

or

PAO

with

modification

of

alone

being

sufficient

to

prevent

the

inhibition

of

MPTP

opening

by

adenine

nucleotides

so

stimulating

pore

opening

-LSB-

-RSB-

Further

evidence

for

an

important

role

for

the

ANT

is

the

ability

of

the

ANT

to

bind

to

CyP-D

in

a

CsA-sensitive

manner

-LSB-

-RSB-

In

addition

when

the

purified

ANT

is

reconstituted

into

proteoliposomes

high

calcium

concentrations

can

induce

the

formation

of

non-specific

channels

-LSB-

-RSB-

and

this

process

is

sensitised

to

-LSB-

-RSB-

by

the

addition

of

purified

cyclophilin

-LSB-

-RSB-

Nevertheless

despite

the

strong

evidence

in

favour

of

the

ANT

being

the

critical

membrane

component

of

the

MPTP

it

is

unlikely

to

be

essential

Thus

in

an

elegant

study

that

has

yet

to

be

confirmed

by

others

mitochondria

from

mouse

livers

in

which

and

had

been

knocked

out

were

found

to

exhibit

MPTP

opening

that

was

inhibited

by

CsA

-LSB-

-RSB-

However

pore

opening

in

the

ANT-knockout

mitochondria

required

much

higher

calcium

concentrations

than

did

wild-type

mitochondria

and

was

no

longer

sensitive

to

adenine

nucleotides

confirming

that

the

ANT

is

at

the

very

least

playing

a

critical

regulatory

role

One

possibility

is

that

the

ANT

is

the

normal

membrane

component

of

the

MPTP

but

that

other

less

abundant

members

of

the

mitochondrial

carrier

family

can

fulfil

this

role

in

the

absence

of

the

ANT

-LSB-

-RSB-

An

alternative

possibility

proposed

by

Lemasters

is

that

unfolded

membrane

proteins

form

the

MPTP

and

that

the

ANT

is

the

most

abundant

of

these

-LSB-

-RSB-

Many

other

proteins

have

been

proposed

to

be

components

of

the

MPTP

including

the

peripheral

benzenediazipine

receptor

creatine

kinase

and

the

voltage

dependent

anion

channel

-LRB-

VDAC

-RRB-

-LSB-

-RSB-

Of

these

perhaps

the

strongest

candidate

is

VDAC

since

an

interaction

between

VDAC

and

the

ANT

has

been

demonstrated

and

there

is

evidence

that

the

MPTP

may

be

associated

with

contact

sites

between

the

inner

and

outer

mitochondrial

membranes

-LSB-

-RSB-

Furthermore

initial

reports

suggested

that

VDAC

might

be

the

locus

for

inhibition

by

ubiquinone

analogues

-LSB-

-RSB-

However

the

recent

demonstration

that

liver

mitochondria

from

mice

lacking

exhibit

normal

MPTP

opening

and

inhibition

by

ubiquinone

analogues

-LSB-

-RSB-

make

an

essential

role

for

unlikely

See

note

added

in

proof

The

MPTP

opening

plays

a

central

role

in

reperfusion

injury

The

MPTP

opens

during

reperfusion

but

not

ischaemia

We

have

developed

a

technique

to

demonstrate

that

the

MPTP

is

kept

firmly

closed

in

the

heart

under

normal

physiological

conditions

but

opens

upon

reperfusion

following

a

period

of

ischaemia

This

technique

involves

measuring

the

extent

of

-LSB-

-RSB-

entrapment

in

mitochondria

that

have

undergone

the

permeability

transition

-LSB-

-RSB-

Others

have

used

fluorescence

microscopy

to

measure

MPTP

opening

in

isolated

cardiac

myocytes

and

have

also

confirmed

that

the

pore

opens

under

conditions

of

simulated

ischaemia

and

reperfusion

-LSB-

-RSB-

It

might

be

expected

that

the

pore

would

also

open

after

prolonged

ischaemia

and

some

have

reported

this

to

be

the

case

-LSB-

-RSB-

However

these

studies

relied

on

cytochrome

c

release

as

a

measure

of

MPTP

opening

but

it

is

known

that

this

can

occur

independently

of

the

MPTP

as

a

result

of

Bax

translocation

to

mitochondria

during

ischaemia

-LSB-

-RSB-

By

contrast

our

own

direct

measurements

-LSB-

-RSB-

and

those

of

others

-LSB-

-RSB-

do

not

support

MPTP

opening

in

ischaemia

Since

the

pore

has

been

shown

to

be

powerfully

inhibited

by

low

pH

-LRB-

-RRB-

-LSB-

-RSB-

and

the

time

course

of

opening

during

reperfusion

correlates

with

the

return

of

pH

to

normal

-LSB-

-RSB-

we

have

suggested

that

it

is

the

low

pH

accompanying

ischaemia

that

prevents

pore

opening

despite

the

conditions

of

oxidative

stress

low

adenine

nucleotide

and

elevated

phosphate

and

calcium

-LSB-

-RSB-

Indeed

as

would

be

predicted

maintaining

a

low

pH

during

the

initial

phase

of

reperfusion

is

known

to

protect

hearts

from

reperfusion

injury

-LSB-

-RSB-

and

may

play

a

role

in

the

cardioprotective

effects

of

inhibitors

of

the

H

exchanger

such

as

amelioride

and

cariporide

-LRB-

see

-LSB-

-RSB-

-RRB-

A

similar

mechanism

has

also

been

implicated

recently

in

the

protection

offered

by

postconditioning

-LSB-

-RSB-

It

has

also

been

proposed

that

the

rapid

energisation

of

mitochondria

at

reperfusion

will

lead

to

electrogenic

calcium

uptake

into

the

mitochondria

Since

the

calcium

has

accumulated

in

the

cytosol

during

ischaemia

this

might

be

expected

to

induce

mitochondrial

calcium

overload

and

hence

MPTP

opening

-LSB-

-RSB-

However

measurements

of

mitochondrial

-LSB-

-RSB-

in

isolated

cardiac

myocytes

imply

that

it

is

the

mitochondrial

-LSB-

-RSB-

at

the

end

of

ischaemia

rather

than

during

reperfusion

that

correlates

better

with

cell

injury

-LSB-

-RSB-

Another

factor

that

will

reinforce

MPTP

opening

at

reperfusion

is

the

surge

of

ROS

production

that

is

known

to

occur

-LSB-

-RSB-

and

this

may

well

be

the

most

important

factor

in

determining

the

outcome

of

reperfusion

-LSB-

-RSB-

Indeed

there

is

increasing

evidence

that

the

extent

of

MPTP

opening

is

a

critical

factor

in

the

transition

from

reversible

to

irreversible

reperfusion

injury

-LSB-

-RSB-

More

accurately

it

is

the

extent

to

which

pores

remain

in

an

open

state

that

correlates

with

damage

since

we

have

demonstrated

that

some

mitochondria

in

which

the

MPTP

opens

initially

subsequently

undergo

pore

closer

as

reperfusion

continues

-LSB-

-RSB-

It

is

possible

that

myocytes

in

which

the

majority

of

mitochondria

have

undergone

such

reversible

MPTP

opening

will

have

swollen

sufficiently

to

release

pro-apoptotic

proteins

such

as

cytochrome

c

and

thus

initiate

apoptosis

as

noted

in

Section

These

myocytes

are

likely

to

be

at

the

periphery

of

the

infarct

where

the

ischaemic

insult

was

incomplete

and

thus

would

account

for

the

ring

of

apoptotic

myocytes

that

have

been

observed

to

surround

the

necrotic

core

of

the

infarct

-LSB-

-RSB-

It

should

be

noted

that

mice

in

which

CyP-D

had

been

knocked

out

demonstrate

normal

development

and

cells

from

them

respond

to

a

range

of

apoptotic

stimuli

in

the

same

manner

as

wild-type

mice

-LSB-

-RSB-

Thus

MPTP

opening

can

not

the

mechanism

by

which

apoptosis

is

mediated

in

the

healthy

animal

but

this

does

not

preclude

apoptosis

being

induced

in

this

way

under

pathophysiological

situations

such

as

those

described

above

-LSB-

-RSB-

Inhibition

of

MPTP

opening

protects

hearts

from

reperfusion

injury

If

the

opening

of

the

MPTP

is

a

critical

event

in

reperfusion

injury

then

it

would

be

predicted

that

inhibition

of

pore

opening

should

protect

hearts

from

injury

Many

studies

have

confirmed

that

this

is

the

case

Protection

was

first

demonstrated

with

CsA

which

when

added

prior

to

global

ischaemia

was

shown

to

improve

haemodynamic

function

and

ATP

levels

on

reperfusion

and

to

decrease

necrotic

damage

as

reflected

in

intracellular

enzyme

release

-LSB-

-RSB-

Subsequent

studies

have

shown

that

other

CsA

analogues

and

the

alternative

CyP-D

inhibitor

sanglifehrin

A

-LRB-

SfA

-RRB-

also

provide

protection

under

these

conditions

-LSB-

-RSB-

whilst

mitochondria

isolated

from

these

hearts

showed

less

pore

opening

in

response

to

a

given

calcium

load

-LSB-

-RSB-

In

addition

CsA

and

SfA

can

reduce

the

infarct

size

of

hearts

in

which

a

coronary

artery

is

occluded

and

then

re-opened

to

mimic

the

treatment

of

a

coronary

thrombosis

In

this

model

of

reperfusion

injury

protection

is

observed

even

when

the

drug

is

added

only

at

reperfusion

-LSB-

-RSB-

Less

direct

approaches

to

inhibit

MPTP

opening

are

also

effective

at

protecting

the

heart

from

reperfusion

injury

Thus

free

radical

scavengers

are

well

known

to

be

protective

and

in

the

case

of

one

such

agent

the

anaesthetic

propofol

we

have

used

the

entrapment

technique

to

confirm

inhibition

of

the

MPTP

directly

-LSB-

-RSB-

In

addition

we

have

demonstrated

that

propofol

provides

protection

from

reperfusion

injury

in

a

pig

model

of

open

heart

surgery

-LSB-

-RSB-

Inhibition

of

the

sodium

proton

exchanger

with

cariporide

is

also

known

to

protect

the

heart

from

reperfusion

injury

-LSB-

-RSB-

and

here

too

inhibition

of

MPTP

opening

correlates

with

this

protection

-LSB-

-RSB-

This

protection

probably

reflects

less

sodium

and

hence

calcium

loading

of

the

cardiac

myocytes

during

ischaemia

coupled

with

a

lower

pH

during

the

initial

phase

of

reperfusion

both

of

which

will

reduce

MPTP

opening

The

most

powerful

agent

for

inhibiting

MPTP

opening

and

protecting

the

heart

is

pyruvate

-LSB-

-RSB-

The

presence

of

mM

pyruvate

during

ischaemia

and

reperfusion

can

almost

totally

protect

rat

hearts

from

min

global

ischaemia

and

this

is

accompanied

by

greatly

reduced

MPTP

opening

followed

by

total

pore

closure

-LSB-

-RSB-

Pyruvate

has

three

beneficial

effects

that

may

contribute

towards

its

inhibition

of

MPTP

opening

it

is

a

free

radical

scavenger

it

maintains

a

lower

intracellular

pH

during

the

early

phase

of

reperfusion

and

it

is

an

excellent

fuel

for

ATP

synthesis

that

bypasses

the

ATP-requiring

steps

of

glycolysis

-LSB-

-RSB-

Protection

by

preconditioning

involves

inhibition

of

MPTP

opening

In

view

of

the

critical

role

of

the

MPTP

in

reperfusion

injury

and

the

proven

ability

of

inhibitors

of

the

MPTP

to

protect

from

injury

it

might

be

predicted

that

ischaemic

preconditioning

-LRB-

IP

-RRB-

would

inhibit

pore

opening

Both

direct

and

indirect

techniques

have

been

used

to

confirm

this

Our

own

experiments

have

used

the

entrapment

technique

to

demonstrate

that

less

MPTP

opening

occurs

at

reperfusion

in

preconditioned

hearts

and

that

as

reperfusion

continues

the

majority

of

mitochondria

that

originally

opened

close

again

in

the

IP

hearts

but

not

in

control

hearts

-LSB-

-RSB-

Others

have

used

confocal

microscopy

to

monitor

mitochondrial

membrane

potential

in

an

isolated

cardiac

myocyte

model

of

preconditioning

and

shown

that

IP

desensitises

the

mitochondria

to

pore

opening

induced

by

oxidative

stress

-LSB-

-RSB-

Recent

experiments

performed

with

two-photon

microscopy

in

the

perfused

heart

have

confirmed

these

data

-LSB-

-RSB-

Yet

other

studies

have

investigated

the

sensitivity

of

isolated

mitochondria

from

control

and

IP

hearts

to

pore

opening

in

response

to

added

calcium

-LSB-

-RSB-

Both

light

scattering

and

mitochondrial

calcium

retention

techniques

demonstrated

that

when

mitochondria

were

isolated

immediately

after

the

preconditioning

stimulus

those

from

IP

hearts

were

not

any

less

sensitive

to

calcium-induced

pore

opening

than

those

from

control

hearts

Indeed

if

anything

they

were

more

sensitive

-LSB-

-RSB-

When

isolated

at

the

end

of

ischaemia

or

during

the

first

few

minutes

of

reperfusion

mitochondria

were

more

sensitive

to

MPTP

opening

than

those

isolated

prior

to

ischaemia

but

at

these

time

points

mitochondria

from

IP

hearts

were

considerably

less

sensitive

to

calcium-activated

pore

opening

than

those

from

control

hearts

-LSB-

-RSB-

These

data

clearly

show

that

the

IP

protocol

alone

is

not

having

a

direct

effect

on

the

MPTP

but

suggest

that

IP

inhibits

a

process

occurring

during

ischaemia

and

reperfusion

that

is

responsible

for

sensitising

the

MPTP

to

calcium

This

is

an

important

fact

to

bear

in

mind

when

evaluating

the

extensive

literature

surrounding

the

mechanisms

by

which

preconditioning

exerts

its

protective

effects

as

will

be

discussed

further

below

-LRB-

Section

-RRB-

Temperature

preconditioning

where

hearts

are

exposed

to

three

brief

hypothermic

-LRB-

°C

-RRB-

episodes

prior

to

normothermic

ischaemia

is

also

thought

to

protect

through

inhibition

of

the

MPTP

-LSB-

-RSB-

as

is

post-conditioning

-LSB-

-RSB-

Thus

mitochondria

isolated

from

such

hearts

during

reperfusion

are

less

sensitive

to

MPTP

than

those

isolated

from

control

hearts

with

temperature

preconditioning

being

even

more

protective

than

IP

-LSB-

-RSB-

Signalling

pathways

linking

preconditioning

to

inhibition

of

the

MPTP

There

is

a

plethora

of

data

implicating

many

different

signalling

pathways

in

preconditioning

and

the

relevant

role

of

each

remains

hotly

debated

-LRB-

see

-LSB-

-RSB-

-RRB-

Many

of

these

studies

have

utilised

pharmacological

agonists

and

antagonists

of

components

of

the

different

signalling

kinase

cascades

and

their

potential

targets

The

complex

interactions

that

exist

between

different

signalling

pathways

makes

interpretation

of

these

data

complicated

and

this

is

made

worse

by

the

questionable

specificity

of

some

of

the

pharmacological

interventions

The

reader

is

referred

elsewhere

for

a

detailed

consideration

of

the

many

potential

pathways

that

have

been

proposed

-LSB-

-RSB-

Here

we

will

only

provide

a

brief

overview

before

focussing

on

how

they

may

exert

their

effects

on

the

mitochondria

The

role

of

protein

kinase

C

There

is

extensive

evidence

that

protein

kinase

C

-LRB-

PKC

-RRB-

plays

a

central

role

in

preconditioning

since

inhibition

of

PKC

has

been

shown

to

block

the

protection

afforded

by

IP

and

pharmacological

activators

of

PKC

are

cardioprotective

-LRB-

see

-LSB-

-RSB-

-RRB-

There

remains

some

controversy

over

which

of

the

many

PKC

isoforms

may

be

involved

in

IP

whether

they

translocate

to

the

particulate

fraction

and

how

they

exert

their

effects

-LSB-

-RSB-

Nevertheless

there

is

a

large

body

of

evidence

to

implicate

PKCε

as

an

important

player

in

IP

-LSB-

-RSB-

Thus

hearts

from

PKCε

knockout

mice

do

not

exhibit

IP

-LSB-

-RSB-

whereas

transgenic

mice

with

cardiac-specific

over-expression

of

PKCε

or

expression

of

an

activator

of

PKCε

are

protected

from

reperfusion

injury

-LSB-

-RSB-

Some

studies

have

reported

PKCε

translocation

to

the

particulate

fraction

including

mitochondria

-LSB-

-RSB-

and

have

suggested

a

direct

inhibition

of

the

MPTP

by

PKCε

involving

phosphorylation

of

components

of

the

MPTP

such

as

the

voltage

dependent

anion

channel

-LRB-

VDAC

-RRB-

-LSB-

-RSB-

However

in

our

own

studies

we

were

unable

to

detect

PKCε

translocation

to

the

mitochondria

of

IP

hearts

-LRB-

Fig.

-RRB-

whilst

others

have

observed

that

PKC

translocation

to

the

particulate

fraction

following

IP

is

transient

and

lost

during

progressive

brief

cycles

of

ischaemia

and

reperfusion

-LSB-

-RSB-

Although

a

role

for

PKCε

in

IP

seems

established

the

mechanism

-LRB-

s

-RRB-

by

which

it

exerts

its

effects

are

less

clear

as

will

become

apparent

below

-LRB-

see

sections

-RRB-

It

is

also

uncertain

how

ischaemic

preconditioning

activates

PKCε

although

several

pathways

may

be

involved

Factors

released

during

the

brief

ischaemic

periods

such

as

adenosine

bradykinin

noradrenaline

and

opioids

may

bind

to

their

G

protein-coupled

receptors

to

stimulate

phospholipase

C

producing

diacylglycerol

that

activates

PKC

Indeed

all

of

these

factors

can

pharmacologically

precondition

the

heart

-LSB-

-RSB-

Accumulating

evidence

supports

a

role

for

the

modest

increase

in

ROS

that

occurs

during

IP

protocol

in

the

activation

of

PKC

-LSB-

-RSB-

Thus

ROS

are

known

to

activate

PKC

in

the

isolated

heart

-LSB-

-RSB-

and

IP

can

be

prevented

if

free

radical

scavengers

are

present

during

the

preconditioning

phase

-LSB-

-RSB-

Oxidation

of

critical

cysteine

residues

on

PKC

isoforms

is

known

to

cause

their

activation

-LSB-

-RSB-

and

thus

provides

a

mechanism

by

which

ROS

could

activate

PKC

The

role

of

nitric

oxide

and

cyclic

GMP

dependent

protein

kinase

Data

from

several

laboratories

have

implicated

activation

of

cyclic

GMP

dependent

protein

kinase

-LRB-

PKG

-RRB-

perhaps

by

nitric

oxide

in

the

signalling

pathway

for

IP

Thus

it

has

been

reported

that

nitric

oxide

donors

and

PKG

activators

can

induce

preconditioning

whilst

NO

scavengers

and

PKG

inhibitors

prevent

preconditioning

-LSB-

-RSB-

Pharmacological

studies

from

Garlid

's

laboratory

have

led

them

to

conclude

that

PGK

and

PKCε

work

in

concert

to

induce

IP

through

an

effect

on

the

putative

mitochondrial

ATP-dependent

potassium

channel

-LRB-

mitoKATP

see

Section

below

-RRB-

They

propose

that

cGMP

activates

PKG

localized

at

the

cytosolic

surface

of

the

mitochondrial

outer

membrane

and

that

this

phosphorylates

some

target

protein

which

in

turn

can

somehow

activate

PKCε

residing

in

the

intermembrane

space

of

mitochondria

This

PKCε

then

would

phosphorylate

the

mitoKATP

channel

to

mediate

preconditioning

-LSB-

-RSB-

In

a

subsequent

paper

they

proposed

that

activation

of

the

mitoKATP

channel

increased

ROS

formation

and

that

this

ROS

activated

a

second

pool

of

PKCε

that

inhibited

the

MPTP

-LSB-

-RSB-

Our

own

data

do

not

support

this

-LRB-

see

Sections

-RRB-

It

should

be

noted

that

an

alternative

or

additional

mechanism

has

been

proposed

by

which

nitric

oxide

may

protect

the

heart

when

present

during

the

initial

stages

of

reperfusion

-LRB-

see

-LSB-

-RSB-

-RRB-

Cytochrome

oxidase

is

competitively

inhibited

by

nitric

oxide

and

this

would

lead

to

an

inhibition

of

respiration

and

hence

a

reduction

in

the

mitochondrial

membrane

potential

It

is

suggested

that

one

consequence

of

this

would

be

less

mitochondrial

calcium

accumulation

and

hence

less

MPTP

opening

However

another

consequence

of

a

lower

mitochondrial

membrane

potential

would

be

impaired

ATP

production

which

would

not

be

predicted

for

hearts

showing

improved

recovery

Similar

arguments

have

been

made

for

the

proposed

depolarisation

caused

by

mitochondrial

KATP

channel

opening

and

these

will

be

considered

in

more

detail

in

Section

The

role

of

pro-survival

kinases

Several

laboratories

have

presented

evidence

to

implicate

activation

of

pro-survival

kinases

such

as

Akt

-LRB-

protein

kinase

B

-RRB-

in

preconditioning

-LSB-

-RSB-

It

is

proposed

that

tyrosine

kinases

are

activated

during

IP

through

some

ill-defined

interaction

with

G-protein

coupled

receptors

such

as

the

adenosine

and

bradykinin

receptors

and

that

this

causes

activation

of

phosphatidyl

inositol

phosphate

kinase

-LRB-

-RRB-

The

resulting

phosphatidylinositol

then

activates

phosphoinositide-dependent

kinase

-LRB-

-RRB-

that

in

turn

phosphorylates

and

activates

Akt

However

it

is

also

possible

that

activation

of

tyrosine

kinases

or

inhibition

of

tyrosine

phosphatases

and

the

phosphatidyl

inositol

phosphatase

PTEN

-LRB-

Phosphatase

and

TENsin

homolog

-RRB-

may

be

mediated

by

the

ROS

produced

during

the

preconditioning

stimulus

-LSB-

-RSB-

In

support

of

this

mechanism

it

has

been

demonstrated

that

phosphorylation

of

Akt

is

enhanced

by

IP

especially

during

reperfusion

and

that

pharmacological

inhibitors

of

either

Akt

or

prevent

preconditioning

-LSB-

-RSB-

It

has

further

been

proposed

that

following

phosphorylation

activated

Akt

phosphorylates

endothelial

nitric

oxide

synthase

to

produces

NO

that

acts

via

PKG

as

noted

above

-LSB-

-RSB-

Sollott

et

al.

-LSB-

-RSB-

have

suggested

that

all

these

kinases

may

converge

to

phosphorylate

and

inhibit

glycogen

synthase

kinase

3β

-LRB-

-RRB-

that

they

report

translocates

to

the

mitochondria

Inhibition

of

by

Akt-induced

phosphorylation

would

then

mediate

protection

Although

it

is

known

that

is

pro-apoptotic

and

that

its

phosphorylation

inhibits

its

activity

and

stimulates

cell

survival

-LSB-

-RSB-

the

mechanism

of

any

mitochondrial

effects

remain

to

be

elucidated

However

it

may

be

significant

that

has

been

reported

to

phosphorylate

VDAC

and

that

this

phosphorylation

is

associated

with

a

sensitisation

to

cytotoxic

drugs

that

stimulate

pore

opening

-LSB-

-RSB-

VDAC

was

widely

regarded

as

an

integral

component

of

the

MPTP

but

recent

data

showing

that

mitochondria

from

the

knockout

mouse

have

a

normal

MPTP

cast

doubt

on

this

-LSB-

-RSB-

See

note

added

in

proof

The

role

of

AMP-activated

protein

kinase

Yet

another

protein

kinase

AMP-activated

protein

kinase

-LRB-

AMPK

-RRB-

is

activated

by

a

large

variety

of

cellular

stresses

that

deplete

ATP

and

increase

AMP

including

glucose

deprivation

hypoxia

ischaemia

oxidative

stress

and

hyperosmotic

stress

-LSB-

-RSB-

It

could

be

argued

that

the

many

pharmacological

agents

known

to

induce

preconditioning

from

KATP

channel

openers

to

uncouplers

and

respiratory

chain

inhibitors

-LRB-

see

Section

-RRB-

might

all

represent

insults

to

the

heart

that

increase

AMP

and

so

activate

AMPK

as

the

focus

of

a

common

signalling

mechanism

to

protect

the

heart

-LSB-

-RSB-

Indeed

transgenic

mice

expressing

a

kinase

dead

form

of

AMPK

in

the

heart

have

been

shown

to

exhibit

greater

myocardial

necrosis

and

apoptosis

after

ischaemia/reperfusion

-LSB-

-RSB-

and

can

not

be

preconditioned

-LSB-

-RSB-

Furthermore

Nishino

et

al.

-LSB-

-RSB-

have

shown

that

PKC

inhibitors

abolished

AMPK

activation

by

ischaemic

episodes

in

the

myocardium

suggesting

that

AMPK

might

be

activated

by

IP

in

a

PKC-dependent

manner

However

our

own

data

fail

to

show

any

abrogation

of

the

IP-induced

protection

by

the

inhibitor

of

AMPK

compound

C

under

conditions

where

the

inhibitor

of

PKC

chelerythrine

did

overcome

the

protection

-LSB-

-RSB-

Taken

together

all

these

data

suggest

that

several

signalling

pathways

may

interact

or

act

in

parallel

to

induce

IP

but

the

ultimate

target

of

their

action

remains

unclear

Since

IP

involves

inhibition

of

the

MPTP

the

signalling

pathway

must

ultimately

inhibit

MPTP

opening

but

this

could

be

achieved

either

by

direct

phosphorylation

of

a

component

of

the

MPTP

or

indirectly

by

influencing

factors

that

enhance

pore

opening

such

as

by

reducing

oxidative

stress

or

calcium

overload

Mechanism

of

inhibition

of

the

MPTP

by

IP

Is

there

evidence

for

regulation

by

phosphorylation

As

noted

above

there

are

several

reports

that

protein

kinases

may

migrate

to

the

mitochondria

in

response

to

IP

suggesting

that

phosphorylation

of

a

component

of

the

MPTP

may

be

responsible

for

its

inhibition

Baines

et

al.

have

reported

that

the

MPTP

of

isolated

heart

mitochondria

was

inhibited

following

incubation

for

min

with

purified

PKCε

plus

phorbol

ester

-LSB-

-RSB-

However

such

experiments

are

hard

to

interpret

since

incubating

isolated

mitochondria

in

vitro

can

change

the

sensitivity

of

MPTP

opening

to

calcium

especially

if

ATP

is

added

since

this

is

an

inhibitor

of

MPTP

opening

in

its

own

right

-LSB-

-RSB-

Similar

criticisms

could

be

levelled

against

experiments

in

which

incubation

of

isolated

rat

liver

mitochondria

with

a

cGMP

analogue

in

the

presence

of

a

cytosolic

extract

and

ATP

caused

inhibition

of

the

calcium-induced

mitochondrial

permeability

transition

-LSB-

-RSB-

although

here

reversal

of

the

effect

by

the

PKG

inhibitor

makes

the

data

more

convincing

However

no

data

are

available

to

show

changes

in

phosphorylation

of

any

mitochondrial

protein

following

IP

that

might

account

for

inhibition

of

the

MPTP

Furthermore

when

we

and

others

measured

MPTP

opening

in

mitochondria

isolated

immediately

after

the

preconditioning

stimulus

we

observed

no

reduction

in

sensitivity

to

calcium

as

might

have

been

predicted

if

a

component

of

the

MPTP

had

been

phosphorylated

-LSB-

-RSB-

Indeed

it

is

unclear

how

activated

cytosolic

protein

kinases

could

cross

the

outer

membrane

to

phosphorylate

and

inhibit

components

of

the

MPTP

let

alone

cross

the

inner

membrane

into

the

matrix

Although

there

are

reports

to

suggest

that

this

occurs

-LSB-

-RSB-

our

own

attempts

to

demonstrate

the

translocation

of

PKCε

or

to

mitochondria

have

consistently

failed

as

illustrated

in

Fig.

A

key

aspect

of

our

experiments

was

the

use

of

Percoll

gradient

centrifugation

to

remove

plasma

membrane

contamination

which

we

have

shown

is

significant

in

less

pure

mitochondrial

fractions

This

was

demonstrated

by

the

presence

of

monocarboxylate

transporter

-LRB-

-RRB-

in

the

crude

but

not

the

purified

mitochondria

Our

inability

to

detect

protein

kinase

translocation

into

the

mitochondrial

matrix

is

entirely

consistent

with

extensive

work

from

this

laboratory

more

than

years

ago

in

which

high

specific

activity

labelling

was

used

to

investigate

the

phosphorylation

of

mitochondrial

matrix

proteins

Such

studies

demonstrated

the

presence

of

two

proteins

within

the

mitochondrial

matrix

that

could

be

phosphorylated

in

Percoll-purified

mitochondria

from

both

heart

and

liver

These

were

pyruvate

dehydrogenase

-LRB-

PDH

-RRB-

and

branched

chain

dehydrogenase

-LRB-

BCDH

-RRB-

-LSB-

-RSB-

This

was

true

whether

the

mitochondria

were

from

control

or

glucagon-treated

-LRB-

raised

cyclic

AMP

-RRB-

rats

-LSB-

-RSB-

yet

mitochondria

from

glucagon-treated

rats

were

found

to

exhibit

enhanced

calcium

retention

characteristic

of

reduced

sensitivity

of

the

MPTP

to

calcium-induced

opening

-LSB-

-RSB-

Although

these

data

do

not

support

a

role

for

protein

kinases

within

the

mitochondrial

matrix

in

the

inhibition

of

the

MPTP

following

preconditioning

they

do

not

exclude

the

possibility

of

regulation

by

phosphorylation

of

proteins

exposed

to

cytosolic

kinases

such

as

those

in

the

outer

mitochondrial

membrane

or

bound

to

it

using

scaffolding

proteins

-LSB-

-RSB-

Indeed

work

from

this

laboratory

many

years

ago

demonstrated

that

when

mitochondria

were

isolated

from

hepatocytes

incubated

with

high

specific

activity

or

isolated

mitochondria

were

incubated

with

γ

-LSB-

-RSB-

ATP

additional

phosphorylated

proteins

were

observed

Two

of

these

proteins

both

kDa

demonstrated

an

increase

in

phosphorylation

with

glucagon

treatment

-LSB-

-RSB-

However

a

weakness

of

the

labelling

technique

is

that

it

will

not

detected

proteins

whose

phosphorylation

turnover

is

small

and

this

may

account

for

the

larger

number

of

phosphorylated

proteins

within

mitochondria

detected

using

a

phosphoprotein-specific

dye

-LSB-

-RSB-

Nevertheless

a

lack

of

turnover

might

suggest

that

such

a

phosphorylation

does

not

play

an

important

role

in

signalling

There

have

been

an

increasing

number

or

reports

in

which

proteomic

and

immunodetection

methods

have

been

used

to

detect

a

variety

of

other

kinases

associated

with

the

mitochondria

-LSB-

-RSB-

However

the

extent

to

which

these

represent

real

mitochondrial

kinases

rather

than

non-specific

mitochondrial

contamination

of

the

mitochondrial

fraction

is

hard

to

assess

We

have

recently

used

a

proteomics

approach

to

identify

any

mitochondrial

proteins

whose

phosphorylation

might

be

altered

by

preconditioning

Mitochondria

were

isolated

from

control

and

IP

hearts

immediately

after

the

preconditioning

stimulus

as

well

as

the

end

of

ischaemia

and

after

three

min

reperfusion

and

phosphor-proteins

detected

using

gel

electrophoresis

and

staining

with

ProQ

Diamond

phospho-protein

fluorescent

dye

Although

a

significant

number

of

phospho-proteins

were

detected

in

addition

to

the

predominant

spots

representing

PDH

and

BCDH

no

consistent

changes

were

detected

in

any

of

these

in

response

to

IP

-LRB-

unpublished

data

of

S.J.

Clarke

I.

Khaliulin

Kate

Heesom

and

A.P.

Halestrap

-RRB-

Typical

data

are

shown

in

Fig.

Overall

our

data

do

not

support

a

role

for

mitochondrial

protein

phosphorylation

in

mediating

the

inhibition

of

MPTP

opening

by

IP

Rather

our

data

suggests

that

the

inhibition

of

the

MPTP

by

preconditioning

may

be

secondary

to

either

diminished

ROS

production

or

calcium

overload

as

described

below

Effects

of

IP

on

ROS

production

and

calcium

loading

during

ischaemia

and

reperfusion

as

potential

mediators

of

MPTP

inhibition

As

noted

above

opening

of

the

MPTP

is

triggered

by

calcium

overload

especially

when

this

is

accompanied

by

oxidative

stress

and

these

two

parameters

appear

critical

determinants

of

the

recovery

of

isolated

heart

cells

from

simulated

ischaemia

Indeed

the

extent

of

recovery

has

been

reported

to

correlate

inversely

with

mitochondrial

matrix

calcium

concentrations

at

the

end

of

ischaemia

-LSB-

-RSB-

and

to

be

triggered

by

the

rise

in

ROS

during

reperfusion

-LSB-

-RSB-

It

is

well

established

that

preconditioning

reduces

ROS

production

both

at

the

end

of

ischaemia

and

during

reperfusion

-LSB-

-RSB-

and

decreases

mitochondrial

calcium

overload

-LSB-

-RSB-

Thus

it

is

quite

possible

to

account

for

the

IP-mediated

inhibition

of

MPTP

opening

at

reperfusion

merely

through

these

indirect

effects

without

having

to

invoke

phosphorylation

of

any

mitochondrial

protein

Our

own

data

are

entirely

consistent

with

this

mechanism

Opening

of

the

MPTP

in

mitochondria

isolated

immediately

following

the

preconditioning

stimulus

showed

the

same

sensitivity

to

calcium

as

in

control

mitochondria

-LSB-

-RSB-

Mitochondria

isolated

at

the

end

of

ischaemia

or

during

reperfusion

were

more

sensitive

to

calcium-induced

MPTP

opening

than

those

isolated

prior

to

ischaemia

However

at

these

times

the

mitochondria

from

preconditioned

hearts

were

less

sensitive

to

MPTP

opening

than

were

mitochondria

isolated

from

control

hearts

at

the

same

time

-LSB-

-RSB-

This

difference

in

sensitivity

to

calcium

correlated

with

the

exposure

of

the

mitochondria

to

oxidative

stress

as

reflected

in

the

carbonylation

of

mitochondrial

proteins

-LSB-

-RSB-

a

surrogate

marker

for

oxidative

stress

-LSB-

-RSB-

These

data

confirm

that

the

oxidative

stress

mitochondria

experience

at

the

end

of

ischaemia

and

during

reperfusion

is

significantly

attenuated

in

preconditioned

hearts

As

such

they

provide

strong

evidence

in

favour

of

IP

exerting

its

primary

effect

on

the

reduction

of

ROS

levels

during

ischaemia

and

reperfusion

with

the

secondary

consequence

that

MPTP

opening

is

diminished

If

this

is

the

case

the

key

signalling

pathways

in

preconditioning

will

be

those

that

mediate

the

decrease

in

ROS

levels

at

the

end

of

ischaemia

and

during

reperfusion

This

could

be

the

result

of

decreased

ROS

production

or

of

improved

ROS

removal

but

the

ability

of

preconditioning

to

protect

against

oxidative

stress

mediated

by

exogenous

hydrogen

peroxide

-LSB-

-RSB-

argues

in

favour

of

the

latter

However

an

additional

effect

of

IP

to

reduce

ROS

production

can

not

be

ruled

out

The

role

of

mitochondrial

potassium

channels

Plasma

membrane

ATP-dependent

potassium

channels

-LRB-

KATP

-RRB-

are

strongly

expressed

in

the

heart

and

have

long

been

associated

with

cardioprotection

-LSB-

-RSB-

It

was

originally

proposed

that

opening

of

the

plasma

membrane

KATP

channel

might

hyperpolarize

the

cell

leading

to

a

shorter

action

potential

duration

-LRB-

APD

-RRB-

and

hence

less

calcium

entry

during

ischaemia

which

would

protect

the

hearts

from

calcium

overload

In

support

of

this

mouse

hearts

whose

KATP

channel

Kir

had

been

knocked

out

exhibited

greater

calcium

overload

and

ischaemia/reperfusion

injury

than

control

hearts

-LSB-

-RSB-

A

role

for

the

sarcolemmal

KATP

channels

in

the

protective

mechanism

exerted

by

preconditioning

was

first

suggested

by

the

observation

that

KATP

channel

blockers

such

as

glibencamide

prevent

preconditioning

-LRB-

see

-LSB-

-RSB-

-RRB-

Further

support

came

from

the

demonstration

that

hearts

from

the

Kir

knockout

mice

were

insensitive

to

either

ischaemic

or

pharmacological

preconditioning

-LSB-

-RSB-

However

despite

this

convincing

evidence

in

favour

of

a

role

for

the

sarcolemmal

KATP

channel

in

preconditioning

other

data

argue

against

such

a

role

In

particular

conditions

were

found

in

which

cardioprotection

could

be

observed

in

the

absence

of

APD

shortening

whilst

a

range

of

KATP

channel

openers

were

reported

to

show

a

poor

correlation

between

their

effect

on

APD

and

their

protective

effects

-LRB-

see

-LSB-

-RSB-

-RRB-

These

data

led

to

a

change

of

emphasis

away

from

the

sarlcolemmal

KATP

channel

towards

a

similar

KATP

channel

proposed

to

reside

in

the

mitochondrial

inner

membrane

The

identity

of

mitochondrial

K

channels

and

their

physiological

roles

The

presence

of

an

electrogenic

mechanism

for

K

entry

into

mitochondria

-LRB-

K

channel

-RRB-

together

with

a

K

H

antiporter

to

pump

K

out

again

has

been

known

for

more

than

years

and

their

relative

activities

are

thought

to

play

a

key

role

in

the

regulation

of

matrix

volume

-LRB-

see

-LSB-

-RSB-

-RRB-

What

is

less

clear

is

the

identity

of

these

channels

and

transporters

since

have

been

purified

sufficiently

to

allow

sequencing

Nor

has

genomic

analysis

revealed

any

plasma

membrane

K

channel

isoform

or

spliced

variant

with

a

mitochondrial

targeting

sequence

-LSB-

-RSB-

Rather

the

presence

and

properties

of

the

K

channels

and

transporters

have

been

surmised

from

their

functional

characterisation

-LRB-

see

-LSB-

-RSB-

-RRB-

Techniques

used

to

characterise

mitochondrial

K

channels

In

isolated

mitochondria

potassium

ion

movements

can

be

measured

directly

using

isotopes

or

K

sensitive

fluorescent

dyes

or

indirectly

by

determining

matrix

volume

changes

-LRB-

see

-LSB-

-RSB-

-RRB-

For

mitoplasts

patch

clamping

has

been

employed

-LSB-

-RSB-

whilst

studies

have

been

performed

on

proteoliposomes

containing

reconstituted

inner

mitochondrial

membrane

proteins

using

electrophysiological

measurement

of

single

channel

current

or

fluorescent

techniques

to

measure

net

K

transport

-LSB-

-RSB-

A

major

concern

with

the

patch

clamping

and

reconstitution

techniques

is

the

possibility

that

any

channels

detected

in

the

mitochondrial

membranes

may

actually

represent

a

small

number

of

K

channels

present

in

contaminating

sarcolemmal

membranes

It

is

known

that

most

preparations

of

isolated

mitochondria

are

extensively

contaminated

with

plasma

membranes

unless

further

purified

by

density

gradient

centrifugation

and

even

then

it

is

difficult

to

achieve

complete

removal

of

contaminating

membranes

-LSB-

-RSB-

However

in

the

majority

of

published

work

no

data

were

presented

to

discount

such

contamination

Thus

it

is

important

to

confirm

that

the

activity

of

any

channels

identified

by

such

techniques

can

be

demonstrated

in

mitochondria

and

this

can

be

achieved

by

measurements

of

mitochondrial

matrix

volume

It

is

widely

agreed

that

the

matrix

volume

is

regulated

by

the

relative

activity

of

the

K

channels

mediating

membrane

potential

driven

potassium

entry

into

mitochondria

and

the

K

H

antiporter

catalysing

proton-linked

K

extrusion

activity

-LSB-

-RSB-

Thus

activation

or

inhibition

of

any

K

channel

should

lead

to

an

increase

or

decrease

in

matrix

volume

respectively

This

can

be

measured

either

directly

using

an

isotopic

technique

based

around

the

permeability

of

into

the

matrix

and

the

exclusion

of

-LSB-

-RSB-

sucrose

or

by

light

scattering

-LRB-

see

-LSB-

-RSB-

-RRB-

The

latter

provides

a

very

sensitive

real-time

measurement

of

matrix

volume

since

as

the

mitochondria

swell

their

refractive

index

decreases

and

they

scatter

less

light

A

wavelength

of

nm

is

frequently

employed

since

this

represents

the

isosbestic

wavelength

at

which

changes

in

the

redox

state

of

the

mitochondrial

cytochromes

cause

no

change

in

absorbance

-LSB-

-RSB-

However

mitochondria

can

exhibit

light-scattering

changes

that

are

independent

of

changes

in

matrix

volume

reflecting

rather

a

change

in

morphology

or

shape

It

has

been

known

for

many

years

that

such

morphological

changes

often

referred

to

in

terms

of

orthodox

and

condensed

conformations

of

mitochondria

can

be

induced

by

ligands

of

the

adenine

nucleotide

translocase

-LRB-

ANT

-RRB-

that

switch

the

carrier

from

the

c

to

the

m

conformation

-LSB-

-RSB-

Thus

externally

added

ATP

ADP

or

bongkrekic

acid

induce

the

m

conformation

of

the

ANT

and

cause

a

contraction

of

the

mitochondria

detected

as

an

increase

in

light

scattering

whilst

carboxyatractyloside

that

induces

the

c

conformation

exerts

the

opposite

effect

Yet

these

light

scattering

responses

all

occur

without

changes

in

matrix

volume

measured

isotopically

-LSB-

-RSB-

Garlid

and

Paucek

dismiss

these

data

stating

that

this

claim

can

not

be

supported

by

any

theoretical

argument

but

the

basis

on

which

they

make

this

assertion

is

unclear

First

the

claim

is

backed

up

by

experimental

data

from

two

different

laboratories

-LSB-

-RSB-

Second

the

curvature

of

the

mitochondria

relative

to

the

wavelength

of

light

determines

its

refractive

index

and

hence

light

scattering

properties

-LSB-

-RSB-

Thus

a

shape

change

without

a

change

in

volume

-LRB-

e.g.

from

a

sphere

to

a

cylinder

-RRB-

will

affect

the

light

scattering

signal

Since

the

ANT

represents

some

or

more

of

the

inner

membrane

protein

in

heart

mitochondria

-LSB-

-RSB-

it

is

not

surprising

that

when

it

switches

its

conformation

it

can

cause

a

change

in

mitochondrial

morphology

and

hence

light

scattering

Third

Garlid

and

Paucek

argue

that

changes

in

light

scattering

induced

by

adenine

nucleotides

must

reflect

mitochondrial

volume

changes

because

they

did

not

observe

them

in

potassium

free

medium

They

go

on

to

say

If

Das

et

al.

had

carried

out

these

simple

control

experiments

they

would

have

avoided

spurious

claims

about

conformational

changes

However

we

-LSB-

-RSB-

and

Brustovetsky

et

al.

-LSB-

-RSB-

have

reported

other

controls

that

confirm

that

the

adenine

nucleotide-induced

light

scattering

changes

do

not

reflect

changes

in

matrix

volume

Thus

additions

of

very

low

concentrations

of

valinomycin

-LRB-

to

pmol

per

mg

mitochondrial

protein

-RRB-

induced

changes

in

matrix

volume

of

that

were

readily

detectable

using

radioisotopes

yet

the

magnitude

of

the

light

scattering

changes

were

similar

to

those

induced

by

ADP

or

ATP

for

which

no

detectable

changes

in

matrix

volume

were

detected

-LRB-

see

Fig.

and

-LSB-

-RSB-

-RRB-

We

have

also

performed

experiments

in

potassium

free

media

as

suggested

by

Garlid

and

Paucek

which

we

report

in

Fig.

These

data

clearly

show

that

such

adenine

nucleotide

induced

changes

in

light

scattering

do

occur

in

potassium

free

medium

and

similar

results

were

obtained

by

Brustovetsky

et

al.

-LSB-

-RSB-

We

suggest

that

experiments

directed

towards

investigating

the

activity

of

K

channels

in

intact

mitochondria

that

rely

entirely

upon

light

scattering

changes

for

measurement

of

matrix

volume

should

be

treated

with

caution

Parallel

isotopic

measurements

of

matrix

volume

provide

an

essential

confirmation

that

such

light

scattering

changes

do

reflect

changes

in

volume

rather

than

morphology

Evidence

for

the

mitochondrial

KATP

channel

Some

years

ago

we

showed

that

adenine

nucleotides

could

inhibit

electrogenic

mitochondrial

K

entry

into

mitochondria

and

that

this

was

probably

mediated

by

their

binding

to

the

ANT

-LSB-

-RSB-

in

agreement

with

earlier

proposals

-LSB-

-RSB-

More

recent

data

obtained

with

brain

mitochondria

have

confirmed

this

proposal

-LSB-

-RSB-

However

the

existence

of

a

mitochondrial

KATP

channel

similar

to

that

found

in

the

plasma

membrane

was

first

formally

proposed

as

a

result

of

data

obtained

using

patch

clamping

of

giant

fused

mitoplasts

from

liver

mitochondria

-LSB-

-RSB-

and

light

scattering

experiments

in

isolated

mitochondria

-LSB-

-RSB-

Subsequently

this

channel

has

been

studied

extensively

in

several

other

laboratories

-LRB-

see

-LSB-

-RSB-

-RRB-

Experiments

using

reconstituted

proteoliposomes

reported

the

characterisation

of

these

channels

with

respect

to

the

specificity

for

nucleotide

inhibition

and

the

activity

of

a

range

of

pharmacological

channel

openers

and

blockers

In

many

cases

the

properties

determined

are

similar

to

those

of

the

plasma

membrane

KATP

channel

lending

support

to

the

possibility

that

it

is

actually

these

channels

present

in

contaminating

plasma

membranes

that

are

being

characterised

rather

than

true

mitochondrial

channels

-LRB-

see

Section

-RRB-

However

there

are

a

number

of

differences

in

the

reported

properties

of

mitochondrial

and

sarcolemmal

KATP

channels

that

argue

against

this

For

example

mitochondrial

KATP

channels

have

been

reported

to

require

the

presence

of

for

ATP

to

inhibit

them

which

is

not

true

of

the

plasma

membrane

KATP

channel

In

addition

ADP

acts

like

ATP

to

inhibit

the

mitochondrial

channel

but

opens

the

sarcolemmal

channel

whilst

GTP

has

been

reported

to

open

the

mitochondrial

KATP

channel

but

not

the

plasma

membrane

one

-LSB-

-RSB-

Furthermore

their

measured

conductances

were

reported

to

be

distinct

-LSB-

-RSB-

and

different

dose

response

curves

for

a

range

of

pharmacological

openers

and

blockers

have

been

described

-LSB-

-RSB-

This

is

especially

true

of

the

KATP

channel

opener

diazoxide

that

has

been

reported

to

show

a

sensitivity

towards

the

mitochondrial

KATP

channel

at

least

three

orders

of

magnitude

greater

than

for

the

sarcolemmal

KATP

channel

whilst

-LRB-

-RRB-

blocks

the

mitochondrial

channel

whilst

having

little

effect

on

the

sarcolemmal

one

-LSB-

-RSB-

However

there

are

other

data

claiming

that

diazoxide

will

open

and

block

plasma

membrane

KATP

channels

at

the

concentrations

frequently

used

to

modulate

mitochondrial

KATP

channels

when

investigating

their

possible

role

in

cardioprotection

-LSB-

-RSB-

Although

no

molecular

identity

has

been

ascribed

to

the

mitochondrial

KATP

channel

at

the

genomic

level

similarities

with

the

components

of

the

plasma

membrane

KATP

channels

have

been

proposed

These

contain

one

of

two

channel

components

-LRB-

kDa

-RRB-

or

-LRB-

kDa

-RRB-

and

the

sulphohylurea

receptors

-LRB-

kDa

-RRB-

or

SUR

-LRB-

kDa

-RRB-

-LSB-

-RSB-

Two

studies

reported

that

antibodies

against

and

detected

proteins

of

the

right

size

-LRB-

about

kDa

-RRB-

in

heart

mitochondria

but

no

rigorous

steps

were

taken

to

remove

sarcolemmal

contamination

-LSB-

-RSB-

In

heart

mitochondria

antibodies

detected

a

protein

of

about

kDa

in

one

study

-LSB-

-RSB-

but

no

protein

in

the

other

study

-LSB-

-RSB-

whilst

a

protein

of

about

kDa

was

labelled

by

-LSB-

-RSB-

glibencamide

-LSB-

-RSB-

Overall

the

available

data

provide

no

strong

evidence

for

the

presence

of

a

KATP

channel

in

mitochondria

that

is

closely

related

to

the

well-characterised

plasma

membrane

KATP

channel

Garlid

's

laboratory

found

proteins

of

kDa

and

kDa

in

a

partially

purified

preparation

from

brain

mitochondrial

inner

membranes

that

showed

KATP

channel

activity

when

reconstituted

into

proteoliposomes

The

protein

showed

labelling

by

a

fluorescent

glibencamide

derivative

-LSB-

-RSB-

leading

them

to

surmise

that

the

protein

represented

the

channel

forming

component

but

no

sequence

data

were

presented

to

identify

either

component

Furthermore

because

only

Coomassie

blue

staining

of

proteins

was

used

it

was

not

established

that

there

were

not

other

minor

components

present

in

the

partially

purified

preparation

used

and

the

high

activity

of

channel

proteins

would

only

require

a

small

contamination

to

produce

significant

channel

activity

Indeed

it

should

be

noted

that

mitochondrial

preparations

used

in

this

study

were

unpurified

on

density

gradient

and

therefore

unavoidably

would

have

contained

significant

contamination

by

fragments

of

plasma

membrane

and

thus

plasmalemmal

KATP

channels

This

might

explain

why

Garlid

and

co-workers

found

to

times

larger

amounts

of

KATP

channel

in

their

preparations

of

isolated

brain

mitochondria

than

reported

in

liver

or

heart

mitochondria

In

view

of

the

problems

associated

with

the

reconstitution

and

patch-clamping

studies

discussed

above

and

the

absence

of

a

molecular

identity

for

the

mitochondrial

KATP

channel

the

best

evidence

for

the

activity

of

mitochondrial

KATP

channels

should

come

from

the

measurement

of

changes

in

matrix

volume

of

isolated

mitochondria

in

response

to

channel

openers

and

blockers

In

this

context

Garlid

's

laboratory

has

performed

extensive

experiments

using

light

scattering

to

detect

matrix

volume

changes

in

response

to

adenine

nucleotides

and

a

variety

of

KATP

channel

openers

and

blockers

However

as

noted

above

-LRB-

Section

-RRB-

we

believe

that

without

direct

measurements

of

matrix

volume

these

data

must

be

treated

with

caution

Thus

although

data

from

both

this

laboratory

and

that

of

Brustovetsky

confirmed

effects

of

adenine

nucleotides

on

light

scattering

that

are

consistent

with

the

presence

of

a

KATP

channel

neither

laboratory

was

able

to

detect

volume

changes

isotopically

despite

demonstrating

with

valinomycin

that

the

technique

was

quite

sensitive

enough

to

do

so

should

they

occur

-LSB-

-RSB-

Furthermore

neither

we

nor

Brustovetsky

et

al.

were

able

to

detect

any

significant

changes

in

light

scattering

with

a

variety

of

KATP

channel

openers

-LRB-

diazoxide

nicorandil

cromakalim

pinacidil

minoxidil

-RRB-

or

blockers

-LRB-

and

glibencamide

-RRB-

-LSB-

-RSB-

Our

own

data

have

led

us

to

conclude

that

if

mitochondrial

KATP

channels

exist

there

is

no

strong

evidence

for

them

being

active

in

isolated

mitochondria

However

this

does

not

preclude

such

channels

being

active

within

the

intact

cell

since

we

might

be

losing

a

factor

during

mitochondrial

isolation

that

is

required

for

their

activity

We

will

consider

if

there

is

any

evidence

for

this

in

the

context

of

studies

investigating

the

role

of

mitochondrial

KATP

channels

in

preconditioning

-LRB-

Section

-RRB-

Evidence

for

a

mitochondrial

calcium-activated

K

channel

Our

own

laboratory

was

the

first

to

suggest

that

calcium

might

regulate

potassium

entry

into

mitochondria

and

hence

matrix

volume

-LRB-

see

-LSB-

-RSB-

-RRB-

We

demonstrated

that

K

entry

into

liver

mitochondria

was

stimulated

by

hormones

that

activated

gluconeogenesis

such

as

glucagon

adrenaline

and

vasopressin

and

that

this

led

to

an

increase

in

matrix

volume

that

was

important

for

the

stimulation

of

mitochondrial

respiration

and

ATP

production

-LSB-

-RSB-

The

mechanism

was

shown

to

involve

a

rise

in

matrix

calcium

that

inhibits

pyrophosphatase

leading

to

a

rise

in

matrix

pyroposphate

-LRB-

PPi

-RRB-

Indeed

elevating

matrix

-LSB-

PPi

-RSB-

independently

of

for

example

by

provision

of

butyrate

also

caused

modest

mitochondrial

swelling

-LSB-

-RSB-

Since

PPi

was

known

to

bind

to

the

ANT

-LSB-

-RSB-

and

adenine

nucleotide

depletion

had

been

reported

to

increase

mitochondrial

K

uptake

-LSB-

-RSB-

we

proposed

that

the

ANT

could

act

as

a

potassium

channel

when

ATP

was

displaced

by

PPi

-LSB-

-RSB-

In

support

of

this

we

confirmed

earlier

data

-LSB-

-RSB-

that

the

permeability

of

the

inner

membrane

to

K

was

increased

in

the

presence

of

atractyloside

which

inhibits

the

ANT

by

trapping

it

in

the

c

conformation

-LSB-

-RSB-

This

mechanism

has

recently

received

support

from

studies

on

brain

mitochondria

where

it

was

shown

that

carboxyatractyloside

could

enhance

potassium

loss

from

de-energised

mitochondria

-LSB-

-RSB-

Our

own

data

were

unable

to

demonstrate

calcium

mediated

increase

in

either

matrix

-LSB-

PPi

-RSB-

or

volume

in

isolated

rat

heart

mitochondria

-LSB-

-RSB-

However

data

from

O'Rourke

's

laboratory

-LSB-

-RSB-

have

suggested

the

presence

of

a

calcium-activated

potassium

channel

-LRB-

KCa

-RRB-

in

these

mitochondria

These

workers

employed

patch-clamped

mitoplasts

to

characterise

mitochondrial

KCa

channels

that

like

the

sarcolemmal

KCa

channels

were

blocked

with

charybdotoxin

Furthermore

antibodies

against

sarcolemmal

KCa

channels

detected

immunoreactive

proteins

in

the

mitoplasts

One

problem

with

these

data

is

that

as

noted

above

it

is

well

established

that

isolated

mitochondria

are

contaminated

with

plasma

membrane

and

thus

it

is

possible

that

the

immunological

and

patch

clamping

data

were

actually

detecting

the

sarcolemmal

KCa

Is

there

good

evidence

for

mitochondrial

K

channels

being

involved

in

the

protective

mechanism

of

preconditioning

The

use

of

pharmacological

agents

is

hampered

by

lack

of

specificity

There

are

numerous

studies

reporting

protection

of

hearts

from

ischaemia

reperfusion

injury

by

drugs

that

are

claimed

to

be

specific

openers

of

the

mitochondrial

putative

mitochondrial

KATP

channel

most

notably

diazoxide

and

the

prevention

of

this

effect

by

supposed

specific

blockers

of

the

channel

in

particular

-LRB-

see

-LSB-

-RSB-

-RRB-

However

when

haemodynamic

performance

of

the

heart

is

used

to

assess

recovery

after

ischaemia/reperfusion

the

effect

of

to

reverse

the

protective

effects

of

diazoxide

is

not

universally

observed

-LSB-

-RSB-

O'Rourke

et

al.

have

suggested

a

role

for

the

putative

mitochondrial

KCa

channels

in

preconditioning

in

addition

to

mitochondrial

KATP

channels

This

proposal

was

based

on

the

observation

that

the

KCa

agonist

decreased

the

infarct

size

upon

reperfusion

after

ischaemia

and

this

was

blocked

by

paxilline

a

KCa

blocker

-LSB-

-RSB-

However

as

noted

above

-LRB-

Section

-RRB-

a

major

problem

associated

with

such

studies

is

the

assumption

that

the

pharmacological

agents

used

are

only

exerting

their

effects

on

the

mitochondrial

KATP

or

KCa

channels

In

the

absence

of

independent

verification

of

this

the

conclusions

drawn

must

be

treated

with

caution

This

is

especially

so

in

the

light

of

increasing

evidence

that

these

agents

have

other

non-specific

effects

on

mitochondria

including

inhibition

of

the

respiratory

chain

and

uncoupling

as

is

discussed

below

Diazoxide

has

been

shown

by

many

laboratories

including

our

own

to

inhibit

succinate

dehydrogenase

at

concentrations

usually

used

-LRB-

μM

-RRB-

to

open

the

mitochondrial

KATP

channel

-LSB-

-RSB-

Because

inhibiting

succinate

dehydrogenase

will

also

block

the

citric

acid

cycle

in

an

intact

cell

this

may

well

perturb

cellular

energy

metabolism

leading

to

mild

depolarisation

of

the

mitochondria

and

flavoprotein

oxidation

It

has

also

been

reported

that

both

diazoxide

and

pinacidil

can

uncouple

mitochondria

although

the

concentrations

-LRB-

μM

-RRB-

required

to

produce

a

significant

depolarisation

are

at

the

high

end

of

the

spectrum

used

in

in

vivo

studies

-LSB-

-RSB-

Interestingly

these

effects

appear

to

require

an

interaction

with

the

ANT

-LSB-

-RSB-

and

modelling

studies

based

around

the

published

structure

of

the

ANT

have

shown

potential

drug

binding

sites

on

this

membrane

protein

-LSB-

-RSB-

The

specificity

of

mitochondrial

KATP

blockers

is

also

doubtful

especially

for

the

most

commonly

used

HD

which

is

a

substituted

fatty

acid

and

has

the

potential

to

be

metabolised

Indeed

our

own

work

from

and

that

of

others

has

confirmed

that

HD

can

be

activated

to

its

CoA

derivative

and

then

further

metabolised

through

the

β-oxidation

pathway

-LSB-

-RSB-

As

such

it

can

act

as

a

poor

respiratory

substrate

In

addition

the

slow

kinetics

of

oxidation

by

dehydrogenase

the

penultimate

step

of

the

pathway

can

produce

a

bottleneck

that

can

inhibit

normal

fatty

acid

oxidation

-LSB-

-RSB-

Interestingly

the

other

commonly

used

mitochondrial

KATP

channel

blocker

glibencamide

also

has

the

potential

to

decrease

fatty

acid

oxidation

by

inhibition

of

palmitoyl

carnitine

transferase

-LSB-

-RSB-

Overall

the

specificity

of

the

mitochondrial

KATP

channel

openers

and

blockers

is

poor

and

the

ability

of

several

of

the

KATP

channel

openers

used

to

act

as

uncouplers

or

inhibitors

of

respiration

is

especially

worrying

This

is

because

it

is

well

established

that

bona

fide

respiratory

chain

inhibitors

-LSB-

-RSB-

succinate

dehydrogenase

inhibitors

-LSB-

-RSB-

and

uncouplers

-LSB-

-RSB-

are

all

cardio-protective

when

applied

before

or

during

ischaemia

even

though

they

have

no

effects

on

mitochondrial

KATP

channels

Thus

in

order

to

establish

a

role

for

mitochondrial

potassium

channels

in

ischaemic

preconditioning

more

direct

evidence

for

their

opening

during

IP

must

be

provided

Direct

measurements

of

mitochondrial

KATP

channel

activation

during

preconditioning

Two

approaches

can

be

taken

to

investigate

whether

mitochondrial

K

channel

opening

is

occurring

during

preconditioning

The

first

is

the

determination

of

matrix

volume

in

situ

and

the

second

is

measurement

of

other

effects

that

an

increase

in

electrogenic

potassium

flux

into

the

matrix

might

have

on

mitochondrial

function

within

the

cell

Measuring

matrix

volume

in

situ

Although

measurement

of

mitochondrial

volume

in

situ

in

the

perfused

heart

is

not

possible

we

have

determined

matrix

volume

in

mitochondria

isolated

very

rapidly

from

the

perfused

heart

-LSB-

-RSB-

Our

data

showed

a

significant

increase

in

matrix

volume

induced

by

both

IP

-LRB-

-RRB-

and

by

μM

diazoxide

-LRB-

-RRB-

which

is

consistent

with

the

opening

of

a

mitochondrial

KATP

channel

However

μM

the

putative

mitochondrial

KATP

channel

blocker

also

induced

an

increase

in

matrix

volume

-LRB-

-RRB-

and

failed

to

prevent

the

increase

induced

by

IP

Indeed

we

found

no

correlation

between

matrix

volume

-LRB-

pre-ischaemic

end

ischaemic

or

during

reperfusion

-RRB-

and

the

haemodynamic

recovery

of

the

heart

-LSB-

-RSB-

Sollott

et

al.

-LSB-

-RSB-

have

used

a

sophisticated

Fourier

analysis

of

transmitted

optics

linescan

imaging

with

a

laser

in

isolated

cardiac

myocytes

to

estimate

changes

in

mitochondrial

diameter

from

which

they

calculate

changes

in

matrix

volume

They

report

a

increase

in

matrix

volume

with

μM

diazoxide

and

μM

pinacidil

consistent

with

opening

of

mitochondrial

KATP

channels

However

their

approach

makes

the

assumption

that

changes

in

diameter

necessarily

mean

a

change

in

matrix

volume

but

as

noted

above

-LRB-

Section

-RRB-

this

is

not

necessarily

the

case

Furthermore

Sollott

et

al.

report

that

a

wide

variety

of

other

agents

known

to

protect

hearts

from

reperfusion

injury

through

quite

distinct

mechanisms

including

cyclosporin

A

the

sarcolemmal

sodium

proton

antiport

inhibitor

and

ligands

of

G

protein

coupled

receptors

such

as

bradykinin

and

Tyr-D-Ala-Gly-Phe-D-Leu

also

cause

similar

increases

in

matrix

volume

-LSB-

-RSB-

They

propose

that

such

an

increase

in

matrix

volume

is

critical

for

providing

a

memory

that

allows

protection

even

after

removal

of

the

primary

stimulus

Their

explanation

as

to

how

this

is

achieved

is

that

swelling

increases

the

threshold

at

which

ROS

induce

the

permeability

transition

However

why

this

should

be

the

case

is

unclear

since

measurements

on

isolated

mitochondria

have

demonstrated

that

MPTP

opening

is

enhanced

at

higher

matrix

volume

-LSB-

-RSB-

Overall

the

published

data

on

measurements

of

mitochondrial

volume

in

situ

provide

no

strong

evidence

either

for

or

against

the

opening

of

mitochondrial

K

channels

Measuring

flavoprotein

oxidation

Another

approach

used

to

investigate

the

activity

of

mitochondrial

K

channels

in

cardiac

myocytes

is

based

on

the

reasoning

that

if

a

K

channel

is

opened

the

entry

of

K

would

cause

depolarisation

of

the

mitochondrial

membrane

potential

Marban

and

colleagues

reported

that

with

diazoxide

this

can

be

detected

indirectly

in

rabbit

cardiac

myocytes

by

an

oxidation

of

mitochondrial

flavoproteins

that

is

reversed

by

-LSB-

-RSB-

Similar

effects

have

been

reported

in

guinea

pig

myocytes

using

to

open

the

putative

mitochondrial

KCa

channel

with

reversal

by

paxilline

-LSB-

-RSB-

However

other

workers

have

failed

to

reproduce

the

effects

of

diazoxide

and

in

guinea

pig

and

rat

cardiac

myocytes

leading

to

the

suggestion

that

the

effects

observed

may

reflect

substrate

deprivation

-LSB-

-RSB-

In

addition

there

are

theoretical

reasons

to

question

whether

opening

of

the

mitochondrial

KATP

channel

would

cause

a

significant

depolarisation

Indeed

Garlid

has

argued

convincingly

that

the

fluxes

of

K

predicted

to

occur

if

the

channel

opens

would

allow

minimal

perturbation

of

the

membrane

potential

even

when

giving

a

significant

increase

in

matrix

volume

-LSB-

-RSB-

In

fact

such

an

increase

in

matrix

volume

might

even

increase

membrane

potential

as

a

result

of

stimulating

the

respiratory

chain

itself

-LSB-

-RSB-

Direct

measurements

of

membrane

potential

with

the

fluorescent

dyes

TMRE

or

failed

to

detect

either

an

increase

or

decrease

-LSB-

-RSB-

There

are

however

other

mechanisms

by

which

changes

in

mitochondrial

membrane

potential

and

thus

flavoprotein

oxidation

could

be

induced

by

putative

mitochondrial

K

channel

openers

and

blockers

that

reflect

other

non-specific

sites

of

their

action

For

example

in

substrate

deprived

myocytes

where

diazoxide

has

been

shown

to

cause

flavoprotein

oxidation

this

could

be

caused

by

the

drug

inhibiting

succinate

dehydrogenase

and

thus

the

citric

acid

cycle

whilst

the

ability

of

HD

to

overcome

this

effect

might

be

the

result

of

the

drug

acting

as

a

respiratory

substrate

as

noted

above

-LRB-

Section

and

-LSB-

-RSB-

-RRB-

Proposed

mechanisms

by

which

opening

of

mitochondrial

K

channels

might

protect

hearts

from

ischaemia

reperfusion

injury

For

mitochondrial

K

channels

to

play

a

role

in

preconditioning

there

would

have

to

be

a

mechanism

by

which

their

opening

might

be

linked

to

improved

recovery

Several

possible

mechanisms

have

been

proposed

Enhanced

mitochondrial

ATP

production

It

is

known

that

an

increase

in

matrix

volume

can

stimulate

respiration

and

oxidative

phosphorylation

-LSB-

-RSB-

and

so

might

improve

ATP

production

during

reperfusion

leading

to

enhanced

haemodynamic

recovery

-LSB-

-RSB-

Although

we

were

able

to

measure

an

increase

in

mitochondrial

volume

and

rates

of

ADP-stimulated

respiration

in

mitochondria

isolated

from

hearts

immediately

following

ischaemic

preconditioning

or

diazoxide

treatment

the

effects

were

largely

lost

during

reperfusion

when

they

are

predicted

to

be

most

important

-LSB-

-RSB-

Furthermore

was

shown

to

exhibit

a

similar

increase

in

matrix

volume

but

was

not

cardioprotective

-LSB-

-RSB-

Garlid

and

colleagues

have

proposed

an

alternative

but

not

unrelated

mechanism

in

which

it

is

the

maintenance

of

the

intermembrane

space

and

the

close

association

between

the

outer

an

inner

membrane

that

is

the

critical

factor

resulting

from

KATP

channel

opening

-LSB-

-RSB-

They

argue

that

during

ischaemia

the

loss

of

membrane

potential

would

decrease

electrogenic

K

entry

into

mitochondria

and

that

this

would

cause

contraction

of

the

matrix

It

is

proposed

that

that

this

would

disrupt

the

interaction

between

VDAC

in

the

outer

membrane

octomeric

creatine

kinase

-LRB-

CrK

-RRB-

in

the

intermembrane

space

and

the

ANT

in

the

inner

membrane

leading

to

faster

permeation

of

ATP

through

VDAC

into

the

intermembrane

space

Further

translocation

of

this

ATP

into

the

matrix

through

the

ANT

would

lead

to

its

hydrolysis

that

would

be

detrimental

to

the

heart

It

is

suggested

that

opening

of

the

mitochondrial

KATP

channel

would

prevent

this

matrix

condensation

and

so

decrease

ATP

breakdown

during

ischaemia

leading

to

less

damage

There

is

some

evidence

that

ATP

decline

during

ischaemia

is

slowed

by

ischaemic

preconditioning

or

diazoxide

treatment

although

the

mechanism

originally

proposed

was

through

inhibition

of

the

ATPase

by

enhanced

binding

of

the

ATPase

inhibitor

protein

-LSB-

-RSB-

Furthermore

other

data

report

the

opposite

effect

of

preconditioning

with

the

decline

in

ATP

and

the

development

of

contracture

being

faster

than

in

control

hearts

-LSB-

-RSB-

Garlid

and

colleagues

further

propose

that

during

reperfusion

keeping

the

mitochondrial

KATP

channel

open

maintains

the

VDAC

CrK

ANT

complex

which

is

vital

to

export

ATP

rapidly

from

the

mitochondria

to

the

cytosol

where

it

is

used

to

drive

contraction

Although

this

may

be

true

our

own

data

showed

that

the

slight

increases

in

matrix

volume

of

diazoxide-treated

and

ischaemic

preconditioned

hearts

compared

to

control

hearts

was

not

significant

at

reperfusion

-LSB-

-RSB-

The

more

probable

determinant

of

the

efficiency

of

myocardial

ATP

production

during

reperfusion

is

the

extent

to

which

the

mitochondria

are

damaged

Mild

uncoupling

leads

to

less

calcium

overload

and

ROS

production

It

has

been

proposed

that

opening

of

K

channels

would

depolarise

the

mitochondria

sufficiently

during

reperfusion

to

reduce

ROS

production

and

calcium

accumulation

and

hence

prevent

MPTP

opening

-LSB-

-RSB-

However

as

noted

above

the

amount

of

depolarisation

predicted

from

K

channel

opening

is

unlikely

to

cause

much

if

any

depolarisation

-LSB-

-RSB-

and

this

is

confirmed

by

direct

measurements

of

mitochondrial

membrane

potential

that

failed

to

detect

any

depolarisation

-LSB-

-RSB-

Furthermore

should

significant

depolarisation

occur

at

reperfusion

ATP

synthesis

would

be

compromised

leading

to

poorer

rather

than

better

recovery

of

the

heart

Yet

NMR

spectroscopy

measurements

have

shown

that

the

bioenergetic

state

of

the

preconditioned

heart

improves

during

reperfusion

consistent

with

the

better

haemodynamic

function

-LSB-

-RSB-

Nevertheless

it

is

not

possible

to

rule

out

the

possibility

that

opening

of

mitoKATP

channels

might

cause

a

minor

depolarisation

during

reperfusion

or

slightly

reduce

the

repolarisation

sufficient

to

reduce

ROS

formation

without

having

a

significant

effect

on

ATP

production

Although

significant

uncoupling

during

the

reperfusion

phase

seems

unlikely

to

be

protective

it

is

known

that

adding

low

doses

of

uncoupler

prior

to

ischaemia

can

precondition

hearts

-LSB-

-RSB-

Indeed

any

interference

with

oxidative

phosphorylation

during

the

preischaemic

phase

seems

able

to

exert

a

similar

protective

effect

whether

it

is

brought

about

by

a

brief

ischaemic

episode

as

in

IP

by

addition

of

a

respiratory

chain

inhibitor

-LSB-

-RSB-

or

a

succinate

dehydrogenase

inhibitor

-LSB-

-RSB-

Since

many

of

the

putative

mitochondrial

K

channel

openers

have

also

been

shown

to

have

direct

uncoupling

effects

-LSB-

-RSB-

or

to

inhibit

components

of

the

respiratory

chain

including

succinate

dehydrogenase

-LSB-

-RSB-

this

provides

a

common

mode

of

action

for

these

agents

How

this

might

be

translated

into

a

protective

effect

at

reperfusion

is

not

clear

although

a

signalling

pathway

involving

AMP-activated

protein

kinase

provides

one

possibility

as

discussed

above

-LRB-

Section

-RRB-

Another

potential

mechanism

would

be

through

increased

levels

of

ROS

production

causing

PKC

activation

and

respiratory

chain

inhibitors

such

as

antimycin

are

known

to

increase

ROS

production

which

is

critical

in

mediating

its

protective

effects

-LSB-

-RSB-

It

has

also

been

reported

that

low

doses

of

uncoupler

can

increase

ROS

production

in

isolated

myocytes

-LSB-

-RSB-

although

how

this

might

occur

is

unclear

since

in

isolated

mitochondria

it

is

well

established

that

even

very

modest

uncoupling

greatly

decreases

ROS

production

-LSB-

-RSB-

In

the

case

of

antimycin

PKCε

translocation

has

been

demonstrated

and

its

protective

effects

shown

to

be

abolished

in

PKCε

knockout

mice

supporting

a

role

for

ROS

and

PKCε

activation

in

its

protective

mechanism

-LSB-

-RSB-

Production

of

ROS

as

a

signalling

mechanism

Some

groups

have

shown

that

diazoxide

and

other

putative

mitochondrial

potassium

channel

openers

cause

an

increase

in

ROS

production

by

isolated

heart

mitochondria

cardiac

myocytes

and

perfused

hearts

and

that

this

can

be

blocked

by

-LSB-

-RSB-

but

this

has

not

been

reproduced

in

other

studies

-LSB-

-RSB-

or

in

our

own

unpublished

experiments

Since

the

protective

effects

of

diazoxide

and

nicorandil

can

be

overcome

by

free

radical

scavengers

such

as

N

-LRB-

-RRB-

glycine

-LSB-

-RSB-

it

has

been

concluded

by

many

workers

that

opening

of

the

KATP

channel

causes

ROS

production

and

that

this

signals

preconditioning

through

the

pathways

discussed

above

Attractive

though

this

hypothesis

may

be

it

is

difficult

to

formulate

a

mechanism

by

which

this

might

occur

As

noted

above

even

if

there

were

modest

uncoupling

this

would

be

predicted

to

decrease

ROS

production

rather

than

increase

it

Another

possible

mechanism

would

be

via

an

increase

in

matrix

volume

causing

a

stimulation

of

electron

flow

from

complex

into

complex

-LSB-

-RSB-

Indeed

there

are

reports

that

low

doses

of

valinomycin

can

increase

ROS

formation

in

isolated

mitochondria

-LSB-

-RSB-

and

myocytes

-LSB-

-RSB-

whilst

pretreatment

of

the

perfused

rabbit

heart

with

valinomycin

substantially

decreased

the

infarct

size

on

reperfusion

-LSB-

-RSB-

However

in

our

own

unpublished

experiments

-LRB-

H.

Buckman

and

A.P.

Halestrap

unpublished

data

-RRB-

we

have

used

Amplex

Red

to

measured

ROS

production

by

isolated

heart

mitochondria

incubated

with

a

variety

of

respiratory

substrates

in

State

and

were

unable

to

detect

an

increase

in

ROS

production

with

any

mitochondrial

potassium

channel

opener

tested

or

when

matrix

volume

was

increased

by

decreasing

the

osmolarity

of

the

incubation

medium

Indeed

in

the

latter

case

we

actually

detected

a

decrease

ROS

production

This

is

consistent

with

the

major

locus

of

ROS

production

being

at

a

highly

reduced

site

on

complex

that

is

oxidised

by

an

increase

in

matrix

volume

as

electron

flow

out

of

complex

and

into

complex

is

stimulated

-LSB-

-RSB-

Another

consequence

of

opening

a

mitochondrial

K

channel

would

be

an

increase

in

the

mitochondrial

pH

gradient

coincident

with

a

decrease

in

membrane

potential

-LSB-

-RSB-

Whereas

the

latter

would

be

predicted

to

decrease

ROS

formation

as

outlined

above

there

is

evidence

to

suggest

than

an

increase

pH

gradient

or

matrix

pH

can

increase

ROS

formation

from

complex

-LSB-

-RSB-

However

in

view

of

the

ability

of

many

of

the

mitochondrial

KATP

channel

openers

to

inhibit

components

of

the

respiratory

chain

-LSB-

-RSB-

its

is

perhaps

more

likely

that

their

ability

to

increase

ROS

is

through

a

direct

interaction

with

a

redox

centre

in

one

of

the

respiratory

complexes

Other

proposed

mechanism

for

inhibiting

the

permeability

transition

pore

in

preconditioning

The

role

of

connexin

Recently

it

has

been

proposed

that

connexion

-LRB-

-RRB-

may

play

a

critical

role

in

linking

preconditioning

to

the

mitochondria

-LSB-

-RSB-

is

the

major

protein

of

the

gap

junction

with

hexameric

assemblies

forming

connexons

on

adjacent

cardiomyocytes

Its

traditional

role

is

to

provide

a

route

for

intercellular

communication

through

the

propagation

of

action

potential

signalling

molecules

and

metabolites

-LSB-

-RSB-

The

permeability

of

the

connexons

can

be

regulated

through

many

mechanisms

including

changes

in

-LSB-

-RSB-

pH

and

phosphorylation

by

several

protein

kinases

-LSB-

-RSB-

including

PKCε

and

PKG

both

of

which

have

been

implicated

in

IP

as

noted

above

-LRB-

see

Section

-RRB-

is

normally

partially

phosphorylated

with

low

conductance

but

progressive

dephosphorylation

occurs

during

ischemia

causing

increased

conductance

It

has

been

proposed

that

this

is

important

in

propagating

injury

from

one

cell

to

another

although

hearts

from

mice

showed

no

difference

in

their

sensitivity

to

reperfusion

injury

-LSB-

-RSB-

Studies

in

both

pig

and

rat

hearts

demonstrated

that

the

dephosphorylation

of

and

electrical

uncoupling

during

ischaemia

were

prevented

by

IP

in

a

PKC-dependent

manner

and

that

IP

increased

the

co-localization

of

several

protein

kinases

with

during

ischemia

-LSB-

-RSB-

Furthermore

hearts

from

mice

showed

no

protection

from

reperfusion

injury

by

either

IP

-LSB-

-RSB-

or

diazoxide

-LSB-

-RSB-

and

in

the

latter

case

the

ability

of

diazoxide

to

increase

ROS

was

also

abolished

These

data

imply

a

link

between

and

preconditioning

and

thus

that

can

in

some

way

influence

the

opening

of

the

MPTP

perhaps

by

a

signalling

mechanism

involving

ROS

Recent

work

has

suggested

that

this

may

be

through

a

translocation

of

to

the

mitochondria

following

preconditioning

-LSB-

-RSB-

A

small

fraction

of

primarily

in

its

phosphorylated

form

was

detected

in

purified

mitochondria

from

rat

mouse

pig

and

human

hearts

and

this

was

increased

following

preconditioning

In

these

studies

considerable

efforts

were

taken

to

show

that

this

was

not

just

the

result

of

sarcolemmal

contamination

of

the

mitochondrial

fraction

and

further

evidence

for

this

was

provided

by

immunofluorescence

confocal

microscopy

and

immuno-gold

electron

microscopy

Furthermore

sub-fractionation

of

mitochondrial

compartments

led

the

authors

to

conclude

that

is

translocated

to

the

inner

mitochondrial

membrane

although

some

may

remain

associated

with

the

outer

membrane

-LSB-

-RSB-

Although

we

have

been

able

to

detect

in

purified

mitochondria

and

in

preliminary

experiments

show

that

this

was

increased

following

preconditioning

we

have

expressed

reservations

about

the

likelihood

of

translocation

to

the

inner

mitochondrial

membrane

-LSB-

-RSB-

Furthermore

following

more

extensive

experiments

in

which

we

determined

the

extent

of

sarcolemmal

contamination

of

mitochondria

by

measuring

the

presence

of

we

were

unable

to

confirm

any

consistent

increase

in

the

associating

with

the

mitochondrial

fraction

following

IP

-LRB-

Fig.

-RRB-

Thus

the

relevance

of

translocation

to

the

mitochondria

remains

uncertain

although

the

loss

of

preconditioning

in

hearts

from

mice

argues

strongly

for

some

pool

of

playing

a

critical

role

-LSB-

-RSB-

Since

myocytes

from

the

mice

did

not

show

any

diazoxide-induced

ROS

formation

-LSB-

-RSB-

this

is

likely

to

involve

a

ROS-mediated

signalling

pathway

although

the

mechanism

linking

to

ROS

production

is

unclear

From

their

data

Garcia-Dorado

and

colleagues

propose

that

recruitment

of

to

the

mitochondria

in

IP

might

be

required

for

opening

of

the

mitochondrial

KATP

channel

leading

to

ROS

production

that

triggers

preconditioning

-LSB-

-RSB-

However

when

they

prevented

this

recruitment

with

geldanamycin

a

blocker

of

heat

shock

protein

protein

translocation

across

the

outer

mitochondrial

membrane

the

cardioprotective

effects

of

IP

were

not

lost

-LSB-

-RSB-

Furthermore

the

cardioprotective

effects

of

both

diazoxide

and

isoprenaline

were

not

accompanied

by

translocation

of

to

the

mitochondria

-LSB-

-RSB-

Thus

the

balance

of

evidence

suggests

that

translocation

to

mitochondria

is

not

essential

for

preconditioning

-LSB-

-RSB-

Transient

MPTP

opening

Hausenloy

and

colleagues

have

reported

that

when

the

MPTP

inhibitors

CsA

or

SfA

are

present

during

IP

or

preconditioning

induced

by

uncoupler

and

diazoxide

cardioprotection

is

abolished

-LSB-

-RSB-

They

conclude

that

transient

opening

of

the

MPTP

may

play

a

critical

role

in

triggering

preconditioning

and

that

this

might

be

caused

by

the

well-documented

rise

in

ROS

that

occurs

during

preconditioning

-LRB-

see

Section

-RRB-

However

we

have

challenged

these

conclusions

both

on

theoretical

and

experimental

grounds

-LSB-

-RSB-

First

our

own

data

using

the

mitochondrial

entrapment

technique

failed

to

detect

any

increase

in

MPTP

opening

immediately

following

the

preconditioning

stimulus

-LSB-

-RSB-

Second

although

the

authors

suggest

that

transient

MPTP

opening

during

the

preconditioning

phase

prevents

mitochondrial

calcium

overload

and

that

this

mediates

protection

it

is

the

calcium

overload

at

the

end

of

ischaemia

and

during

reperfusion

that

mediates

the

MPTP

opening

responsible

for

reperfusion

injury

Third

the

authors

argue

that

uncoupler

also

preconditions

hearts

by

opening

the

MPTP

but

uncouplers

will

depolarize

mitochondria

independently

of

MPTP

opening

Thus

any

protection

mediated

by

uncoupler

that

involves

a

decrease

in

mitochondrial

calcium

loading

should

be

insensitive

to

both

SfA

and

CsA

but

this

is

not

what

the

authors

observed

-LSB-

-RSB-

Hence

it

seems

probable

that

CsA

and

SfA

are

overcoming

IP

independently

of

any

effects

on

MPTP

opening

This

might

involve

roles

for

cyclophilins

in

the

preconditioning

signalling

pathway

similar

to

those

responsible

for

the

immunosuppressive

actions

of

these

drugs

-LSB-

-RSB-

Conclusions

and

future

directions

The

ability

of

preconditioning

-LRB-

and

post-conditioning

-RRB-

to

inhibit

the

opening

of

the

MPTP

during

reperfusion

and

so

provide

cardioprotection

seems

to

be

well

established

What

is

less

clear

is

the

mechanism

-LRB-

s

-RRB-

by

which

this

is

achieved

We

end

this

review

by

presenting

our

own

views

on

how

preconditioning

is

most

likely

to

be

mediated

These

are

summarised

schematically

in

Fig.

The

observation

that

mitochondria

isolated

immediately

after

the

preconditioning

protocol

show

no

protection

from

MPTP

opening

-LSB-

-RSB-

argues

against

a

signalling

pathway

that

causes

phosphorylation

of

some

regulatory

component

of

the

MPTP

as

an

early

event

Nor

have

we

been

able

to

observe

any

consistent

migration

of

protein

kinases

to

the

mitochondria

or

changes

in

phosphorylation

of

a

mitochondrial

protein

-LRB-

see

Sections

and

-RRB-

By

contrast

mitochondria

isolated

at

the

end

of

ischaemia

or

during

reperfusion

do

show

a

decreased

sensitivity

to

calcium-mediated

MPTP

opening

-LSB-

-RSB-

Here

too

we

were

unable

to

detect

consistent

migration

of

protein

kinases

to

the

mitochondria

or

changes

in

phosphorylation

of

a

mitochondrial

protein

arguing

against

this

protection

being

mediated

by

phosphorylation

-LRB-

see

Section

-RRB-

However

the

changes

in

sensitivity

of

the

MPTP

do

correlate

with

the

extent

of

protein

carbonylation

a

surrogate

indicator

of

the

oxidative

stress

the

mitochondria

have

experienced

-LSB-

-RSB-

Since

it

is

well

established

that

oxidative

stress

sensitises

the

MPTP

to

calcium

this

would

seem

to

provide

an

adequate

explanation

as

to

how

preconditioning

inhibits

MPTP

opening

at

reperfusion

-LRB-

see

Section

-RRB-

Furthermore

this

is

entirely

consistent

with

the

decrease

in

ROS

production

late

in

ischaemia

and

during

reperfusion

that

has

been

observed

directly

in

preconditioned

hearts

-LSB-

-RSB-

If

this

explanation

is

correct

then

the

focus

of

research

should

shift

towards

investigating

the

mechanisms

underlying

the

decreased

levels

of

ROS

seen

in

preconditioned

hearts

at

the

end

of

ischaemia

and

during

reperfusion

This

will

involve

consideration

of

both

the

sources

of

ROS

which

may

include

mitochondria

NAD

-LRB-

P

-RRB-

H

oxidase

or

xanthine

oxidase

and

the

pathways

that

remove

them

including

superoxide

dismutase

glutathione

peroxidase

and

catalase

-LSB-

-RSB-

The

many

signalling

pathways

identified

as

potential

mediators

of

preconditioning

including

ROS

PKCε

and

PKG

-LRB-

see

Section

-RRB-

may

converge

on

one

or

more

of

these

steps

to

decrease

ROS

levels

and

hence

reduce

activation

of

the

MPTP

by

thiol

oxidation

The

pharmacological

agents

used

to

implicate

these

pathways

may

interact

specifically

with

their

proposed

target

but

they

may

also

have

less

specific

effects

such

as

on

mitochondrial

respiratory

chain

components

-LRB-

see

Section

-RRB-

to

produce

a

modest

increase

in

ROS

levels

that

stimulate

signalling

pathways

rather

than

cause

damage

Indeed

it

does

seem

that

any

stress

to

the

myocyte

's

energy

status

be

it

through

respiratory

chain

inhibition

uncoupling

or

ischaemia

causes

preconditioning

This

may

also

implicate

the

energy

sensing

protein

kinase

AMP-activated

protein

kinase

-LRB-

AMPK

-RRB-

-LSB-

-RSB-

in

the

signalling

pathway

of

preconditioning

and

there

are

data

supporting

such

a

role

for

AMPK

in

both

the

heart

-LSB-

-RSB-

and

the

liver

-LSB-

-RSB-

In

addition

to

the

protection

seen

early

in

reperfusion

that

could

be

explained

through

the

mechanisms

described

above

there

is

good

evidence

for

ongoing

protection

against

MPTP

opening

being

important

as

reperfusion

progresses

Thus

inhibition

of

MPTP

opening

with

CsA

or

SfA

provides

effective

cardioprotection

as

measured

by

decreased

infarct

size

even

if

the

drug

is

not

present

at

the

start

of

reperfusion

provided

it

is

added

within

the

first

min

-LSB-

-RSB-

Furthermore

there

is

a

considerable

body

of

evidence

to

suggest

that

activation

of

survival

kinase

pathways

during

reperfusion

is

critical

for

protection

during

this

phase

of

the

insult

and

that

these

may

converge

on

Akt

and

to

mediate

their

effects

-LSB-

-RSB-

It

is

possible

to

bring

these

observations

together

by

invoking

the

established

phenomenon

of

MPTP-induced

ROS

production

-LSB-

-RSB-

Once

MPTP

opening

has

occurred

during

the

initial

phase

of

reperfusion

the

resulting

ROS

production

would

lead

to

a

progressive

increase

in

pore

opening

in

other

mitochondria

and

thus

an

escalating

number

of

necrotic

cells

as

reflected

in

the

ongoing

LDH

release

and

increased

infarct

size

CsA

or

SfA

will

prevent

this

ongoing

opening

but

so

too

might

the

survival

kinase

cascades

by

reducing

the

ROS

production

or

increasing

their

removal

as

described

below

It

is

well

established

that

MPTP

opening

causes

mitochondrial

swelling

and

rupture

of

the

outer

mitochondrial

membrane

-LSB-

-RSB-

This

leads

to

release

of

pro-apoptotic

factors

including

cytochrome

c

that

activates

caspase

and

hence

caspase

-LSB-

-RSB-

In

addition

it

has

been

shown

that

the

pro-apoptotic

factor

Bax

can

translocate

to

the

mitochondria

during

ischaemia

-LSB-

-RSB-

and

this

in

conjunction

with

cleaved

Bid

-LRB-

tBid

-RRB-

might

cause

the

cytochrome

c

release

observed

during

prolonged

ischaemia

-LSB-

-RSB-

despite

there

being

no

evidence

of

MPTP

opening

-LSB-

-RSB-

The

resulting

loss

of

cytochrome

c

will

slow

electron

transfer

out

of

complex

and

thus

potentially

cause

increased

ROS

production

in

either

complex

or

in

complex

An

additional

mechanism

that

may

lead

to

increased

ROS

production

during

ischaemia

and

reperfusion

is

through

caspase

mediated

cleavage

of

the

component

of

complex

-LSB-

-RSB-

Protection

from

the

rise

in

ROS

that

accompanies

such

cytochrome

c

release

could

be

mediated

by

survival

kinase

cascades

in

two

ways

They

might

stimulate

ROS

removal

as

described

above

for

the

ischaemic

phase

or

they

could

reduce

the

Bax-induced

cytochrome

c

release

Indeed

it

is

well

established

that

survival

kinases

can

block

apoptosis

by

inhibiting

cytochrome

c

release

-LSB-

-RSB-

This

is

brought

about

by

Akt-mediated

phosphorylation

of

the

pro-apoptotic

family

member

Bad

-LSB-

-RSB-

and

via

phosphorylation

and

stabilisation

of

the

anti-apoptotic

family

member

-LSB-

-RSB-

Note

added

in

proof

Since

submission

of

this

article

it

has

been

reported

that

the

properties

of

the

mitochondrial

permeability

transition

pore

in

mitochondria

devoid

of

all

VDAC

isoforms

are

the

same

as

in

mitochondria

from

wild-type

mitochondria

-LRB-

C.P.

Baines

R.A.

Kaiser

T.

Sheiko

W.J.

Craigen

J.D.

Molkentin

Voltage-dependent

anion

channels

are

dispensable

for

mitochondrial-dependent

cell

death

Nat

Cell

Biol

-LRB-

-RRB-

-RRB-

This

confirms

that

VDAC

is

not

an

essential

component

of

the

MPTP

bcdh, branched chain 2-oxoacid dehydrogenase

mitokatp, mitochondrial atp-dependent potassium channels

mptp, mitochondrial permeability transition pore

pdk1, phosphoinositide-dependent kinase 1

pi-3-kinase, phosphatidyl inositol 3 phosphate kinase

pkg, cyclic gmp-dependent protein kinase

ppiase, peptidyl-prolyl cis-trans isomerase