Mitochondria and cardioprotection

Fabio Di Lisa Æ Marcella Canton Æ Roberta Menabo ÆNina Kaludercic Æ Paolo Bernardi

Published online: 22 May 2007

� Springer Science+Business Media, LLC 2007

Abstract Major factors linking mitochondrial dysfunc-

tion with myocardial injury are analyzed along with pro-

tective mechanisms elicited by endogenous processes and

pharmacological treatments. In particular, a reduced rate of

ATP hydrolysis and a slight increase in ROS formation

appear to represent the prevailing components of self-de-

fense mechanisms, especially in the case of ischemic pre-

conditioning. These protective processes are activated by

signaling pathways, which converge on mitochondria

activating the mitochondrial KATP channels and/or inhib-

iting the mitochondrial permeability transition pore. These

pathways can also be stimulated by pharmacological

treatments. Another major goal for cardioprotection is

decreasing the burst in mitochondrial ROS formation that

characterizes post-ischemic reperfusion. Finally, mito-

chondrial targets for therapeutic intervention may include

the switch of substrate being utilized, because inhibition of

fatty acid oxidation is associated with cardioprotective

effects.

Keywords Mitochondria � Metabolism � Ischemia �Permeability Transition � Reactive oxygen species

Abbreviations

AIF Apoptosis inducing factor

BH BCL-2 homology

CPT Carnitine palmitoyl transferase

CsA Cyclosporin A

Cx43 Connexin 43

CyP Cyclophilin

Dp Protonmotive force

Dwm Mitochondrial membrane potential

ER Endoplasmic reticulum

FAO Fatty acid oxidation

IMM Inner mitochondrial membrane

IMS Intermembrane space

IPC Ischemic preconditioning

MAO Monoamine oxidase

mitoKATP Mitochondrial KATP channel

Pi Inorganic phosphate

OMM Outer mitochondrial membrane

PDH Pyruvate dehydrogenase

PTP Permeability transition pore

ROS Reactive oxygen species

SOD Superoxide dismutase

PLA2 Phospholipase A2

TPP Triphenylphosphonium

Introduction

The heart is especially vulnerable to mitochondrial

derangements owing to its strict dependence on aerobic

metabolism [1–3]. A wide variety of cardiomyopathies

Fabio Di Lisa and Paolo Bernardi are recipients of grants from CNR

and MIUR.

F. D. Lisa (&) � M. Canton � N. Kaludercic

Dipartimento di Chimica Biologica, Universita di Padova, Viale

G. Colombo 3, Padova 35121, Italy

e-mail: dilisa@civ.bio.unipd.it

P. Bernardi

Dipartimento di Scienze Biomediche Sperimentali, Universita di

Padova, Viale G. Colombo 3, Padova 35121, Italy

F. D. Lisa � R. Menabo � P. Bernardi

Istituto di Neuroscienze del CNR, Universita di Padova, Viale G.

Colombo 3, Padova 35121, Italy

123

Heart Fail Rev (2007) 12:249–260

DOI 10.1007/s10741-007-9028-z

have been reported as direct consequences of inborn errors

of mitochondrial metabolism [4–6]. In addition, alterations

of mitochondrial bioenergetics are likely to play a crucial

role in myocardial failure [7–9]. However, most of the

current knowledge on mitochondrial contribution to myo-

cardial pathology derives from studies on ischemia/reper-

fusion injury. The present article analyzes the mechanisms

of cardioprotection underlying both self-defense processes

and therapeutic strategies, and tries to sort causes from

consequences of mitochondrial impairment in the ischemic

heart.

Mitochondrial derangements in ischemia and

reperfusion

Oxygen is the main acceptor of electrons harvested from

nutrients, mainly carbohydrates and lipids through the

activity of NAD- and FAD-dependent dehydrogenases. The

reduced forms of these coenzymes are reoxidized at the

level of Complex I and II of the respiratory chain and the

electrons are funneled to complex IV where more than 90%

of the intracellular oxygen is reduced to water. Thus,

oxygen is for the most part utilized in a single reaction and

this explains the profound metabolic changes occurring

when oxygen supply is no longer sufficient for the activity

of cytochrome oxidase. In fact, the very first and immediate

consequence of an insufficient cellular oxygenation is the

inhibition of electron flow along the respiratory chain that

inevitably impairs both energy conservation and oxidative

metabolism (Fig. 1).

(i) Impairment of energy metabolism. The energy re-

leased from the flux of electrons from reducing to oxidizing

components of the respiratory chain is converted into the

extrusion of protons from the mitochondrial matrix into the

intermembrane space generating a protonmotive force (Dp)

that is composed of membrane potential (Dwm) and pH

gradient (DpHm) components. The utilization of this pro-

tonmotive force allows ATP synthesis, maintenance of ion

homeostasis and protein import for mitochondrial biogen-

esis. Therefore, by inhibiting electron flow the lack of

oxygen prevents ADP phosphorylation into ATP at the

level of F1Fo ATP synthase. Notably, this enzyme catalyzes

a reversible process of coupling between proton move-

ments across the inner mitochondrial membrane and ATP

synthesis or hydrolysis. In fact, if the proton gradient is not

generated by the respiratory chain, F1Fo ATP synthase

couples ATP hydrolysis with proton pumping to maintain

the mitochondrial membrane potential. This process ex-

plains why this enzyme is also termed ATPase, and this

reverse operation resembles the activity of other cellular

ATPases that convert ATP hydrolysis into active transport

of ions against concentration and/or electrical gradients.

The net result is that mitochondria cease to represent the

main source of intracellular ATP and become a powerful

system for hydrolyzing glycolytically-produced ATP [10–

13]. As detailed in the following paragraphs, under anoxic/

ischemic conditions the inhibition of ATP hydrolysis is

likely a major component of myocardial protection elicited

by both self-defense mechanisms and pharmacological

treatments [12].

(ii) Impairment of oxidative metabolism. The degrada-

tive oxidation of nutrients catalyzed by anaerobic dehy-

drogenases is made possible by the reduction of the

oxidized forms of pyridine and flavin nucleotides (i.e.,

NAD(P)+ and FAD). Once reduced, these coenzymes must

be immediately reoxidized, since their intracellular con-

tents are not abundant. In addition, they cannot be

replenished by extracellular sources since they cannot

permeate the cell membrane, at least at the turnover rates

dictated by catabolic pathways. Since the main pathway for

NADH(H+) and FADH2 reoxidation is the respiratory

chain, its inhibition hampers the oxidation of all substrates.

On the other hand, in the presence of respiratory chain

inhibition NADH(H+) oxidation is mostly catalyzed by

lactate dehydrogenase. This allows the prosecution of

cytosolic (anaerobic) glycolysis which is practically the

only pathway for ATP (re)synthesis under anoxic condi-

tions, especially considering that phosphocreatine is rap-

idly depleted at the onset of ischemia [14, 15]. The arrest of

fatty acid oxidation results in the accumulation of poten-

tially toxic intermediates, such as long chain acyl-CoAs

and long chain acylcarnitines [16–18]. In addition, the

formation of these metabolites exacerbates metabolic

impairments by reducing the availability of unesterified

CoASH and carnitine.

These primary modifications cause the occurrence of a

wide array of changes. Among the latter processes, a

tnemriapmiaC fo +2

sisatsoemoh

desaercniSOR

noitcudorp

PTAnoitelped

fo noitibihnilairdnohcotim

niahc yrotaripser

aimehcsi

PTA decudersisehtnys

PTA desaercnisisylordyh

gninepo PTP

Ktm PTA gninepo

fo egamad evitadixo sdipil lairdnohcotim

snietorp dna

fo esaelercitotpopa-orp

snietorp

sisorcensisotpopa

Fig. 1 Mitochondrial sequence of events triggered by ischemia. The

scheme summarizes the processes analyzed in the text. mtKATP,

mitochondrial KATP channels; PTP, mitochondrial permeability

transition pore

250 Heart Fail Rev (2007) 12:249–260

123

prominent role is attributed to impaired Ca2+ homeostasis

and increased ROS formation.

(iii) [Ca2+] increase. Since both mitochondrial Ca2+

uptake and release depend on Dp [19, 20], respiratory chain

inhibition results in profound alterations of mitochondrial

Ca2+ homeostasis that can trigger and be further exacer-

bated by opening of the mitochondrial permeability tran-

sition pore (PTP), as discussed in the following paragraphs

and in many reviews (see for instance [19, 21–24]). It is

generally accepted that during ischemia and/or reperfusion

mitochondrial [Ca2+] ([Ca2+]m) increases, yet it must be

pointed out that quite different mechanisms underlie

[Ca2+]m rise or overload. In fact, during ischemia Dp is

abolished because of respiratory chain inhibition and

[Ca2+]m may passively follow the occurrence of elevations

of cytosolic Ca2+. On the other hand, upon reperfusion, Dp

is recovered along with the capacity of mitochondria to

take up huge amounts of Ca2+. Therefore, only under the

latter conditions active accumulation of Ca2+ takes place

within the matrix, which is more likely to cause PTP

opening, mitochondrial depolarization, impairment of ATP

synthesis and initiation of massive hydrolysis of ATP. In

any case [Ca2+]m elevation is the consequence of a pre-

ceding rise in cytosolic [Ca2+] ([Ca2+]c). Therefore, the

abrupt release of Ca2+ from an overloaded matrix is not

likely to determine per se intracellular Ca2+ overload. The

lack of adequate pharmacological or genetic tools to inhibit

mitochondrial Ca2+ uptake in situ has so far prevented a

clear assessment of its role in myocardial physiology and

pathology. Relevant questions, such as mitochondrial

involvement in excitation-contraction coupling or the

relationships between mitochondrial Ca2+ uptake and cell

injury, remain indeed unanswered. Nevertheless, strategies

aimed at decreasing cytosolic Ca2+ overload are likely to

prevent or delay alterations in mitochondrial structure and

function.

(iv) Increased ROS formation. Reactive oxygen specie

(ROS) are formed within mitochondria under physiological

and pathological conditions, especially during post-ische-

mic reperfusion [25–27]. The common estimate that 2–4%

of oxygen utilized by the respiratory chain undergoes

univalent reduction becoming superoxide anion (O2–•) is

probably too high, and it has been suggested that a correct

estimate might be one order of lower magnitude [28, 29].

O2–• formed at the level of Complex I and III is rapidly

transformed into hydrogen peroxide (H2O2) by a family of

metalloenzymes, superoxide dismutases (SOD) [30]. The

inhibition of the respiratory chain favors ROS formation

lending support to the concept that respiratory complexes,

especially Complex I, are the main intracellular sites for

ROS formation. However, mitochondrial sites other than

the inner mitochondrial membrane are capable of gener-

ating H2O2 at significant rates. A relevant role in this re-

spect is likely played by monoamine oxidases (MAO).

These outer membrane flavoproteins catalyze electron

transfer from various amine compounds (including cate-

cholamines) to O2, thus producing large amounts of H2O2

[31, 32].

Another example of mitochondrial ROS generation at

sites other than the respiratory chain is provided by

p66Shc. This protein, which localizes in part within mito-

chondria, catalyzes electron transfer from cytochrome c to

oxygen. The consequent ROS formation has been linked to

PTP opening and apoptosis [33]. Indeed, fibroblasts lacking

p66Shc displayed a reduced formation of ROS in response

to oxidative stress-inducing agents. Conversely, p66Shc

overexpression resulted in enhanced stress-induced apop-

tosis [33–35]. Interestingly, life span is extended by ab-

lating p66Shc gene expression [36]. More recent work

suggests that PKC b induced phosphorylation of p66Shc on

Ser36 could cause its translocation to mitochondria [37].

Therefore, the increase in mitochondrial ROS formation

caused by p66Shc appears to amplify PKC b signaling

triggered by an initial oxidative stress.

The above-mentioned alterations trigger and underlie all

the deleterious consequences of ischemia, and their rele-

vance is likely to be crucial for any myocardial disease.

Considering mechanisms involved in cell death, PTP

opening and release of apoptogenic proteins represent the

most relevant processes.

(v) PTP opening. A central role in cell death is attributed

to PTP opening as related to a wide range of pathologies

extending far beyond myocardial injury ([21] and refer-

ences therein). This paragraph briefly summarizes the rel-

evance of PTP in myocardial ischemia. Additional details

have been addressed by numerous reviews [10, 22–24, 38]

The permeability transition describes a sudden increase of

the inner membrane permeability to solutes with molecular

weights up to 1500 Daltons (reviewed in [19, 20, 39, 40])

caused by the opening of a voltage-dependent, high-con-

ductance channel located in the inner mitochondrial

membrane defined as the permeability transition pore

(PTP). PTP opening can be induced by all the factors

analyzed above, i.e., [Ca2+]m increase, Dwm fall and oxi-

dative stress, and represents a major cause of mitochondrial

dysfunction creating conditions hardly compatible with cell

survival. This especially holds true in the case of prolonged

openings [41] that cause collapse of Dwm, depletion of ATP

and NAD+, matrix swelling, rupture of the outer membrane

followed by release of proteins of the intermembrane

space, such as cytochrome c.

The PTP opening is favored by [Ca2+]m elevation,

depolarization, increases in ROS and inorganic phosphate

(Pi). These factors are counteracted by physiological PTP

antagonists, such as elevated values of Dwm and high

concentrations of H+, Mg2+ and adenine nucleotides,

Heart Fail Rev (2007) 12:249–260 251

123

especially ADP [19, 23]. This explains the evidence ob-

tained in many laboratories that PTP opening occurs during

post-ischemic reperfusion. In fact during ischemia, intra-

cellular acidosis along with high levels of Mg2+ and ADP

overrides the PTP promoting conditions established by

Dwm decrease and increases in Ca2+ and Pi levels. Con-

versely, upon reperfusion the recovery of pH together with

a burst in ROS formation in the presence of high-matrix

concentrations of Ca2+ and Pi create the perfect scenario

for PTP opening despite the antagonizing effect of Dwm

recovery (Fig. 2).

In isolated mitochondria PTP opening is usually ob-

tained by additions of Ca2+ exceeding 0.1 mM. Such high

Ca2+ concentrations are hardly achieved in viable cells

suggesting that Ca2+ does not cause PTP opening directly,

and PTP is sensitized to Ca2+ by several processes, such as

formation of ROS or metabolites generated by Ca2+-

dependent enzymes. For instance, a recent report suggests

that PTP onset is due to a deadly mixture of ROS gener-

ation and pH normalization, whereas intracellular Ca2+

overload would be the consequence and not the cause of

bioenergetic failure [42]. On the other hand, the activation

of Ca2+-dependent enzymes could signal the occurrence of

cell damage to mitochondria by converting and amplifying

initial Ca2+-related signals into Ca2+-dependent derange-

ments. Among the possible signaling pathways we have

characterized the activation of cytosolic phospholipase A2

resulting in the formation of arachidonic acid which in-

duces apoptosis by promoting PTP opening [43]. Several

reports indicate the relevance of calpains. The relationship

between these Ca2+ dependent proteinases and cell death

involves multiple processes and cellular sites [44, 45]. The

involvement of mitochondria is suggested by evidence of

intramitochondrial localization especially of the l isoform

[46], and by the reports that calpains may be involved in

PTP opening [47], mitochondrial release of the apoptosis

inducing factor (AIF) [48] and cleavage of Bid, a proa-

poptotic member of the BCL-2 family [49]. Interestingly,

calpain activation within mitochondria appears to be trig-

gered by interaction with its natural activator, acyl CoA

binding protein [46]. Therefore, counteracting [Ca2+]i rise

and/or activation of Ca2+ dependent enzymes should rep-

resent an effective strategy for preventing PTP opening

(Fig. 3).

(vi) Release of proapoptotic proteins. Besides changes

occurring at the level of IMM and matrix space, changes in

the permeability of the outer mitochondrial membrane

(OMM) allow the efflux of proteins of the intermembrane

space (IMS) into the cytosol [50, 51]. Several of these

proteins, such as cytochrome c, AIF, endonuclease G,

SMAC-Diablo are pivotal factors for the executioner phase

of apoptosis [51–53].

The mechanisms linking impairment of mitochondrial

bioenergetics with structural changes causing IMS protein

release are subjects of ongoing studies and debates. Two

major hypotheses have been proposed highlighting pro-

cesses occurring at the IMM or at the OMM, respectively.

As far as IMM is concerned, PTP-induced mitochondrial

swelling could cause OMM rupture allowing the release of

IMS proteins. This sequence of events is easily detectable

in isolated mitochondria, but it might not be a necessary

consequence of PTP opening in situ, especially in the case

of transient openings [54]. Nevertheless, PTP-dependent

swelling of mitochondria has been observed in ischemic

tissues [55, 56]. Notably, matrix swelling is likely to be

required for releasing a large fraction of cytochrome c [57].

In fact, under physiological conditions the majority of

cytochrome c is sequestered within spaces delimited by

mitochondrial cristae, so that in the absence of matrix

swelling OMM permeabilization per se would result in the

release of only a minor fraction (about 15%) of cytochrome

c [54, 58, 59].

Hypotheses supporting a pivotal role of OMM permea-

bilization are centered on a large group of proteins related

to BCL-2 that includes pro and antiapoptotic members.

This family of proteins share up to four regions known as

BCL-2 homology (BH) domains [50, 51]. Antiapoptotic

members, such BCL-2 and BCL-XL contain all four do-

DESOLC NEPO

Hp wolgM hgih +2

SOR wol

ψ∆ m llaf

esaercni HpaC[ +2 esaercni ]

SOR hgihiP hgih

gM[ +2 esaerced ]

DESOLC NEPO

ψ∆ m yrevocer

AIMEHCSI NOISUFREPER

Fig. 2 PTP agonists vs. antagonists in ischemia and reperfusion

ralullecartnIaC[ +2 daolrevo ]

gninepo PTP

ralullecartni thgilSaC[ +2 noitavele ]

gninepo PTP

fo noitavitcAaC +2 semyzne tnedneped-

ytivitisnes desaercnIaC ot PTP fo +2

TCERID TCERIDNI

SOR

Fig. 3 Relationships between intracellular [Ca2+] elevation and PTP

opening

252 Heart Fail Rev (2007) 12:249–260

123

mains. Based upon structural features, the proapoptotic

group is divided into multidomain members that contain

BH1–3 domains, and BH3-only members. The former

subgroup includes BAX and BAK that are required for

ensuing apoptosis. In fact, in the absence of these proteins

cells become resistant to apoptogenic stimuli [60]. The

proapoptotic action of BAX and BAK appears to be

facilitated by BH3-only proteins and counteracted by an-

tiapoptotic members.

Far from being mutually exclusive, IMM and OMM

processes are likely to be coordinated and integrated. For

instance, an initial OMM permeabilization can be followed

by or trigger a PTP-dependent widening of tubular cristae

allowing rapid and complete release of IMS proteins. In

addition, it has to be pointed out that several BCL-2 family

members are also located at cellular sites other than the

OMM. Antiapoptotic members have been reported to re-

duce [Ca2+] within the endoplasmic reticulum (ER),

whereas proapoptotic members appear to promote ER Ca2+

release. Due to the close proximity of ER and mitochon-

dria, the latter event may lead to an increased probability of

PTP opening [61]. Additional evidence that BCL-2 pro-

teins and the PTP are connected is provided by the

observation that PTP is sensitized to opening in isolated

mitochondria and cells treated with compounds that share

the ability to bind the BH3 domain of BCL-2 and thus

displace BAX causing apoptosis [62]. These compounds

may be beneficial in the treatment of leukemia, suggesting

that the opposite strategy, i.e., stimulating BCL-2 interac-

tions with proapoptotic members could be effective in

decreasing the occurrence of apoptosis in diseased hearts.

Mitochondrial self-defense mechanisms

Owing to their multifaceted relationship with cell death,

mitochondria appear to be ideal targets for interventions

aimed at preserving cardiomyocyte viability. Obviously, in

the absence of oxygen mitochondrial ATP synthesis cannot

be restored or increased, yet protection results from

decreasing ATP hydrolysis. This goal can be achieved

through several mechanisms that are also a part of self-

defense mechanisms triggered by ischemic preconditioning

(IPC). For instance, mitochondrial depolarization promotes

F1Fo ATPase binding to its natural inhibitor IF(1). This

direct inhibition has been proposed to play a relevant role

in both classical and pharmacological IPC [63, 64]. Mito-

chondrial ATP hydrolysis also appears to be modulated by

BCL-2, since its overexpression in mouse hearts was found

to both increase cardiomyocyte survival upon reperfusion

and reduce the decline in ATP during ischemia. This effect

was attributed to a direct inhibition of F1Fo ATPase, since

the addition of oligomycin did not cause any additional

effect [65]. This finding extends the role of BCL-2 to

modulation of mitochondrial metabolism [12]. Interest-

ingly, BCL-2 is upregulated in preconditioned hearts, while

it is downregulated by ischemia/reperfusion [66]. In addi-

tion, its downregulation by means of antisense oligonu-

cleotides abolished IPC cardioprotection in perfused rat

hearts [67].

ATP hydrolysis could also be prevented by PTP inhi-

bition, a beneficial effect that would add the ATP sparing

effect to a wider range of protective actions, such as pre-

vention of NAD+ depletion, preservation of Ca2+ homeo-

stasis and lack of release of proapoptotic proteins [22–24].

The probability of PTP opening is reduced in precondi-

tioned hearts upon post-ischemic reperfusion which confers

a stress-tolerant state to cardiomyocytes [68, 69]. The re-

duced probability of PTP opening in preconditioned car-

diomyocytes is likely to be contributed by the activation of

signaling pathways [70–72]. The analysis of these pro-

cesses goes beyond the scope of this review and is covered

by other articles in this same issue.

In addition to the protection afforded by PTP inhibition,

numerous studies attribute a protective role to opening of

the mitochondrial KATP channel (mitoKATP) [73–75]. Upon

mitoKATP opening the electrophoretic influx of K+ into the

matrix causes mitochondrial depolarization along with

matrix swelling and alkalinization [76]. It has been sug-

gested that the matrix contraction caused by the Dwm de-

crease occurring during anoxia would be compensated by

the matrix swelling resulting from mitoKATP opening. This

type of swelling might both increase the efficiency of en-

ergy metabolism in the normoxic myocyte and accelerate

the recovery of optimal ATP concentrations during post-

ischemic reperfusion by preventing the loss of substrate

channeling otherwise caused by matrix contraction and

expansion of the intermembrane space [77].

Since cardioprotection is elicited by both PTP inhibition

and mitoKATP opening, the two processes are likely to be

related. Mitochondrial depolarization and ROS formation

have been suggested as plausible links rendering mitoKATP

an additional mode for modulating the PTP. The decrease

in mitochondrial membrane potential caused by mitoKATP

opening could reduce the driving force for mitochondrial

Ca2+ uptake, thus decreasing the probability of PTP

opening [78, 79]. On the other hand, matrix alkalinization

has been suggested to be responsible for the increase in

H2O2 formation observed in cardiomyocytes treated with

the mitoKATP opener diazoxide [80–82]. The increased

formation of ROS might reduce the probability of PTP

opening by affecting the redox state of critical thiol groups

[83]. In addition, a recent report suggests that PKCe acti-

vates mitoKATP resulting in a slight elevation of ROS

which eventually cause PTP inhibition [84], as also docu-

mented in a previous study [85]. The ROS-mediated link

Heart Fail Rev (2007) 12:249–260 253

123

between PKCe and mitoKATP might involve additional

factors. For instance, the increase in ROS formation in-

duced by diazoxide is blunted in cardiomyocytes and hearts

of heterozygous connexin 43 deficient (C·43+/–) mice [81].

This protein that forms gap junctions within the sarco-

lemma has been reported to accumulate within the IMM as

a result of IPC [86], while IPC protection is lost in C·43+/–

mice [87]. Therefore, it is tempting to speculate that C·43

favors PKCe interaction with mitoKATP or modulates di-

rectly mitoKATP activity.

Mitochondrially targeted cardioprotective approaches

Besides self-defense mechanisms pharmacological ap-

proaches have been developed to tackle myocardial injury

directly at the mitochondrial level. The majority of com-

pounds proposed so far can be grouped into three major

cardioprotective strategies, namely PTP inhibition, de-

creased ROS formation, and inhibition of fatty acid oxi-

dation (FAO).

PTP inhibition

Opening of the PTP is inhibited by cyclosporin (Cs) A after

binding to cyclophilin (CyP)-D, a matrix peptidyl-prolyl

cis-trans isomerase [88]. The very high-affinity binding

CsA to CyP-D is displaced by Ca2+ [89] implying that

under conditions of intracellular Ca2+ overload CsA might

not prevent PTP opening. In fact, in mitochondria devoid

of CyP-D, CsA sensitivity is lost, yet PTP opening can be

obtained although at higher [Ca2+] [90–92]. A relevant

implication of these results is that the effect of CsA is best

described as ‘‘desensitization’’ rather than inhibition of the

PTP [21]. This concept holds valid also for all the other

available PTP ‘‘inhibitors,’’ such as Sanglifehrin A and

NIM811 [93, 94]. Despite the fact that it is not a PTP

blocker, CsA represented an invaluable tool for the

advancement in our knowledge of PTP role in myocardial

ischemia. Indeed, CsA largely attenuates reperfusion in-

jury. The initial evidence obtained by using a pharmaco-

logical approach [95, 96] has also been validated by

developing direct assays for detecting PTP opening in

isolated cells and perfused hearts [97–100]. More recently,

PTP inhibition has been shown to limit the loss of viability

even when administered only at the time of reperfusion

[68, 101]. Definite support to the role of PTP desensitiza-

tion through modulation of its interaction with CyP-D was

recently provided by the reduced susceptibility to ischemic

injury observed in mice lacking CyP-D [90, 102]. All

together these findings demonstrate that PTP opening is

causally related to the loss of viability and represents a

crucial target for cardioprotection.

Antioxidants and MAO inhibition

Any antioxidant treatment is likely to be cardioprotective at

the mitochondrial level based upon the deleterious roles of

ROS on mitochondrial function and structure. However,

since mitochondria are the main intracellular source of

ROS, antioxidants acting at other cellular sites might have

a limited efficacy. This possible limitation has prompted

the development of ‘‘mitochondriotropic’’ compounds

[103, 104]. The strategy adopted in the design of these

molecules resembles that used for assessing mitochondrial

membrane potential whereby fluorescent lipophilic cations

are electrophoresed into the negatively charged mito-

chondrial matrix [105]. An example of this strategy is the

synthesis of triphenylphosphonium (TPP)-based com-

pounds, which were demonstrated to be effective antioxi-

dants in several cell types [106–108]. A TPP+-linked

analogue of ubiquinone, MitoQ, decreased reperfusion-in-

duced heart dysfunction, cell death, and mitochondrial

damage, whereas ubiquinone or TPP+ were ineffective

[109].

Besides scavenging ROS once they are formed an

alternative and perhaps more efficacious strategy is to

prevent their generation or at least the large increase

occurring under pathological conditions. This goal can be

achieved by mild uncoupling. In fact, mitochondrial

depolarization decreases ROS generation that is exponen-

tially dependent on Dwm [110, 111]. A decreased ROS

formation associated with myocardial protection has been

demonstrated by using uncouplers [112] or overexpressing

uncoupling proteins [113, 114]. On the other hand deple-

tion of uncoupling proteins reduces IPC protection and

increases ROS formation in cardiac myoblasts [115]. It

must be pointed out that also a severe uncoupling reduces

the loss of viability [116, 117]. This paradoxical finding

has been explained as resulting from a decreased avail-

ability of ATP for the generation of irreversible hyper-

contracture, which jeopardizes sarcolemma integrity upon

post-ischemic reperfusion [1, 118–121].

A third possibility for reducing mitochondrially gener-

ated oxidative stress is to inhibit mitochondrial processes

generating ROS at sites other than the respiratory chain.

For instance, MAO-A has been shown to represent an

important source of hydrogen peroxide in rat heart [122],

and MAO-A induced ROS formation resulted in cardio-

myocyte apoptosis [123, 124]. Quite interestingly, a recent

study demonstrated that MAO-A inhibition reduces myo-

cardial damage induced by 30 min of ischemia followed by

60 min of reperfusion in the rat heart [125]. Both in the

case of isolated cardiomyocytes and intact heart MAO-A

stimulation has been attributed to an increased availability

of its substrate serotonin. Besides investigating whether

MAO-A activity is modulated by factors other than an

254 Heart Fail Rev (2007) 12:249–260

123

increased supply of exogenous substrates, the specificity of

MAO inhibitors should be ascertained by comparing

pharmacological evidence with results from a genetic ap-

proach based on loss of function studies.

Metabolic approaches

The inhibition of fatty acid oxidation is the common

denominator of several, if not the majority of metabolic

approaches aimed at protecting the ischemic heart. The

expected goal of this strategy is the stimulation of pyruvate

dehydrogenase (PDH). In fact, acetyl-CoA produced in

large amounts by b-oxidation inhibits PDH thus hampering

the oxidation of pyruvate, while favoring its reduction to

lactate. It must be pointed out that this scenario is not likely

to occur under conditions of anoxia or severe ischemia,

since the absence of oxygen prevents b-oxidation. How-

ever, when oxygen supply no longer matches oxygen de-

mand or upon post-ischemic reperfusion when b-oxidation

and anaerobic (cytosolic) glycolysis are stimulated, the

excess FAO results in an increased lactate formation at the

expense of a decrease in the complete oxidation of glucose

into CO2 and H2O [126, 127] (Fig. 4).

It has been proposed that lactate accumulation by means

of intracellular pH decrease plays a relevant role in func-

tional and structural alterations of cardiomyocytes [126,

128]. Under these conditions, FAO inhibition decreases

lactate formation by restoring pyruvate oxidation [129].

This metabolic shift is likely to explain the protective ef-

fects of compounds reducing the mitochondrial uptake of

long chain acyl-CoAs by means of inhibition of carnitine

palmitoyl transferase (CPT, [130–132]) or by inhibiting

enzymes of b-oxidation [133–135]. At present, the greatest

clinical experience is with trimetazidine [136, 137] which

inhibits the last step of b-oxidation, namely b-keto acyl

CoA thiolase [133].

Alternatively, PDH can be directly stimulated by di-

chloroacetate [138, 139] or its inhibition can be removed

by reducing the acyl-CoA/CoASH ratio. This goal is effi-

ciently achieved by L-carnitine. It might seem paradoxical

that a substance known for its essential role in promoting

FAO mimics the protective effects of FAO inhibitors.

However, it is frequently overlooked that L-carnitine is the

substrate of several transferases that also transforms med-

ium-chain and short-chain acyl-CoAs into the corre-

sponding acylcarnitines. For instance, an increased supply

of carnitine promotes pyruvate oxidation by transforming

acetyl-CoA into acetylcarnitine which does not affect PDH

oxidation [140, 141]. This explains the apparent paradox

whereby the administration of L-carnitine to perfused rat

heart increases glucose oxidation while decreasing FAO

[142]. This biochemical rationale underlies the clinical

evidence of L-carnitine protection [143–145].

The maintenance of an optimal acyl-CoA/CoASH ratio

expands the role of carnitine in the metabolism of phos-

pholipids adding anabolic functions to the abovementioned

involvement in the oxidative catabolism of energy sub-

strates. Indeed, membrane phospholipid fatty acid turnover

and repair depend on LCACoA availability. In this respect

the role of carnitine and CPT is two fold: provide acyl-units

at no ATP-cost and buffer the harmful decrease in cellular

CoASH levels [146]. Supporting this concept, CPT inhi-

bition has been shown to result in a marked depression of

the reacylating capability in both human erythrocytes and

neuronal cells [147, 148].

The functions of carnitine in both energy metabolism

and phospholipid turnover indicate a general role of this

compound in the maintenance of cell viability. This cyto-

protective role is likely to be contributed by the inhibitory

effects on ceramide synthesis [149] and caspase activities

[150]. In cardiac myocytes exposed to doxorubicin, carni-

tine administration reduced the degree of apoptotic death

by preventing the increase in intracellular levels of cera-

mide, a powerful endogenous promoter of apoptosis [149].

The inhibition of ceramide production is the result of two

different actions of carnitine. In fact the subtraction of

palmitoyl CoA which is diverted from ceramide synthesis

to oxidative metabolism [151] is reinforced by the inhibi-

tory effect on acid sphyngomyelinase, which generates

ceramide in response to a host of apoptotic stimuli [149]. In

addition, carnitine has been shown to inhibit the activity of

caspases 3 and 8 [150], which act as initiator and execu-

tioner of apoptosis, respectively. Since the action of car-

nitine is reversed by palmitoylcarnitine. The free/esterified

AoCACL

enitinraclyteca

enitinrac-L

AoC-lytecaetavuryp

OAFsrotibihni

β-ox

idat

ion

HDP

esoculg

etatcal

AoCACL

OC 2 H + 2O

raCACL

AFF

I TPCsrotibihni

ACD

Fig. 4 Interventions favoring glucose oxidation while reducing

lactate formation. Inhibited processes are indicated by dashed lines.

CPT, carnitine palmitoyl transferase; DCA, dicholoroacetate; FAO,

fatty acid oxidation; FFA, free fatty acids; LCACoA, long-chain acyl-

Coa; LCACar, long-chain acylcarnitines; PDH, pyruvate dehydroge-

nase

Heart Fail Rev (2007) 12:249–260 255

123

carnitine ratio has been suggested to play a relevant role in

the cell commitment to apoptosis [150].

Concluding remarks

Although countless cellular processes are involved, mito-

chondria take center stage in determining the fate of

ischemic cardiomyocytes. It is now widely accepted that

both necrosis and apoptosis can be antagonized pharma-

cologically by drugs acting primarily or exclusively at the

level of mitochondria. In this respect, the PTP appears to be

the most relevant target to prevent the detrimental conse-

quences of myocardial ischemia. Although the significant

growth of information on mitochondrial processes related

to cell death and the availability of protective compounds

encourage some optimism, several relevant issues are far

from being solved. For instance, the molecular identity of

mitochondrial channels is still elusive hindering their

characterization as pharmaceutical targets. As discussed

above, the compounds available for reducing the proba-

bility of PTP opening act by interacting with Cyp-D and

not with pore components. On the other hand, our current

understanding of the role of mitoKATP is undermined by

lack of direct methods for cellular studies and scarce

specificity of available agonists and antagonists.

Elucidating the mitochondrial switches that control the

evolution of the ischemic injury toward recovery or loss of

viability remains an exciting challenge. The results will

help translating experimental strategies into the clinical

arena.

Acknowledgements This work was supported by grants from CNR,

FIRB, and MIUR.

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