SPOTLIGHT REVIEW
Cardiovascular Research (2010) 88, 30–39
doi:10.1093/cvr/cvq225
The SR–mitochondria interaction: a new player
in cardiac pathophysiology
Marisol Ruiz-Meana *, Celia Fernandez-Sanz, and David Garcia-Dorado
Laboratorio de Cardiologı́a Experimental, Area del Cor, Vall d’Hebron University Hospital and Research Institute, Universitat Autonoma de Barcelona, Pg. Vall d’Hebron, 119-129, 08035
Barcelona, Spain
Received 19 April 2010; revised 14 June 2010; accepted 28 June 2010; online publish-ahead-of-print 8 July 2010
Mitochondria are essential for energy supply and cell signalling and may be triggers and effectors of cell death. Mitochondrial respiration is
tightly controlled by the matrix Ca2+ concentration, which is beat-to-beat regulated by uptake and release mainly through the mitochondrial
Ca2+ uniporter and Na+/Ca2+ exchanger, respectively. Recent studies demonstrate that mitochondrial Ca2+ uptake is more dependent on
anatomo-functional microdomains established with the sarcoplasmic reticulum (SR) than on cytosolic Ca2+. This privileged communication
between SR and mitochondria is not restricted to Ca2+ but may involve ATP and reactive oxygen species, which has important implications
in cardiac pathophysiology. The disruption of the SR –mitochondria interaction caused by cell remodelling has been implicated in the deterioration of excitation –contraction coupling of the failing heart. The SR– mitochondria interplay has been suggested to be involved in the
depressed Ca2+ transients and mitochondrial dysfunction observed in diabetic hearts as well as in the genesis of certain arrhythmias, and
it may play an important role in myocardial reperfusion injury. During reperfusion, re-energization in the presence of cytosolic Ca2+ overload
results in SR-driven Ca2+ oscillations that may promote mitochondrial permeability transition (MPT). The relationship between MPT and
Ca2+ oscillations is bidirectional, as recent data show that the induction of MPT in Ca2+-overloaded cardiomyocytes may result in mitochondrial Ca2+ release that aggravates Ca2+ handling and favours hypercontracture. A more complete characterization of the structural arrangements responsible for SR–mitochondria interplay will allow better understanding of cardiac (patho)physiology but also, and no less
important, should serve as a basis for the development of new treatments for cardiac diseases.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Calcium microdomains † Ischaemia–reperfusion † Heart failure † Diabetes † Ageing
-----------------------------------------------------------------------------------------------------------------------------------------------------------
This article is part of the Review Focus on: Mitochondria in Cardiac Disease: Emerging Concepts and Novel Therapeutic
Targets
1. Introduction
Mitochondria have long been recognized as critical players in eukaryotic cell metabolism and energetics, but it was not until recent years
that they have re-emerged as determinants of cell death or survival,
with great relevance in cardiovascular (patho)physiology. The possibility that they form local privileged communication with other cell
organelles, in particular with endo(sarco)plasmic reticulum, has
aroused a new area of interest among cardiovascular scientists.
1.1 Mitochondria made complexity
possible
1.1.1 Energy suppliers and cell signalling mediators
The ability of cells to produce energy from atmospheric oxygen is
possible only because of mitochondria, and this feature is in the
basis of multicellular life. Electrons from dietary substrates are transferred by the respiratory complexes to the oxygen, generating an
H+ electrochemical gradient (DCm), whose energy is used by the
F1Fo-ATP synthase to drive ATP synthesis. Remarkably, as the
DCm dissipates, i.e. under hypoxic conditions or when H+ influx
follows an alternative route not coupled to energy production, mitochondria can act as ATP consumers by reversing the activity of ATP
synthase into ATP hydrolase in an attempt to maintain their own
DCm.1 The endosymbiotic evolutionary origin of mitochondria may
provide the clues to understanding their molecular response under
this and other stressful conditions. Mitochondrial energy production
requires the coordination of several sequential steps tightly interconnected and suited to cellular requirements. However, even under
well-coupled respiration, up to 1% of electron flux results in an incomplete reduction of molecular oxygen and production of superoxide
that is subsequently converted to hydrogen peroxide.2 Thus, mitochondria are the main source of reactive oxygen species (ROS) production within cells. Whereas a mild increase in oxidative stress acts
as a cell signalling mechanism required to trigger several stress
responses,3 the insidious increase in free radical production or a
burst of oxidative damage may risk mitochondrial integrity and exacerbate cell damage during different processes, from ageing to ischaemia –reperfusion.2,4 Superoxide production is very sensitive to the
* Corresponding author. Tel: +34 93 4894038; fax: +34 93 4894032, Email: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: [email protected].
Pathophysiological role of SR – mitochondria microdomains
DCm, being strongly decreased by mild uncoupling. It has been proposed that the modulation of the inner membrane H+ permeability
(by uncoupling proteins, mitochondrial ion channels, and fatty acids,
among others) may be an important mechanism of cardioprotection
due to its effect on ROS production by mitochondria.5
1.1.2 Contribution of mitochondria to ionic homeostasis
From the pioneering studies of Carafoli and Lehninger6 and Carafoli
et al.,7 mitochondria have been recognized as organelles capable of
accumulating large quantities of calcium. The physiological significance
and the specific routes of mitochondrial calcium uptake pathways,
however, have been much debated. Mitochondria take up calcium primarily through the calcium uniporter,8 whose isolation and purification
has not yet been accomplished, in a manner that is dependent on DCm
at the physiological range of cytosolic calcium concentration. Calcium
extrusion from the matrix may take place even after DCm dissipation
through the mitochondrial Na+/Ca2+ exchanger (mNCX),9 through
reversal of the uniporter,10 or by permeability transition of mitochondrial membranes.11 – 13 Mitochondrial calcium controls the rate of
energy production14 and plays an instrumental role in cell apoptosis
and necrosis.15 – 17 Moreover, mitochondrial calcium uptake and
release participates in the regulation of the amplitude and spatiotemporal pattern of intracellular calcium signals in different cell types,
including contracting cardiomyocytes.8 – 20
Among the several mitochondrial membrane ion channels and
exchangers, K channels (either ATP-sensitive or calcium-activated)
have been extensively investigated for their potential cardioprotective
roles. The opening of mitochondrial K-ATP channels21,22 or calciumactivated K channels23 has been shown to be beneficial for cell survival
under certain stress conditions, including ischaemia–reperfusion and
heart failure. The precise mechanism of this protective effect has not
been elucidated, but it is postulated that the opening of these channels
produces both an increase in mitochondrial volume24 and a certain
degree of mitochondrial uncoupling which, in turn, reduces calcium
overload.25 The regulation of matrix volume is crucial for the correct
functioning of mitochondria and is accomplished indirectly by a
number of specific exchangers, uniporters, and ion channels present
in both membranes. Moderate matrix swelling has been shown to
exert a protective function against myocardial ischaemic– reperfusion
damage.24 In particular, voltage-dependent anion channel or mitochondrial porin, the most abundant protein of the outer membrane, is a
large, water-filled pore that allows charged molecules to cross freely
to the intermembrane space.26 In the last years, aquaporins have
been found associated with mitochondrial inner membranes, where
they may constitute the molecular pathway underlying osmotic movement of water to the matrix.27 Connexin 43, a protein constitutively
forming sarcolemmal hemichannels and gap junction channels in ventricular myocytes, is also present at the inner mitochondrial membranes28,29 where it contributes to mitochondrial K+ influx.30 Resting
intramitochondrial pH has been estimated at 7.7– 8, is controlled by
several inner membrane transporters and exchangers, and may influence cytosolic pH. Thus, reverse operation of FoF1 ATPase has been
implicated in cytosolic acidification (concomitant with mitochondrial
alkalinization), and represents a triggering event in the mitochondriadependent pathway for caspase activation and apoptosis.31
1.1.3 Mitochondrial death pathways
Mitochondria are the central regulators of cell fate during exposure to
stress, as they contain the specific and latent mechanisms for
31
activating both the intrinsic apoptotic pathway and necrotic death in
a variety of cell types.19,32 The intrinsic apoptotic pathway involves
the activation and subsequent translocation to mitochondria of the
members of the Bcl2 family (Bax, Bak, and Bid), following the canonical mechanism of programmed cell death.32,33 These pro-apoptotic
proteins insert into the mitochondrial outer membrane where they
can behave as death channels, allowing the release of apoptogenic
proteins from the intermembrane space that are responsible for the
activation of the signal cascade that eventually leads to cell death
through caspase-9 and -3-dependent proteolysis. It has been
described that the intrinsic and extrinsic pathways (receptor-mediated
apoptosis) may be interconnected by the activation and subsequent
translocation of the protein Bid (a substrate of caspase 8), eventually
leading to mitochondrial permeabilization.34 An irreversible loss of
mitochondrial function or the release of some mitochondrial death
effectors, like apoptosis-inducing factor, may initiate a suicide programme by a caspase-independent pathway. Other cell death effectors
released by mitochondria are cytochrome C, second mitochondrialderived activator of caspase (Smac), and endonuclease G, among
others.32
Mitochondria-induced cell death may also be initiated by the development of a sudden increase in the permeability of the mitochondrial
inner membrane, the so-called permeability transition (MPT), which
allows the passage of ions and solutes with molecular masses up to
1500 Da.35 This phenomenon leads to the dissipation of DCm,
uncoupling of oxidative phosphorylation (ATP depletion), and
matrix swelling and is triggered by oxidative stress and matrix
calcium overload. MPT is modulated by voltage, adenine nucleotide
concentration and pH,35 and by pharmacological agents.36,37 The
exact molecular nature of MPT remains elusive,38,39 but its contribution to the pathophysiology of multiple cardiac diseases is unequivocal and has been extensively reviewed.39
1.2 The connection between mitochondria
and endoplasmic reticulum
The endoplasmic reticulum (ER) is a complex, folded membrane
network composed of tubules, vesicles, and cisternae expanded
within the cytoplasm of eukaryotic cells. It is divided into rough
(ribosome-containing) ER, smooth ER, and nuclear membrane, and
it fulfils several cellular functions, including synthesis of lipids and
steroids, metabolism of carbohydrates, manufacturing and folding of
proteins, regulation of calcium concentration, drug detoxification,
and attachment of receptors on cell membrane proteins.40 The
lumen of the ER contains the highest calcium concentration within
the cell, which is due to the activity of calcium ATPases, and a very
rich oxidative environment necessary for the formation of protein disulfide bonds and proper protein folding.41 As a result, the ER is particularly sensitive to disturbances in redox regulation and calcium
homeostasis, which can promote the unfolded protein response, an
activation of a signal transduction event aimed at accelerating degradation of defective proteins.42 Upon persistent noxious stimulus,
the ER can initiate cell death via mitochondria-dependent and
-independent apoptotic pathways.43 Thus, there is a close interconnection between ER stress and cell survival. The Bcl-2 family proteins,
first described by their actions on mitochondrial death pathway, may
also interact with ER membranes, and they have been described to
modulate ER calcium44 and protect mitochondria through regulation
of ER–mitochondria communication.45
32
Mitochondria also have an important role as intracellular reservoirs
of calcium. Interestingly, there is a clear discrepancy between mitochondrial calcium kinetics observed in in vitro preparations and in
intact cells. Studies with calcium microelectrodes or with fluorescent
markers have established that energized, isolated mitochondria may
take up significant amounts of calcium when exposed to high
calcium concentrations—in the high micromolar range—through
the low-affinity calcium uniporter, without suffering permeability transition.46,47 However, the cytosolic calcium concentration in resting
cells is well below this level, and even maximal calcium concentrations
reached after physiological stimulation (1–3 mM) appear to be insufficient for mitochondrial calcium uptake, according to the described
calcium affinity of the mitochondrial calcium uniporter. The studies
from Rizzuto et al.48,49 using chimeric recombinant aequorin targeted
to mitochondria established that the mitochondrial calcium concentration increases rapidly and transiently upon the stimulation of ER
calcium release (IP3 agonists) in different quiescent cell types, although
the speed and amplitude of the rise in mitochondrial calcium levels
was not accounted for by the relatively small increases in mean cytosolic calcium. These and other similar experimental approaches
showed that mitochondria respond more efficiently to calcium
increases in the specific sites located close to the ER calcium channels
than to increases in general cytosolic calcium, and that only a fraction
of mitochondria are exposed to these microanatomical sites.50 The
existence of a physical coupling between ER and mitochondria has
been demonstrated in experiments in which limited proteolysis with
trypsin or proteinase K significantly modified mitochondrial calcium
signalling evoked by IP3 receptor-mediated calcium release.51 Crosstalk between mitochondria and ER has been confirmed in many cell
types, including astrocytes,18 hepatocytes,52 and cardiac myocytes53,54
and has been unequivocally established by electron microscopy using
fixed samples.55 According to electronic tomography measurements,
the distance between the outer mitochondrial membrane and the ER
in situ has been calculated to be 10–50 nm.51 These findings agree
with the existence of local calcium microdomains that allow privileged
signalling between the ER and mitochondria that are responsible for
the microheterogeneity of cellular calcium.56 Mitochondria would
act as calcium sensors in a discrete number of functional microdomains (in close juxtaposition with the ER) and would allow the diffusion and amplification of the calcium signal following a
well-coordinated pattern. This concept has been proved in experiments in which high-speed mitochondrial calcium imaging revealed
propagating intramitochondrial calcium waves in intact cells that
were susceptible to interruption by overexpression of the mitochondrial fission factor Drp-1.57 A handful of biochemical and functional
results have implicated several proteins in sustaining ER–mitochondria coupling.58 – 61 The preferential communication between ER and
mitochondria has emerged as a concept with great biological relevance in a variety of tissues, and more specifically in those cells
with a high degree of specialized functions.62,63
1.3 The case of cardiac myocytes:
communication between mitochondria
and sarcoplasmic reticulum
1.3.1 Specific characteristics of mitochondria and
sarcoplasmic reticulum in cardiac cells
Cardiac myocytes are the paradigm of highly compartmentalized cells,
with a terminally differentiated morphology and extremely specialized
M. Ruiz-Meana et al.
functions. Their large size, as well as the uninterrupted mechanical
work they exert throughout life, makes cardiac myocytes the most
energy-demanding cells in the body. The development of contractile
activity is also possible by a precise and perfectly coupled calcium
oscillatory movement. These requirements have impacted on the
specialization and abundance of cardiac mitochondria and sarcoplasmic reticulum (SR), the two organelles that coordinate energy production and calcium control. Cardiac mitochondria occupy as much
as 40% of cell volume and are unique, in that they can be morphologically and biochemically differentiated into two subpopulations: subsarcolemmal mitochondria, located beneath the plasma membrane, and
interfibrillar mitochondria, located between myofibrils.64 Morphological differences in the two types of mitochondria are subtle, but respiratory activity and the capacity to retain calcium are significantly higher
in interfibrillar mitochondria.65 It has been recently described that
connexin 43, a protein whose translocation to mitochondrial membranes has been associated with cardioprotection during ischaemic
preconditioning,28,29 is present only in subsarcolemmal mitochondria.66 Similarly, functional decline associated with ageing is predominantly manifested in interfibrillar mitochondria,67 and diabetic
interfibrillar mitochondria have been shown to display a greater propensity for undergoing apoptosis when compared with subsarcolemmal mitochondria.68 It can be speculated that biochemical differences
between the two mitochondrial subpopulations may be the result of
their different interaction with other intracellular organelles.
The SR shares several morphofunctional features with the ER, like
the complex tubular structure and cisternae, but they differ in some
biochemical characteristics and interactions with other cell structures.
In striated skeletal and cardiac cells, the SR coexists with the classical
ER-containing ribosomal network. The SR constitutes the main intracellular calcium store in striated muscle and plays a crucial role in excitation–contraction coupling. Owing to its high degree of functional
specialization, the vast majority of SR protein content in cardiac
cells corresponds to calcium transporters SERCA at the longitudinal
region, and calcium release channels (ryanodine receptors, or RyR)
at the region juxtaposed to T-tubules.56,69 SERCA belongs to a
family of ATP-dependent calcium transporters that maintain intraluminal calcium concentration 3–4 orders of magnitude greater than
cytosolic calcium,70 and it is regulated by phospholamban, a 22 kDa
protein present in the longitudinal fraction of SR.71 SERCA2 is the
only isoform expressed in cardiac cells. RyR mediate the release of
calcium from SR to myofibrils during cell contraction. RyR channels
are blocked by micromolar concentrations of ryanodine and activated
by millimolar concentrations of caffeine, and they are the main components responsible for calcium-induced calcium release, a cycle of
positive feedback, in cardiac cells.72 RyR2 is the only isoform
expressed in myocardial tissue. RyR have a complex regulatory mechanism, and several factors have been described to modify their open
probability apart of calcium, like oxidation of critical residues, phophorylation/dephosphorylation events, and protein interactions
(mainly with calmodulin, FKBP12.6, and calsequestrin).3,73,74 Moreover, SR calcium storage capacity is regulated by a series of intraluminal, low-affinity calcium-binding proteins. During calcium transients,
only 40 –60% of total SR calcium is released.75,76 Calsequestrin, the
most abundant intraluminal protein, acts as a calcium sensor
capable of inhibiting RyR activity at low calcium concentrations and
terminating SR calcium release during normal cell contraction.77
Several other less-represented SR proteins also participate in SR
calcium regulation.78 Efficient delivery of calcium and ATP to the
33
Pathophysiological role of SR – mitochondria microdomains
sarcomere is possible because of an intimate structural interaction
between T-tubules, RyR channels, myofilaments, and mitochondria.
Calcium uptake by mitochondria located in close proximity to SR
calcium release channels participates in the modulation of the
calcium transient and contraction.79
1.3.2 Functional microdomains between mitochondria
and SR
In cardiac myocytes, mitochondria form a dynamic and continuous
network surrounding myofibrils and the SR network, within which
the inter-organelle distance has been estimated to be 10–50 nm.51
SR– mitochondria communication occurs through anatomical and
functional cell microdomains and is in part responsible for the heterogeneous distribution of calcium across the cytosol of cardiac cells. It
has been demonstrated that mitochondrial calcium uptake is more
dependent on high cytosolic Ca2+ microdomains around the
contact sites with SR than on the cytosolic Ca2+ concentration.54
Although it is clear that the interplay between SR and mitochondria
allows a rapid exchange of molecules between both organelles,50
the mechanism of such diffusion is less well understood. According
to the recent evidence, SR–mitochondrial communication is supported by a physical coupling resistant to purification procedures
that cause demolition of cytoskeletal structure.50 By means of
conventional transmission electron microscopy as well as electron
tomography, it has been possible to visualize tethering structures
(electron-dense bridges) between the terminal cisternae of the SR
and mitochondria80 (Figure 1). In any case, the mitochondrial
calcium uniporter is exposed to local high calcium concentrations,
since RyR-mediated calcium propagation to mitochondria persists
even after cytosolic calcium chelation with BAPTA in permeabilized
cardiac myocytes.81
Cross-talk between mitochondria and SR is not only a determinant
of calcium handling but for a proper matching of energy supply and
demand and regulation of mitochondrial respiration.82,83 More
recently, the concept of ‘ROS microdomains’ has been proposed to
describe a spatially restricted ROS generation with different physiological effects, depending on their localization.84 Because mitochondria are the major producers of superoxide, it can be speculated
that mitochondrial ROS production may specifically impact SR function and calcium microdomains. In fact, both SERCA pumps and
RyR channels contain multiple, potentially redox-sensitive cysteine
residues, and cysteine thiol oxidation has been described to increase
RyR channel activity.3 Therefore, it is not surprising that photostimulation of mitochondrial ROS production is able to induce a transient
increase in SR calcium sparks in rat cardiac myocytes.85 Remarkably,
the relationship between ROS and calcium at the locally restricted
cell domains may take place in both directions. A recent report86
has shown that local increases in cytosolic calcium concentration,
initiated by calcium release from the SR, increased ROS production
and favoured MPT. Thus, the existence of a mutual relationship
between calcium and ROS signalling is favoured by the SR–mitochondria interface and probably represents one of the most important signalling mechanisms in cardiac cells.
2. Mitochondria and SR in
cardiovascular pathophysiology
The interplay between SR and mitochondria has some genuine
characteristics that go beyond the individual role of each organelle.
Functional consequences of the close anatomical interaction
between SR and mitochondria are increasingly recognized as playing
important roles in the pathophysiology of several conditions. Truncation or modification of the physiological behaviour of one of the components of these microdomains may impact on the other, amplify cell
injury, and even induce necrotic or apoptotic death.87,88 Defects in
the intercommunication between SR and mitochondria make
cardiac myocytes particularly vulnerable to problems related to
ATP availability, sarcomeric calcium, and oxidative stress.
2.1 SR – mitochondria communication may
amplify ischaemia – repefusion injury
Cell death secondary to transient myocardial ischaemia occurs mainly
within the first minutes of restoration of blood flow in the form of
necrosis. The mechanisms of reperfusion-induced cardiomyocyte
death have been reviewed elsewhere.89 Although they have not been
completely elucidated, calcium overload and altered calcium handling
play a prominent role by inducing calpain-mediated proteolysis, hyperactivation of the contractile machinery, and MPT. The pathological hallmark of reperfusion necrosis is the presence of contraction bands,
reflecting cardiomyocyte hypercontracture.90,91 Energy-dependent
hypercontracture92 contributes to cell death, as demonstrated by
studies in which pharmacological inhibition of contractility at the
Figure 1 Electro-dense tethering structures are observed between SR and mitochondria in cardiac myocytes (small arrows). SR is located between
mitochondria and T-tubules (TT). Mitochondria are positioned on the SR site opposite to that bearing RyR-feet (arrowheads). (A), (B) and (C) correspond to same cell with different magnification. Adapted from Boncompagni et al.,80 with permission.
34
onset of reperfusion reduced infarct size.93 – 95 Previous studies have
indicated that hypercontracture can be triggered by SR calcium
cycling96,97 induced by restoration of ATP in the presence of cytosolic
calcium overload. The reactivation of SERCA causes SR calcium
uptake beyond maximal SR storage capacity, resulting in subsequent
calcium release through RyR and an increase in the concentration of
cytosolic calcium, reinitiating the process. This abnormal SR behaviour
gives rise to cytosolic calcium oscillations that propagate as calcium
waves and may induce arrhythmias and myofibrillar hypercontracture.96,97 The prevention or attenuation of calcium oscillations by
drugs interfering with SR calcium uptake or release96,98 or reducing
cytosolic calcium overload 99,100 has been proved to be effective strategies to protect against hypercontracture and cell death during
reperfusion.
The role of mitochondria in myocardial reperfusion injury has been
the object of extensive research and has been reviewed in detail previously.101 Oxidative stress, mitochondrial Ca2+ overload, and low
ATP concentration—circumstances that concur upon myocardial
reperfusion after prolonged ischaemia—promote MPT in different
experimental models.101 – 103 Pharmacological and genetic inhibition
of MPT effectively reduced infarct size in several experimental
models,36,37,104 and two proof-of-concept clinical trials have demonstrated that targeting MPT with postconditioning105 or cyclosporine
A106 at the moment of angioplasty improves contractile function
and reduces cell death in patients with acute myocardial infarction.
However, hypercontracture is an energy-dependent response,
whereas MPT causes energy dissipation. One interesting possibility
is the coexistence within the same cell of an intact subpopulation of
mitochondria, capable of sustaining ATP synthesis, with more severely
M. Ruiz-Meana et al.
damaged mitochondria undergoing membrane permeabilization. The
anatomo-functional interplay between the SR and mitochondria predicts, in fact, a different tolerance to ischaemic damage of subsarcolemmal and interfibrillar mitochondria, since only the latter
subpopulation is in close contact with the SR. The coexistence of
damaged and intact mitochondria within the same cell during reperfusion has been tested in a model of laser-induced mitochondrial permeabilization in calcium-overloaded cadiomyocytes.107 In this study,
experimental induction of MPT impaired cytosolic calcium overload,
favoured SR-driven calcium oscillations, and eventually led to hypercontracture, supporting the existence of heterogeneously damaged
mitochondria within the same cell. The relationship between
SR-driven calcium oscillations and MPT appears to be bidirectional,
and SR calcium cycling may increase the susceptibility for MPT in a
fraction of mitochondria located in areas of close contact with the
SR. In a recent study,108 pharmacological blockade of SR calcium
load with thapsigargin/ryanodine slowed mitochondrial calcium
uptake in intact cells but had no effect in isolated mitochondria
where the contribution of SR is negligible. The inhibition of SR
calcium uptake and release also reduced MPT, hypercontracture,
and cell death during reperfusion, and this protective effect was abrogated when anatomical interaction between the SR and mitochondria
was partially disrupted with colchicine108 (Figure 2). The prevention of
cytosolic calcium oscillations did not reduce the total cytosolic
calcium concentration during the first minutes of reperfusion, supporting the hypothesis that there is a component of cell death triggered by a local mechanism not related to total cellular calcium
load.108 Figure 3 illustrates the concept of the role of SR –mitochondria microdomains in reperfusion injury.
Figure 2 Three-dimensional reconstruction of a rat cardiac myocyte in which SR (ER-tracker red) and mitochondria (Mito-tracker green) were
simultaneously stained, before (A) and after (B) addition of 1 mM colchicine. Colchicine reduced SR and mitochondria co-localization, and this
effect was associated with a loss of protection afforded by SR calcium blockers ryanodine/thapsigargin against mitochondrial calcein release (indicative
of MPT) (C) and cell death (lactate dehydrogenase release) (D) during simulated reperfusion. Modified from Ruiz-Meana et al.,108 with permission.
35
Pathophysiological role of SR – mitochondria microdomains
studies integrating the role of excitation –contraction coupling and
mitochondrial bioenergetics showed that mitochondrial ROS production triggers DCm oscillations and shortens the duration of the
cell action potential.120 The possibility that altered SR function may
locally modify mitochondrial energetics deserves further investigation.
2.3 Disruption of mitochondria –SR
interaction in the failing heart
Figure 3 Pathophysiological role of SR– mitochondria functional
units on lethal reperfusion injury. Calcium overload and
re-energization cause calcium oscillations. ROS favour oscillations
and trigger MPT. mNCX, mitochondrial Na/Ca exchanger; MCU:
mitochondrial calcium uniporter.
2.2 Arrhythmias may be the consequence
of a defective SR –mitochondria interplay
Imbalanced SR– mitochondria communication may have important
functional consequences in cardiac arrhythmias. Mitochondrial
calcium is the main regulatory factor for the orchestration of mitochondrial energetics with variations in cardiac workload and excitation– contraction coupling.79,109,110 Some studies have linked
mitochondrial dysfunction to certain types of cardiac arrhythmias
and sudden death.111 Atrial myocyte energetics has been reported
to be perturbed in patients and animal models of atrial fibrillation,112
and mitochondrial depolarization during reperfusion—secondary to
calcium uptake—could facilitate mitochondrial network disruption
and the occurrence of ventricular fibrillation.113 Whether mitochondrial dysfunction is the cause or consequence of SR dysfunction
remains to be elucidated. It is postulated that calcium-dependent
arrhythmias are generally associated with the instability of SR
calcium release, whereas heart failure is related to a reduction in systolic SR calcium release.114 The pathophysiological mechanism of SR
dysfunction is extremely complex and multiple molecular disturbances, like abnormal RyR2 behaviour with increased SR calcium
leak115 or failure of RyR2 gating,116 have been described. Moreover,
conditions causing an increase in the cytosolic calcium concentration,
i.e. myocardial ischaemia/reperfusion, may lead to the generation of
spontaneous SR calcium waves, which can give rise to sustained
tachyarrhythmias117 and promote excessive activation of the contractile machinery, leading to hypercontracture and cell death.96,97 The SR
has been pointed out as the major contributor of the contractile dysfunction in human atrial cardiomyocytes obtained from patients with
atrial fibrillation by a mechanism involving hyperphosphorylation of
the RyR2.118 This effect could be responsible for a continuous SR
calcium leak and arrhythmogenesis. Alternatively, the increase in mitochondrial ROS production described in human atrial fibrillation119
could be in part responsible for SR dysfunction. Computer simulation
Advanced heart failure has been consistently associated with a progressive decline in mitochondrial respiratory activity,121 reduced
ATP generation,122 and mitochondrial structural abnormalities.123
These mitochondrial alterations, together with reduced amplitude
of SR-driven calcium transients,124,125 are the most prominent disturbances described to contribute to cardiac contractile dysfunction.
A large variety of defects in the activity of individual components of
the mitochondrial respiratory chain have been proposed to participate in the pathogenesis of heart failure.126 – 128 It is controversial
whether the reduction in the respiratory rates may be attributed to
a mere decrease in mitochondrial function or to a reduced number
of mitochondria.129 Studies from Rosca et al.121,130 propose that the
pronounced variability of mitochondrial electron transport defects
reported thus far in severe acquired cardiomyopathies may be
explained by a sequential mechanistic pathway in which defects in
supramolecular assembly (respirasomes)—rather than in the individual components of oxidative phosphorylation—are the primary
event responsible for the progression of heart failure. Recently, it
has also been suggested that changes in mitochondrial protein
dynamics can contribute to the cell loss and the progression of
cardiac failure.131 Moreover, in some specific conditions like heart
failure secondary to diabetic cardiomyopathy, interfibrillar and
subsarcolemmal mitochondria are selectively affected.68,132
Mitochondria-induced apoptotic death of cardiac myocytes has
been described to play a role in the progression of cardiac dysfunction, since it is persistently activated in patients with decompensated
heart failure.133
Because in cardiac cells, the matching of energy supply and demand
is strongly dependent on local, metabolically active microdomains, the
impairment of the local ATP/ADP ratio is one of the molecular disturbances described in failing myocardium, probably developing as a
consequence of a cytoarchitectural perturbation.134,135 Indeed,
heart failure is associated with structural remodelling of various subcellular organelles,136 and it has been described that local energy
transfer between SR and mitochondria is affected in mice with genetically altered subcellular organization.137 Mouse hearts deficient in
muscle LIM protein exhibit a defective nucleotide channelling from
mitochondria to SR, despite having normal mitochondrial oxidative
capacity, an effect that likely accounts for the energetic and contractile
dysfunction.137 Therefore, there is a tight relationship between the
disorganization of several cellular structural components and the
impairment of SR–mitochondria intercommunication, with concomitant deterioration in the excitation– contraction coupling.135,137
Although the specific role of SR– mitochondria interplay in the pathogenesis of the different cardiomyopathies (dilated vs. hypertrophic)
has not been elucidated, subcellular microanatomical remodelling
seems to be dependent on the differences in cardiac load and
cardiac failure stage.135
Disarrangement between SR and mitochondria is not the only
mechanism responsible for the defective biochemical interaction
36
among both organelles. Alterations of structural or regulatory proteins on any side of the functional unit may have consequences on
its counterpart. Thus, the modification of the expression of the
uncoupling protein 2 not only has an intrinsic mitochondrial effect
on DCm and ROS production but also exerts a deleterious influence
on excitation– contraction coupling and calcium handling.138 Conversely, calcium/calmodulin-dependent protein kinase II, a protein that
plays a role in regulating SR calcium release, may reduce DCm and
increase mitochondrial susceptibility to undergoing membrane permeabilization by a mechanism related to local rise in cytosolic
calcium and stimulation of mitochondrial ROS generation.86 Overall,
these studies emphasize the critical role of the disruption of functional
units, particularly those established by SR and mitochondria interplay,
in the progression of heart failure.
2.4 Role of mitochondria –SR
microdomains in diabetes
Several lines of evidence support the notion that mitochondrial
defects play a critical role in the progression of diabetes.139,140 Similarly, a number of studies have found altered SR function with disturbed calcium handling in the cardiomyopathic phenotype
associated with the diabetic state.141,142 Cardiac myocytes submitted
to hyperglycaemic conditions are exposed to increased oxidative
stress and mitochondrial calcium overload, which can lead to a
reduction in mitochondrial respiration and increased susceptibility
to undergoing MPT.143 Mitochondrial perturbations have been attributed to impaired myocardial insulin signalling144 or to a fragmentation
of the mitochondrial network.145 Defective nitric oxide production
might also deteriorate mitochondrial biogenesis in the metabolic syndrome.146 Indirect evidence suggests an interaction between SR and
mitochondrial disturbances in the progression of diabetes. Reduced
insulin action promoted a rapid decline in mitochondrial fatty acid oxidative capacity.147 Interestingly, the expression levels of SERCA2 were
concomitantly reduced.147 A different study148 indicated that chronic
exposure to high fatty acid levels adversely affected mitochondrial
function (reduced DCm and stimulated ROS production), which, in
turn, was associated with reduced SR calcium content and depressed
cytosolic calcium transients. It cannot be ruled out that SR (or ER)
stress mediates mitochondrial damage by a cell signalling mechanism
rather than by a direct local effect. Miki et al.149 described a
reduced threshold for MPT associated with ER stress in the myocardium of type 2 diabetic rats by an impaired phospho-GSK3b-mediated
suppression of MPT, an effect that may underlie the failure of cytoprotective strategies against ischaemia– reperfusion injury in diabetic
hearts. Dysregulation in the formation of ROS microdomains could
play a role in the pathophysiology of diabetes due to the existence
of a tight interplay between ROS and calcium, but, to date, the specific
contribution of SR–mitochondria interaction in the aetiopathogenesis
of diabetic disease has not yet been explicitly investigated.
2.5 Might ageing be a disease of SR –
mitochondria communication?
Ageing is a complex, multifactorial process that is far from being deciphered. It is usually linked to degenerative diseases, including cardiac
diseases, but the nature of this link remains speculative.150 An overwhelming amount of experimental data links mitochondrial dysfunction with cell senescence and lifespan.151 The mitochondrial theory
of ageing assumes three main mediators of cell damage: increased
M. Ruiz-Meana et al.
ROS accumulation, progressive mitochondrial DNA damage, and insidious failure of respiratory complexes.151,152 However, to our knowledge, the possibility that different populations of mitochondria age at
different rates depending on their interrelation with the SR, orchestrating cell senescence, has not been investigated thus far. An intriguing observation is that subsarcolemmal and interfibrillar
mitochondria experience different functional decline during ageing,
with a selective reduction in oxidative phosphorylation affecting interfibrillar mitochondria.153 The capacity to retain calcium before undergoing membrane permeabilization is also specifically reduced in the
interfibrillar mitochondria from myocardium of old rats.154 Remarkably, interfibrillar mitochondria are exposed to continuous SR
calcium oscillations throughout the life in the beating heart. This is
probably the biological reason for their higher respiratory capacity
and a more efficient calcium uptake system.65 Because the SR –mitochondria interaction is bidirectional, the possibility exists that progressive mitochondrial decline in oxidative phosphorylation
observed in interfibrillar mitochondria deteriorates the closely juxtaposed, energy-dependent SR, providing the pathophysiological basis
for the progression of age-related degenerative diseases in which
calcium homeostasis is lost, i.e. heart failure. Ageing has been
shown to account for an increased sensitivity to ischaemia–repefusion injury mediated in part by a higher diastolic calcium concentration,
although the mechanisms leading to this persistent loss of calcium
control have not been identified.155 The activity of SR calcium transporters is very sensitive to redox state,3 and functional units regulating
local SR calcium release and the calcium-induced-calcium-release
response in skeletal and cardiac cells have been shown to lose their
plasticity during ageing.156 However, what the molecular machinery
that induces such alterations might be is not clear at present, nor is
the cause–consequence relationship between them, and the role of
the SR–mitochondria interaction in the cellular functional decline
associated with ageing is still an unexplored field of research.
2.6 Implications
The concept that mitochondria and SR behave in many respects as a
functional unit has potentially important implications. First, it may
allow a better understanding of the pathophysiology and evolution
of cardiovascular diseases and their modulation by ageing. Secondly,
and most importantly, it may help to identify and develop new therapeutic strategies. It should allow the protection of mitochondria by
targeting SR molecules or treatment of SR dysfunction by mitochondrial interventions. Finally, it opens a new field of research on the
potential value of strategies aimed at modifying SR –mitochondria
communication itself.
Conflict of interest: none declared.
Funding
Partially supported by RECAVA-RETICS (RD060014/0025, ISCIII, Spanish
Ministry of Science), FIS (PS09/02034, Spanish Ministry of Science), and
SAF (2008-03067, Spanish Ministry of Education).
References
1. Di Lisa F, Blank PS, Colonna R, Gambassi G, Silverman HS, Stern MD et al. Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed
to anoxia or metabolic inhibition. J Physiol 1995;486:1 –13.
2. Brand MD, Buckingham JA, Esteves TC, Green K, Lambert AJ, Miwa S et al. Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production. Biochem Soc Symp 2004;71:203 –213.
Pathophysiological role of SR – mitochondria microdomains
3. Zima AV, Blatter LA. Redox regulation of cardiac calcium channels and transporters.
Cardiovasc Res 2006;71:310 – 321.
4. Yoshida T, Maulik N, Engelman RM, Ho YS, Das DK. Targeted disruption of the
mouse Sod I gene makes the hearts vulnerable to ischemic reperfusion injury. Circ
Res 2000;86:264 –269.
5. Speakman JR, Talbot DA, Selman C, Snart S, McLaren JS, Redman P et al. Uncoupled
and surviving: individual mice with high metabolism have greater mitochondrial
uncoupling and live longer. Aging Cell 2004;3:87–95.
6. Carafoli E, Lehninger AL. Binding of adenine nucleotides by mitochondria during
active uptake of CA++. Biochem Biophys Res Commun 1964;16:66 –70.
7. Carafoli E, Rossi CS, Lehninger AL. Uptake of adenine nucleotides by respiring mitochondria during active accumulation of Ca2+ and phosphate. J Biol Chem 1965;240:
2254 –2261.
8. Rizzuto R, Bernardi P, Pozzan T. Mitochondria as all-round players of the calcium
game. J Physiol 2000;529:37 –47.
9. Saotome M, Katoh H, Satoh H, Nagasaka S, Yoshihara S, Terada H et al. Mitochondrial membrane potential modulates regulation of mitochondrial Ca2+ in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 2005;288:H1820 –H1828.
10. Montero M, Alonso MT, Albillos A, Garcia-Sancho J, Alvarez J. Mitochondrial
Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter. Mol Biol Cell
2001;12:63–71.
11. Gunter TE, Gunter KK, Sheu SS, Gavin CE. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol 1994;267:C313 – C339.
12. Bernardi P, Petronilli V. The permeability transition pore as a mitochondrial calcium
release channel: a critical appraisal. J Bioenerg Biomembr 1996;28:131 – 138.
13. Ichas F, Jouaville LS, Mazat JP. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 1997;89:1145 – 1153.
14. McCormack JG, Halestrap AP, Denton RM. Role of calcium ions in regulation of
mammalian intramitochondrial metabolism. Physiol Rev 1990;70:391 –425.
15. Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T et al. BAX
and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis.
Science 2003;30:135 –139.
16. Nakayama H, Chen X, Baines CP, Klevitsky R, Zhang X, Zhang H et al. Ca2+- and
mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart
failure. J Clin Invest 2007;117:2431 –2444.
17. Halestrap AP. Mitochondria and reperfusion injury of the heart—a holey death but
not beyond salvation. J Bioenerg Biomembr 2009;41:113 –121.
18. Boitier E, Rea R, Duchen MR. Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes. J Cell Biol 1999;145:
795 –808.
19. Babcock DF, Herrington J, Goodwin PC, Park YB, Hille B. Mitochondrial participation in the intracellular Ca2+ network. J Cell Biol 1997;136:833 –844.
20. Aon MA, Cortassa S, Marban E, O’Rourke B. Synchronized whole cell oscillations in
mitochondrial metabolism triggered by a local release of reactive oxygen species in
cardiac myocytes. J Biol Chem 2003;278:44735 –44744.
21. Maack C, Dabew ER, Hohl M, Schafers HJ, Bohm M. Endogenous activation of mitochondrial KATP channels protects human failing myocardium from hydroxyl
radical-induced stunning. Circ Res 2009;105:811 –817.
22. Murata M, Akao M, O’Rourke B, Marban E. Mitochondrial ATP-sensitive potassium
channels attenuate matrix Ca(2+) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ Res 2001;89:891 –898.
23. Xu W, Liu Y, Wang S, McDonald T, Van Eyk JE, Sidor A et al. Cytoprotective role of
Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science
2002;298:1029 –1033.
24. Garlid KD, Dos SP, Xie ZJ, Costa AD, Paucek P. Mitochondrial potassium transport:
the role of the mitochondrial ATP-sensitive K(+) channel in cardiac function and
cardioprotection. Biochim Biophys Acta 2003;1606:1–21.
25. Kang SH, Park WS, Kim N, Youm JB, Warda M, Ko J et al. Mitochondrial Ca2+activated K+ channels more efficiently reduce mitochondrial Ca2+ overload in rat
ventricular myocytes. Am J Physiol Heart Circ Physiol 2007;293:H307 –H313.
26. Blachly-Dyson E, Forte M. VDAC channels. IUBMB Life 2001;52:113 –118.
27. Calamita G, Ferri D, Gena P, Liquori GE, Cavalier A, Thomas D et al. The inner
mitochondrial membrane has aquaporin-8 water channels and is highly permeable
to water. J Biol Chem 2005;280:17149– 17153.
28. Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres P, Morente M, Ruiz-Meana M
et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its
importance for cardioprotection. Circ Res 2006;99:93 –101.
29. Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P
et al. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res 2005;67:234 – 244.
30. Miro-Casas E, Ruiz-Meana M, Agullo E, Stahlhofen S, Rodriguez-Sinovas A,
Cabestrero A et al. Connexin43 in cardiomyocyte mitochondria contributes to
mitochondrial potassium uptake. Cardiovasc Res 2009;83:747 –756.
31. Matsuyama S, Llopis J, Deveraux QL, Tsien RY, Reed JC. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2000;2:318 –325.
37
32. Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell
death. Physiol Rev 2007;87:99 –163.
33. Gustafsson AB, Gottlieb RA. Heart mitochondria: gates of life and death. Cardiovasc
Res 2008;77:334 –343.
34. Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B et al. Bid-deficient mice are
resistant to Fas-induced hepatocellular apoptosis. Nature 1999;400:886 –891.
35. Zorov DB, Juhaszova M, Yaniv Y, Nuss HB, Wang S, Sollott SJ. Regulation and
pharmacology of the mitochondrial permeability transition pore. Cardiovasc Res
2009;83:213–225.
36. Argaud L, Gateau-Roesch O, Muntean D, Chalabreysse L, Loufouat J, Robert D et al.
Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury. J Mol Cell Cardiol 2005;38:367–374.
37. Clarke SJ, McStay GP, Halestrap AP. Sanglifehrin A acts as a potent inhibitor of the
mitochondrial permeability transition and reperfusion injury of the heart by binding
to cyclophilin-D at a different site from cyclosporin A. J Biol Chem 2002;277:
34793–34799.
38. Halestrap AP. What is the mitochondrial permeability transition pore? J Mol Cell
Cardiol 2009;46:821 –831.
39. Baines CP. The cardiac mitochondrion: nexus of stress. Annu Rev Physiol 2010;72:
61 –80.
40. English AR, Zurek N, Voeltz GK. Peripheral ER structure and function. Curr Opin Cell
Biol 2009;21:596 – 602.
41. Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum stress: cell life and death
decisions. J Clin Invest 2005;115:2656 –2664.
42. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein
response. Nat Rev Mol Cell Biol 2007;8:519–529.
43. Minamino T, Kitakaze M. ER stress in cardiovascular disease. J Mol Cell Cardiol 2009;
48:1105 –1110.
44. Schendel SL, Montal M, Reed JC. Bcl-2 family proteins as ion-channels. Cell Death
Differ 1998;5:372 –380.
45. Thomenius MJ, Distelhorst CW. Bcl-2 on the endoplasmic reticulum: protecting the
mitochondria from a distance. J Cell Sci 2003;116:4493 –4499.
46. Argaud L, Gateau-Roesch O, Chalabreysse L, Gomez L, Loufouat J, Thivolet-Bejui F
et al. Preconditioning delays Ca2+-induced mitochondrial permeability transition.
Cardiovasc Res 2004;61:115 –122.
47. Javadov SA, Clarke S, Das M, Griffiths EJ, Lim KH, Halestrap AP. Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the
reperfused rat heart. J Physiol 2003;549:513 –524.
48. Rizzuto R, Brini M, Murgia M, Pozzan T. Microdomains with high Ca2+ close to
IP3-sensitive channels that are sensed by neighboring mitochondria. Science 1993;
262:744 –747.
49. Rizzuto R, Bastianutto C, Brini M, Murgia M, Pozzan T. Mitochondrial Ca2+ homeostasis in intact cells. J Cell Biol 1994;126:1183 –1194.
50. Garcia-Perez C, Hajnoczky G, Csordas G. Physical coupling supports the local Ca2+
transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart
muscle. J Biol Chem 2008;283:32771 – 32780.
51. Csordas G, Renken C, Varnai P, Walter L, Weaver D, Buttle KF et al. Structural and
functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol 2006;174:915 –921.
52. Hajnoczky G, Robb-Gaspers LD, Seitz MB, Thomas AP. Decoding of cytosolic
calcium oscillations in the mitochondria. Cell 1995;82:415 –424.
53. Duchen MR, Leyssens A, Crompton M. Transient mitochondrial depolarizations
reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes.
J Cell Biol 1998;142:975 –988.
54. Szalai G, Csordas G, Hantash BM, Thomas AP, Hajnoczky G. Calcium signal transmission between ryanodine receptors and mitochondria. J Biol Chem 2000;275:
15305–15313.
55. Pezzati R, Meldolesi J, Grohovaz F. Ultra rapid calcium events in electrically stimulated frog nerve terminals. Biochem Biophys Res Commun 2001;285:724 –727.
56. Rizzuto R, Pozzan T. Microdomains of intracellular Ca2+: molecular determinants
and functional consequences. Physiol Rev 2006;86:369 – 408.
57. Szabadkai G, Simoni AM, Chami M, Wieckowski MR, Youle RJ, Rizzuto R.
Drp-1-dependent division of the mitochondrial network blocks intraorganellar
Ca2+ waves and protects against Ca2+-mediated apoptosis. Mol Cell 2004;16:59 –68.
58. Simmen T, Aslan JE, Blagoveshchenskaya AD, Thomas L, Wan L, Xiang Y et al.
PACS-2 controls endoplasmic reticulum-mitochondria communication and Bidmediated apoptosis. EMBO J 2005;24:717 – 729.
59. Szabadkai G, Bianchi K, Varnai P, De Stefani D, Wieckowski MR, Cavagna D et al.
Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+
channels. J Cell Biol 2006;175:901 –911.
60. Hayashi T, Su TP. Sigma-1 receptor chaperones at the ER-mitochondrion interface
regulate Ca(2+) signaling and cell survival. Cell 2007;131:596–610.
61. de Brito OM, Scorrano L. Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 2008;456:605 –610.
62. Tinel H, Cancela JM, Mogami H, Gerasimenko JV, Gerasimenko OV, Tepikin AV et al.
Active mitochondria surrounding the pancreatic acinar granule region prevent
spreading of inositol trisphosphate-evoked local cytosolic Ca(2+) signals. EMBO J
1999;18:4999 –5008.
38
63. Rusinol AE, Cui Z, Chen MH, Vance JE. A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains
pre-Golgi secretory proteins including nascent lipoproteins. J Biol Chem 1994;269:
27494– 27502.
64. Palmer JW, Tandler B, Hoppel CL. Biochemical properties of subsarcolemmal and
interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 1977;252:
8731 –8739.
65. Palmer JW, Tandler B, Hoppel CL. Biochemical differences between subsarcolemmal
and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations. Arch Biochem Biophys 1985;236:691 –702.
66. Boengler K, Stahlhofen S, van de Sand A, Gres P, Ruiz-Meana M, Garcia-Dorado D
et al. Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria. Basic Res Cardiol 2009;104:141 –147.
67. Judge S, Jang YM, Smith A, Hagen T, Leeuwenburgh C. Age-associated increases in
oxidative stress and antioxidant enzyme activities in cardiac interfibrillar mitochondria: implications for the mitochondrial theory of aging. FASEB J 2005;19:419 –421.
68. Dabkowski ER, Williamson CL, Bukowski VC, Chapman RS, Leonard SS, Peer CJ
et al. Diabetic cardiomyopathy-associated dysfunction in spatially distinct mitochondrial subpopulations. Am J Physiol Heart Circ Physiol 2009;296:H359–H369.
69. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem 1987;56:395 –433.
70. Pozzan T, Rizzuto R, Volpe P, Meldolesi J. Molecular and cellular physiology of intracellular calcium stores. Physiol Rev 1994;74:595 –636.
71. MacLennan DH, Asahi M, Tupling AR. The regulation of SERCA-type pumps by
phospholamban and sarcolipin. Ann N Y Acad Sci 2003;986:472 –480.
72. Endo M. Calcium release from the sarcoplasmic reticulum. Physiol Rev 1977;57:
71 –108.
73. Eisner DA, Kashimura T, Venetucci LA, Trafford AW. From the ryanodine receptor
to cardiac arrhythmias. Circ J 2009;73:1561 –1567.
74. Maier LS, Bers DM. Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat
to heartbeat and beyond. J Mol Cell Cardiol 2002;34:919 –939.
75. Bassani JW, Bassani RA, Bers DM. Twitch-dependent SR Ca accumulation and
release in rabbit ventricular myocytes. Am J Physiol 1993;265:C533 –C540.
76. Delbridge LM, Satoh H, Yuan W, Bassani JW, Qi M, Ginsburg KS et al. Cardiac
myocyte volume, Ca2+ fluxes, and sarcoplasmic reticulum loading in
pressure-overload hypertrophy. Am J Physiol 1997;272:H2425 –H2435.
77. Gyorke S, Terentyev D. Modulation of ryanodine receptor by luminal calcium and
accessory proteins in health and cardiac disease. Cardiovasc Res 2008;77:245 –255.
78. Treves S, Vukcevic M, Maj M, Thurnheer R, Mosca B, Zorzato F. Minor sarcoplasmic
reticulum membrane components that modulate excitation-contraction coupling in
striated muscles. J Physiol 2009;587:3071 –3079.
79. Maack C, Cortassa S, Aon MA, Ganesan AN, Liu T, O’Rourke B. Elevated cytosolic
Na+ decreases mitochondrial Ca2+ uptake during excitation-contraction coupling
and impairs energetic adaptation in cardiac myocytes. Circ Res 2006;99:172 –182.
80. Boncompagni S, Rossi AE, Micaroni M, Beznoussenko GV, Polishchuk RS,
Dirksen RT et al. Mitochondria are linked to calcium stores in striated muscle by
developmentally regulated tethering structures. Mol Biol Cell 2009;20:1058 –1067.
81. Sharma VK, Ramesh V, Franzini-Armstrong C, Sheu SS. Transport of Ca2+ from sarcoplasmic reticulum to mitochondria in rat ventricular myocytes. J Bioenerg Biomembr 2000;32:97 –104.
82. Liu T, O’Rourke B. Enhancing mitochondrial Ca2+ uptake in myocytes from failing
hearts restores energy supply and demand matching. Circ Res 2008;103:279 – 288.
83. Seppet EK, Kaambre T, Sikk P, Tiivel T, Vija H, Tonkonogi M et al. Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum
in muscle cells. Biochim Biophys Acta 2001;1504:379 –395.
84. Davidson SM, Duchen MR. Calcium microdomains and oxidative stress. Cell Calcium
2006;40:561 –574.
85. Yan Y, Liu J, Wei C, Li K, Xie W, Wang Y et al. Bidirectional regulation of Ca2+
sparks by mitochondria-derived reactive oxygen species in cardiac myocytes. Cardiovasc Res 2008;77:432 –441.
86. Odagiri K, Katoh H, Kawashima H, Tanaka T, Ohtani H, Saotome M et al. Local
control of mitochondrial membrane potential, permeability transition pore and reactive oxygen species by calcium and calmodulin in rat ventricular myocytes. J Mol Cell
Cardiol 2009;46:989 –997.
87. Chami M, Oules B, Szabadkai G, Tacine R, Rizzuto R, Paterlini-Brechot P. Role of
SERCA1 truncated isoform in the proapoptotic calcium transfer from ER to mitochondria during ER stress. Mol Cell 2008;32:641 –651.
88. Diwan A, Matkovich SJ, Yuan Q, Zhao W, Yatani A, Brown JH et al. Endoplasmic
reticulum-mitochondria crosstalk in NIX-mediated murine cell death. J Clin Invest
2009;119:203 –212.
89. Piper HM, Garcia-Dorado D, Ovize M. A fresh look at reperfusion injury. Cardiovasc
Res 1998;38:291 –300.
90. Miyazaki S, Fujiwara H, Onodera T, Kihara Y, Matsuda M, Wu DJ et al. Quantitative
analysis of contraction band and coagulation necrosis after ischemia and reperfusion
in the porcine heart. Circulation 1987;75:1074 –1082.
91. Barrabes JA, Garcia-Dorado D, Ruiz-Meana M, Piper HM, Solares J, Gonzalez MA
et al. Myocardial segment shrinkage during coronary reperfusion in situ. Relation
to hypercontracture and myocardial necrosis. Pflugers Arch 1996;431:519–526.
M. Ruiz-Meana et al.
92. Altschuld RA, Wenger WC, Lamka KG, Kindig OR, Capen CC, Mizuhira V et al.
Structural and functional properties of adult rat heart myocytes lysed with digitonin.
J Biol Chem 1985;260:14325 –14334.
93. Garcia-Dorado D, Theroux P, Duran JM, Solares J, Alonso J, Sanz E et al. Selective
inhibition of the contractile apparatus. A new approach to modification of infarct
size, infarct composition, and infarct geometry during coronary artery occlusion
and reperfusion. Circulation 1992;85:1160 –1174.
94. Sumida T, Otani H, Kyoi S, Okada T, Fujiwara H, Nakao et al. Temporary blockade of
contractility during reperfusion elicits a cardioprotective effect of the P38 MAP
kinase inhibitor SB-203580. Am J Physiol Heart Circ Physiol 2005;288:H2726 –H2734.
95. Tani M, Hasegawa H, Suganuma Y, Shinmura K, Kayashi Y, Nakamura Y. Protection
of ischemic myocardium by inhibition of contracture in isolated rat heart. Am J Physiol
1996;271:H2515 –H2519.
96. Siegmund B, Schlack W, Ladilov YV, Balser C, Piper HM. Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture. Circulation 1997;96:
4372–4379.
97. Abdallah Y, Gkatzoflia A, Gligorievski D, Kasseckert S, Euler G, Schluter KD et al.
Insulin protects cardiomyocytes against reoxygenation-induced hypercontracture
by a survival pathway targeting SR Ca2+ storage. Cardiovasc Res 2006;70:346 – 353.
98. Vila-Petroff M, Salas MA, Said M, Valverde CA, Sapia L, Portiansky E et al. CaMKII
inhibition protects against necrosis and apoptosis in irreversible ischemiareperfusion injury. Cardiovasc Res 2007;73:689 –698.
99. Ladilov Y, Haffner S, Balser-Schafer C, Maxeiner H, Piper HM. Cardioprotective
effects of KB-R7943: a novel inhibitor of the reverse mode of Na+/Ca2+ exchanger.
Am J Physiol 1999;276:H1868 –H1876.
100. Inserte J, Garcia-Dorado D, Ruiz-Meana M, Padilla F, Barrabes JA, Pina P et al. Effect
of inhibition of Na(+)/Ca(2+) exchanger at the time of myocardial reperfusion on
hypercontracture and cell death. Cardiovasc Res 2002;55:739–748.
101. Duchen MR, McGuinness O, Brown LA, Crompton M. On the involvement of a
cyclosporin a sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res 1993;27:1790 –1794.
102. Di Lisa F, Menabo R, Canton M, Barile M, Bernardi P. Opening of the mitochondrial
permeability transition pore causes depletion of mitochondrial and cytosolic NAD+
and is a causative event in the death of myocytes in postischemic reperfusion of the
heart. J Biol Chem 2001;276:2571 –2575.
103. Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during
cardiac ischaemia, but open upon reperfusion. Biochem J 1995;307:93–98.
104. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA et al. Loss of
cyclophilin D reveals a critical role for mitochondrial permeability transition in cell
death. Nature 2005;434:658 –662.
105. Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L’Huillier I et al. Postconditioning the
human heart. Circulation 2005;112:2143 –2148.
106. Piot C, Croisille P, Staat P, Thibault H, Rioufol G, Mewton N et al. Effect of cyclosporine on reperfusion injury in acute myocardial infarction. N Engl J Med 2008;359:
473– 481.
107. Ruiz-Meana M, Abellan A, Miro-Casas E, Garcia-Dorado D. Opening of mitochondrial permeability transition pore induces hypercontracture in Ca2+ overloaded
cardiac myocytes. Basic Res Cardiol 2007;102:542 – 552.
108. Ruiz-Meana M, Abellan A, Miro-Casas E, Agullo E, Garcia-Dorado D. Role of sarcoplasmic reticulum in mitochondrial permeability transition and cardiomyocyte death
during reperfusion. Am J Physiol Heart Circ Physiol 2009;297:H1281 –H1289.
109. Cortassa S, Aon MA, Marban E, Winslow RL, O’Rourke B. An integrated model of
cardiac mitochondrial energy metabolism and calcium dynamics. Biophys J 2003;84:
2734–2755.
110. Maack C, O’Rourke B. Excitation-contraction coupling and mitochondrial energetics.
Basic Res Cardiol 2007;102:369 –392.
111. Brown DA, Aon MA, Frasier CR, Sloan RC, Maloney AH, Anderson EJ et al. Cardiac
arrhythmias induced by glutathione oxidation can be inhibited by preventing mitochondrial depolarization. J Mol Cell Cardiol 2010;48:673 – 679.
112. Kalifa J, Maixent JM, Chalvidan T, Dalmasso C, Colin D, Cozma D et al. Energetic
metabolism during acute stretch-related atrial fibrillation. Mol Cell Biochem 2008;
317:69–75.
113. Garcia-Rivas GJ, Carvajal K, Correa F, Zazueta C. Ru360, a specific mitochondrial
calcium uptake inhibitor, improves cardiac post-ischaemic functional recovery in
rats in vivo. Br J Pharmacol 2006;149:829 –837.
114. Gyorke S, Carnes C. Dysregulated sarcoplasmic reticulum calcium release: potential
pharmacological target in cardiac disease. Pharmacol Ther 2008;119:340 –354.
115. Kubalova Z, Terentyev D, Viatchenko-Karpinski S, Nishijima Y, Gyorke I,
Terentyeva R et al. Abnormal intrastore calcium signaling in chronic heart failure.
Proc Natl Acad Sci USA 2005;102:14104 –14109.
116. Belevych A, Kubalova Z, Terentyev D, Hamlin RL, Carnes CA, Gyorke S. Enhanced
ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure. Biophys J 2007;93:4083 –4092.
117. Venetucci LA, Trafford AW, O’Neill SC, Eisner DA. The sarcoplasmic reticulum and
arrhythmogenic calcium release. Cardiovasc Res 2008;77:285 –292.
118. Neef S, Dybkova N, Sossalla S, Ort KR, Fluschnik N, Neumann K et al. CaMKIIdependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial
myocardium of patients with atrial fibrillation. Circ Res 2010;106:1134 –1144.
Pathophysiological role of SR – mitochondria microdomains
119. Mihm MJ, Yu F, Carnes CA, Reiser PJ, McCarthy PM, Van Wagoner DR et al.
Impaired myofibrillar energetics and oxidative injury during human atrial fibrillation.
Circulation 2001;104:174 – 180.
120. Zhou L, Cortassa S, Wei AC, Aon MA, Winslow RL, O’Rourke B. Modeling cardiac
action potential shortening driven by oxidative stress-induced mitochondrial oscillations in guinea pig cardiomyocytes. Biophys J 2009;97:1843 – 1852.
121. Rosca MG, Vazquez EJ, Kerner J, Parland W, Chandler MP, Stanley W et al. Cardiac
mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation. Cardiovasc Res 2008;80:30 –39.
122. Shen W, Asai K, Uechi M, Mathier MA, Shannon RP, Vatner SF et al. Progressive loss
of myocardial ATP due to a loss of total purines during the development of heart
failure in dogs: a compensatory role for the parallel loss of creatine. Circulation
1999;100:2113 –2118.
123. Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura K et al. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts
after myocardial infarction. Circ Res 2001;88:529 – 535.
124. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N et al. PKA
phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine
receptor): defective regulation in failing hearts. Cell 2000;101:365 – 376.
125. Bers DM. Altered cardiac myocyte Ca regulation in heart failure. Physiology
(Bethesda) 2006;21:380 –387.
126. Marin-Garcia J, Goldenthal MJ, Moe GW. Abnormal cardiac and skeletal muscle
mitochondrial function in pacing-induced cardiac failure. Cardiovasc Res 2001;52:
103 –110.
127. Jarreta D, Orus J, Barrientos A, Miro O, Roig E, Heras M et al. Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy. Cardiovasc
Res 2000;45:860 –865.
128. Ide T, Tsutsui H, Kinugawa S, Utsumi H, Kang D, Hattori N et al. Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing
myocardium. Circ Res 1999;85:357 –363.
129. Garnier A, Fortin D, Delomenie C, Momken I, Veksler V, Ventura-Clapier R.
Depressed mitochondrial transcription factors and oxidative capacity in rat failing
cardiac and skeletal muscles. J Physiol 2003;551:491–501.
130. Rosca MG, Hoppel CL. New aspects of impaired mitochondrial function in heart
failure. J Bioenerg Biomembr 2009;41:107 –112.
131. Chen L, Gong Q, Stice JP, Knowlton AA. Mitochondrial OPA1 apoptosis heart
failure. Cardiovasc Res 2009;84:91 –99.
132. Williamson CL, Dabkowski ER, Baseler WA, Croston TL, Always SE, Hollander JM.
Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of subcellular spatial location. Am J Physiol Heart Circ Physiol 2010;298:H633–H642.
133. Olivetti G, Abbi R, Quaini F, Kajstura J, Cheng W, Nitahara JA et al. Apoptosis in the
failing human heart. N Engl J Med 1997;336:1131 –1141.
134. Joubert F, Wilding JR, Fortin D, Domergue-Dupont V, Novotova M, Ventura-Clapier R
et al. Local energetic regulation of sarcoplasmic and myosin ATPase is differently
impaired in rats with heart failure. J Physiol 2008;586:5181 –5192.
135. Hein S, Kostin S, Heling A, Maeno Y, Schaper J. The role of the cytoskeleton in heart
failure. Cardiovasc Res 2000;45:273–278.
136. Dhalla NS, Saini-Chohan HK, Rodriguez-Leyva D, Elimban V, Dent MR, Tappia PS.
Subcellular remodelling may induce cardiac dysfunction in congestive heart failure.
Cardiovasc Res 2009;81:429 – 438.
137. Wilding JR, Joubert F, de Araujo C, Fortin D, Novotova M, Veksler V et al. Altered
energy transfer from mitochondria to sarcoplasmic reticulum after cytoarchitectural
perturbations in mice hearts. J Physiol 2006;575:191 –200.
39
138. Turner JD, Gaspers LD, Wang G, Thomas AP. Uncoupling protein-2 modulates
myocardial excitation-contraction coupling. Circ Res 2010;106:730 –738.
139. Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science 2005;
307:384 –387.
140. Szabadkai G, Duchen MR. Mitochondria mediated cell death in diabetes. Apoptosis
2009;14:1405 –1423.
141. Belke DD, Dillmann WH. Altered cardiac calcium handling in diabetes. Curr Hypertens Rep 2004;6:424 –429.
142. Shao CH, Wehrens XH, Wyatt TA, Parbhu S, Rozanski GJ, Patel KP et al. Exercise
training during diabetes attenuates cardiac ryanodine receptor dysregulation. J Appl
Physiol 2009;106:1280 –1292.
143. Oliveira PJ. Cardiac mitochondrial alterations observed in hyperglycaemic rats—
what can we learn from cell biology? Curr Diabetes Rev 2005;1:11 –21.
144. Boudina S, Bugger H, Sena S, O’Neill BT, Zaha VG, Ilkun O et al. Contribution of
impaired myocardial insulin signaling to mitochondrial dysfunction and oxidative
stress in the heart. Circulation 2009;119:1272 –1283.
145. Yu T, Sheu SS, Robotham JL, Yoon Y. Mitochondrial fission mediates high
glucose-induced cell death through elevated production of reactive oxygen
species. Cardiovasc Res 2008;79:341 –351.
146. Nisoli E, Clementi E, Carruba MO, Moncada S. Defective mitochondrial biogenesis: a
hallmark of the high cardiovascular risk in the metabolic syndrome? Circ Res 2007;
100:795 –806.
147. Sena S, Hu P, Zhang D, Wang X, Wayment B, Olsen C et al. Impaired insulin signaling
accelerates cardiac mitochondrial dysfunction after myocardial Infarction. J Mol Cell
Cardiol 2009;46:910 –918.
148. Fauconnier J, Andersson DC, Zhang SJ, Lanner JT, Wibom R, Katz A et al. Effects of
palmitate on Ca(2+) handling in adult control and Ob/Ob cardiomyocytes: impact
of mitochondrial reactive oxygen species. Diabetes 2007;56:1136 –1142.
149. Miki T, Miura T, Hotta H, Tanno M, Yano T, Sato T et al. Endoplasmic reticulum
stress in diabetic hearts abolishes erythropoietin-induced myocardial protection
by impairment of phospho-glycogen synthase kinase-3beta-mediated suppression
of mitochondrial permeability transition. Diabetes 2009;58:2863 –2872.
150. Lane NA. Unifying view of ageing and disease: the double-agent theory. J Theor Biol
2003;225:531–540.
151. Alexeyev MF. Is there more to aging than mitochondrial DNA and reactive oxygen
species? FEBS J 2009;276:5768 –5787.
152. Dufour E, Larsson NG. Understanding aging: revealing order out of chaos. Biochim
Biophys Acta 2004;1658:122 – 132.
153. Fannin SW, Lesnefsky EJ, Slabe TJ, Hassan MO, Hoppel CL. Aging selectively
decreases oxidative capacity in rat heart interfibrillar mitochondria. Arch Biochem
Biophys 1999;372:399 – 407.
154. Hofer T, Servais S, Seo AY, Marzetti E, Hiona A, Upadhyay SJ et al. Bioenergetics and
permeability transition pore opening in heart subsarcolemmal and interfibrillar mitochondria: effects of aging and lifelong calorie restriction. Mech Ageing Dev 2009;130:
297– 307.
155. Howlett SE, Grandy SA, Ferrier GR. Calcium spark properties in ventricular myocytes are altered in aged mice. Am J Physiol Heart Circ Physiol 2006;290:
H1566– H1574.
156. Weisleder N, Brotto M, Komazaki S, Pan Z, Zhao X, Nosek T et al. Muscle aging is
associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+
release. J Cell Biol 2006;174:639 –645.