See related article, pages 153–159
The Cytoplasm
No Longer a Well-Mixed Bag
James N. Weiss, Paavo Korge
U
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
energy of ATP hydrolysis.2,3 In heart, this role of PC in
mediating crosstalk between ATP production and ATP utilization has been shown for both contractile function and
sarcolemmal function.3–5
But if the norm for other signaling pathways is a high
degree of compartmentalization, perhaps energy consumption
is not as democratic a process as once assumed. In this issue
of Circulation Research, Kaasik et al6 address the following
important question: does ATP, once generated, diffuse rapidly to wherever it is needed, or is it channeled directly and
preferentially to nearby energy-consuming processes? This is
not a new question, but it has been difficult to answer
convincingly for two reasons. First, imaging tools to track
energy production/consumption directly at the subcellular
level are still not as well developed as they are, for example,
for imaging Ca2⫹ microdomains or hot spots of protein kinase
activity using fluorescence techniques. Second, both the
traditional “grind-and-bind” biochemical methods and global
measures of metabolic function, such as nuclear magnetic
resonance, are based on averaged cytoplasmic values of
various metabolites and do not easily lend themselves to
investigating subcellular compartmentalization of metabolism. Thus, the evidence for metabolic compartmentalization
has largely rested on studies of functional responses to
metabolic interventions.
There is a long history of functional studies suggesting that
energy production and consumption are colocalized at the
subcellular level. A general theme in both muscle and
nonmuscle cells has been that glycolytically derived ATP is
used preferentially to support surface membrane–related
functions (such as ion transporters and channels), whereas
mitochondrially generated ATP is used preferentially for
supporting functions in the cytoplasm (such as muscle contraction) (Figure). Several of the major observations supporting this idea are the following. Selective inhibition of anaerobic glycolysis and selective inhibition of mitochondrial
oxidative phosphorylation have different functional effects,
which cannot be explained by changes in global tissue
high-energy phosphate levels.7–9 Manipulations of anaerobic
glycolysis markedly affect functional performance and recovery during ischemia/reperfusion or hypoxia/reoxygenation,
again in a manner that does not correlate with changes in
global-tissue high phosphate levels.10 –14 Finally, substrates
for glycolysis and substrates for mitochondrial oxidative
phosphorylation or ATP regeneration via CK have differential efficacies at supporting specific membrane or contractile
functions in heart and other tissues.2,7,15–17
In this issue of Circulation Research, Kaasik et al6 take the
latter approach to examine the relationship between the
source of ATP and the efficacy of Ca2⫹ uptake by the SR.
ntil the last decade or so, many intracellular signaling
pathways were viewed as processes in which second
messengers diffused uniformly through a well-mixed
milieu of the cell’s cytoplasm to reach their targets. Although
it was recognized that the cell’s interior was compartmentalized, this compartmentalization was believed to be largely
defined by internal membranes, such as the nuclear envelope,
endoplasmic reticulum (ER), sarcoplasmic reticulum (SR),
and mitochondria. But like the joke about the person who has
lost his keys in the dark but looks for them under the street
lamp because the light is better, this view of the cytoplasm as
a well-mixed milieu was less of a proven fact than a
simplifying assumption. Over the last decade, advances in
subcellular imaging have dramatically upset this view, so that
now a high degree of compartmentalization of signaling
pathways within the cytoplasm is considered the norm rather
than the exception. It is now clear that the cytoplasm has a
highly organized cytoskeleton and sophisticated molecular
trafficking mechanisms that direct and tether proteins into
macroaggregates at specific locations to facilitate localized
signaling. The cytoplasm is now viewed as a system of
microdomains with restricted diffusion (eg, hierarchical Ca2⫹
signaling) and direct channeling of substrates to enzymes (eg,
protein kinases/phosphatases cascades).
In the field of metabolism, subcellular compartmentation
of energy production has been a well-accepted fact ever since
mitochondria were identified as the engines driving aerobic
high-energy phosphate production. In addition, glycolytic
enzymes complexes are well-known to be associated with
specific intracellular structures, such as the SR.1 On the flip
side, however, energy consumption by the cell has traditionally been viewed as a fairly democratic process, with highenergy phosphates freely diffusing throughout the cytoplasm
to be consumed wherever they are needed. In tissues with
high energy requirements, such as muscle, the creatine kinase
(CK) system has been viewed as the essential equalizer in this
design, with phosphocreatine (PC) shuttling rapidly to regenerate ATP from ADP wherever CK is located, maintaining
free ADP concentration at low levels to maximize the free
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Cardiovascular Research Laboratory and the Departments of
Medicine (Cardiology) and Physiology, University of California Los
Angeles School of Medicine, Los Angeles, Calif.
Correspondence to James N. Weiss, MD, 3645 MRL Building,
UCLA School of Medicine, Los Angeles, CA 90095-1760. E-mail
[email protected]
(Circ Res. 2001;89:108-110.)
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
108
Weiss and Korge
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
Hypothetical schema of metabolic energy channeling in cardiac
myocytes. The study by Kaasik et al6 provides evidence that
mitochondria (Mito) channel ATP directly to SERCA pumps in
the SR (arrow 1) as well as via the CK-mediated PC shuttle
(arrow 2), which also delivers ATP to myofilaments (MyoF).
Other evidence suggests that, in addition to anaerobic ATP, glycogenolysis may provide pyruvate (Pyr) to mitochondria (arrow
3) to generate ATP preferentially for SERCA pumps and myofilaments (MyoF), whereas exogenous glucose may preferentially
generate ATP for sarcolemmal (SL) ion pumps and channels via
glycolytic enzymes (GE) associated with the SL (arrow 4). Cr
indicates creatine.
Using permeabilized cardiac muscle fibers, they show that
when mitochondria are inhibited to prevent them from generating ATP locally, exogenously supplied ATP is greatly
inferior as a substrate for SR Ca2⫹ uptake, as estimated from
tension development when accumulated SR Ca2⫹ is released
with caffeine. Under the same conditions, provision of
exogenous creatine phosphate plus ADP to permit local ATP
generation via CK was nearly as effective as endogenously
generated mitochondrial ATP. This observation is consistent
with the idea that CK plays an essential role in distributing
ATP throughout the cytosol. However, Kaasik et al6 take the
matter a step further by exploring the observation that
genetically engineered mice lacking mitochondrial and cytosolic CK have nearly normal cardiac performance, up to at
least moderate workloads. Yet in these CK-deficient mice,
they demonstrated that exogenously supplied ATP remained
markedly inferior to mitochondrially generated ATP at supporting SR function, as in wild-type mice. The implication is
that their mitochondrially generated ATP is being directly
channeled to the SR independently of the CK system. If not,
then CK-deficient mice should have had markedly impaired
cardiac energetics. In wild-type mice, Kaasik et al6 estimate
that at normal cardiac workload, approximately one third of
ATP used by the SR Ca2⫹ pump is directly channeled from
the mitochondria, and about two thirds is provided by the CK
system. In CK-deficient mice, mitochondria effectively compensated for the lack of CK. However, there was a cost. The
hearts of these mice showed significant cytoarchitectural
changes in addition to mild hypertrophy. Interestingly, the
myocardium demonstrated increased splitting of myofibrils,
as if the heart were trying to decrease the average distance
Energy Compartmentalization
109
between mitochondria and myofilaments to compensate for
the lack of CK-facilitated ATP regeneration.
In all functional studies of cellular metabolism, there are
valid concerns about interpretation. Metabolic inhibitors are
neither completely effective nor completely specific. This is
especially true with respect to inhibition of glycolysis, although not a factor in the present study. Also, metabolic
inhibition has global consequences, with a wide array of
effects on many cellular processes, so that distinguishing the
primary effects of metabolic inhibition from its secondary
consequences is often very difficult. Finally, in studies in
which global tissue high-energy phosphate levels are used to
track metabolic inhibition, these levels reflect a net effect on
metabolism but do not provide direct information about how
the underlying metabolic fluxes have been affected unless
supplemented by other techniques.
Some of these criticisms apply to the study by Kaasik et
al.6 However, these investigators were careful to use more
than one method of inhibiting mitochondrial function and
showed that simply omitting mitochondrial substrates produced similar results as direct mitochondrial inhibition with
Na⫹ azide. Importantly, they also showed that the ATP
synthase inhibitor oligomycin did not attenuate the effects of
mitochondrial inhibition, ruling out enhanced ATP consumption by depolarized mitochondria as the explanation for the
lower efficacy of exogenous ATP. However, it should be
noted that other investigators have reported different results.
Altschuld et al18 measured SR Ca2⫹ uptake directly in an
otherwise similar experimental protocol and did not find
evidence for preferential use of mitochondrial versus exogenous ATP. The reasons for this discrepancy are unclear.
In conclusion, despite some limitations, this elegant study
makes an important contribution to our understanding of
cardiac metabolism by providing additional evidence for
localized crosstalk between energy production and energy
consumption in the cardiac cell. Although direct visualization
of this energy channeling will require development of better
metabolic imaging tools, the functional picture emerging is
that metabolism shares features in common with other compartmentalized cellular signaling mechanisms: namely, the
cytoplasm is far from being a well-mixed bag, in which
high-energy phosphates diffuse readily and indiscriminately
to various cellular ATPases. Like other signaling molecules,
ATP is emerging as a local cellular currency with PC as its
global facilitator. A very close physical relationship has been
demonstrated between the mitochondrial and ER networks,19
and mitochondria participate in very localized Ca2⫹ signaling
with ER and SR membranes,20 so a similar relationship with
respect to ATP transfer by mitochondria is not surprising.
The study by Kaasik et al6 also raises interesting issues
about upstream events in metabolism, such as the extent to
which substrate delivery to mitochondria is compartmentalized. For example, does the previously described association
of glycogenolytic enzyme complexes with the SR1 imply that
glycogen, rather than exogenous glucose, is the preferential
upstream source of pyruvate for oxidative phosphorylation, as
proposed in vascular smooth muscle?7 Also, how do free fatty
acids, the preferred myocardial substrate, participate in this
energy channeling? The implications extend beyond merely
110
Circulation Research
July 20, 2001
physiological interest. Additional progress in this area should
lead to an improved understanding of how metabolic pathways can be manipulated during ischemia and reperfusion to
protect the heart and other tissues from injury.
Acknowledgments
The authors thank Tan Duong for help preparing the figure.
References
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
1. Entman ML, Kaniike K, Goldstein MA, Nelson TE, Bornet EP, Futch
TW, Schwartz A. Association of glycogenolysis with cardiac sarcoplasmic reticulum. J Biol Chem. 1976;251:3140 –3146.
2. Bessman SP, Geiger PJ. Transport of energy in muscle: the phosphorylcreatine shuttle. Science. 1981;211:448 – 452.
3. Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM. Intracellular compartmentation, structure and function of creatine kinase
isoenzymes in tissues with high and fluctuating energy demands: the
‘phosphocreatine circuit’ for cellular energy homeostasis. Biochem J.
1992;281:21– 40.
4. Saks VA, Lipina NV, Sharov VG, Smirnov VN, Chazov E, Grosse R. The
localization of the MM isozyme of creatine phosphokinase on the surface
membrane of myocardial cells and its functional coupling to ouabaininhibited (Na, K)-ATPase. Biochim Biophys Acta. 1977;465:550 –558.
5. Sasaki N, Sato T, Marbán E, O’Rourke B. ATP consumption by
uncoupled mitochondria activates sarcolemmal KATP channels in cardiac
myocytes. Am J Physiol. 2001;280:H1882–H1888.
6. Kaasik A, Veksler V, Boehm E, Novotova M, Minajeva A, VenturaClapier R. Energetic crosstalk between organelles: architectural integration of energy production and utilization. Circ Res. 2001;89:153–159.
7. Lynch RM, Paul RJ. Compartmentation of glycolytic and glycogenolytic
metabolism in vascular smooth muscle. Science. 1983;222:1344 –1346.
8. Doorey AJ, Barry WH. The effects of inhibition of oxidative phosphorylation and glycolysis on contractility and high-energy phosphate content
in cultured chick heart cells. Circ Res. 1983;53:192–201.
9. Weiss J, Hiltbrand B. Functional compartmentation of glycolytic versus
oxidative metabolism in isolated rabbit heart. J Clin Invest. 1985;75:
436 – 447.
10. McDonald TF, MacLeod DP. Metabolism and the electrical activity of
anoxic ventricular muscle. J Physiol. 1973;229:559 –583.
11. Bricknell OL, Opie LH. Effects of substrates on tissue metabolic changes
in the isolated rat heart during underperfusion and on release of lactate
dehydrogenase and arrhythmias during reperfusion. Circ Res. 1978;43:
102–115.
12. Higgins TJC, Bailey PJ. The effects of cyanide and iodoacetate intoxication and ischaemia on enzyme release from the perfused rat heart.
Biochim Biophys Acta. 1983;762:67–75.
13. Runnman EM, Lamp ST, Weiss JN. Enhanced utilization of exogenous
glucose improves cardiac function in hypoxic rabbit ventricle without
increasing total glycolytic flux. J Clin Invest. 1990;86:1222–1233.
14. Jeremy RW, Koretsune Y, Marban E, Becker LC. Relation between
glycolysis and calcium homeostasis in postischemic myocardium. Circ
Res. 1992;70:1180 –1190.
15. Parker JC, Hoffman JF. The role of membrane phosphoglycerate kinase
in the control of glycolytic rate by active cation transport in human red
blood cells. J Gen Physiol. 1967;50:893–916.
16. Weiss JN, Lamp ST. Glycolysis preferentially inhibits ATP-sensitive K⫹
channels in isolated guinea pig cardiac myocytes. Science. 1987;238:
67– 69.
17. Hoerter JA, Ventura-Clapier R, Kuznetsov A. Compartmentation of
creatine kinases during perinatal development of mammalian heart. Mol
Cell Biochem. 1994;133–134:277–286.
18. Altschuld RA, Wenger WC, Lamka KG, Kindig OR, Capen CC, Mizuhira
V, Vander Heide RS, Brierley GP. Structural and functional properties of
adult rat heart myocytes lysed with digitonin. J Biol Chem. 1985;260:
14325–14334.
19. Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM,
Tuft RA, Pozzan T. Close contacts with the endoplasmic reticulum as
determinants of mitochondrial Ca2⫹ responses. Science. 1998;280:
1763–1766.
20. Hajnóczky G, Csordás G, Madesh M, Pacher P. The machinery of local
Ca2⫹ signalling between sarco-endoplasmic reticulum and mitochondria.
J Physiol. 2000;529:69 – 81.
KEY WORDS: metabolism 䡲 bioenergetics 䡲 compartmentalization
sarcoplasmic reticulum Ca2⫹ ATPase 䡲 high-energy phosphates
䡲
The Cytoplasm: No Longer a Well-Mixed Bag
James N. Weiss and Paavo Korge
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
Circ Res. 2001;89:108-110
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/89/2/108
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/