© 2001 American Heart Association, Inc.
Editorial |
The Cytoplasm
No Longer a Well-Mixed Bag
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 jweiss{at}mednet.ucla.edu
Key Words: metabolism • bioenergetics • compartmentalization • sarcoplasmic reticulum Ca2+ ATPase • high-energy phosphates
Until 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 high-energy 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 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
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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. 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 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 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.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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