doi: 10.1161/CIRCULATIONAHA.106.653485
(Circulation. 2006;114:1452-1454.)
© 2006 American Heart Association, Inc.
Editorial |
Exploring Mitochondria in the Intact Ischemic Heart
Advancing Technologies to Image Intracellular Function
From the Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.
Correspondence to Michael N. Sack, MD, PhD, Cardiology Branch, NHLBI/NIH, Building 10-CRC, Room 5-3150, 10 Center Dr, Bethesda, MD 20892-1454. E-mail sackm{at}nhlbi.nih.gov
Key Words: Editorials • imaging • ischemia • metabolism
In describing human nature, the Irish author and satirist Jonathan Swift noted that "vision is the art of seeing what is invisible to others." In the biological sciences, "vision" is transformed into a "science" as progress in imaging technology enables the uncovering of previously "invisible" intracellular programs. This capacity to "see inside cells" is improving in lock-step with advancing technologies that enable fluorophore-labeling of genes, proteins, cells, substrates, and metabolites. Thus, biomedical imaging is expanding our understanding of cellular function and disease pathophysiology. In this issue of Circulation, the practical application of 2-photon scanning laser microscopy is used to directly assess the mitochondrial inner membrane potential in the intact rat heart in response to cardiac ischemia and reperfusion.1
Article p 1497
Before discussing this study, I will digress for a moment to review the relevance of mitochondrial function and the proposed role of the inner mitochondrial membrane potential on cardiac function and in its response to ischemia and reperfusion. The mitochondrion is central to cardiac function, as it modulates cardiac energetics, reactive radical biology, calcium homeostasis, and apoptosis.2 The inner mitochondrial membrane potential in turn reflects a composite of mitochondrial functioning, which means the maintenance of this electrochemical potential requires: (1) the functional integrity of electron transfer redox centers of oxidative phosphorylation; (2) the catalytic integrity of enzymes of ß-oxidation and the Kreb’s cycle; and (3) the appropriate functioning of transport mechanisms linking the cytosol and the mitochondrial matrix.
The mitochondrial inner membrane potential is not static; rather, the modulation of this electrochemical gradient directly controls mitochondrial adenosine triphosphate generation, Ca2+ flux, and the production and control of reactive oxygen species (ROS).3 Interestingly, modest modulation of the mitochondrial membrane potential appears to confer cellular adaptations that enhance tolerance to ischemic and redox stress.4–7 These adaptive events are thought to be initiated by the consequences of the modulation of the mitochondrial membrane and may result in part from mitochondrial depolarization–mediated reduction in ROS production during ischemia and reperfusion.3 Interestingly, mitochondria are also proposed to directly orchestrate nuclear regulation, metabolic pathways, and cell survival programs via a process termed retrograde signaling.3,8,9 As retrograde signaling from the mitochondria to nucleus and cytosol is thought to occur partially via ROS and Ca2+ signaling,8 and as these signaling events can be modulated by alterations in the mitochondrial membrane potential,3 it is intriguing to speculate whether the modest modulation of the mitochondrial membrane potential may play an important role in retrograde signaling. Conversely, in response to oxidative stress and other death signals, more robust changes in the mitochondrial membrane potential are evident. Here,10 a large depolarization of the inner membrane potential is evident after enhanced permeability of the mitochondrial membranes, which results in swelling of the mitochondrial matrix, disruption of the outer mitochondrial membrane, and dissipation of the mitochondrial inner membrane potential. This phenomenon is termed the mitochondrial permeability transition (MPT) and signifies the onset of cellular injury and death.10 Together, these data demonstrate that the mitochondrial membrane potential can be considered a comprehensive measure reflecting "global" mitochondrial function and/or dysfunction.
In this issue, Matsumoto-Ida et al1 utilize the robust changes in the mitochondrial membrane potential during the MPT to explore the practical application of real-time 2-photon laser scanning microcopy (TPLSM). TPLSM is a technology whereby molecular excitation by the simultaneous absorption of 2 photons provides enhanced 3-dimensional spatial resolution of photochemistry within biological systems.11,12 The advantages of this approach over single-photon confocal microscopy in the intact heart include increased penetration of the excitation beam to allow the visualization of photochemistry in refractory tissue up to a depth of approximately 50 µm and reduced overall photo bleaching and photo damage, thereby enabling extended viability of cardiomyocytes during longer-term imaging.13 Matsumoto-Ida et al1 used ischemia and reperfusion in the isolated Langendorff perfused rat heart that had been loaded with a fluorescent indicator that measures the inner mitochondrial membrane potential to assess the MPT using TPLSM. Ischemia/reperfusion induces redox stress and calcium overload, thereby increasing the permeability of the mitochondrial inner membrane, resulting in the collapse of the electrochemical gradient across the inner mitochondrial membrane.10 The major findings of this study demonstrate that (1) the MPT occurrence is heterogeneous, ie, some cardiomyocytes exhibit MPT during ischemia, some during reperfusion, and some not at all; (2) the kinetics of the collapse of the mitochondrial membrane potential is the same, irrespective of whether the MPT is triggered during ischemia or during reperfusion; and (3) the cardiomyocyte cell survival program of ischemic preconditioning14 attenuates the number of cells undergoing the MPT but not the rate of depolarization of mitochondria in cells "committed" to MPT. Additional observations support that theory that the MPT antagonist cyclosporine does diminish and alter the rate of depolarization and that mitochondrial collapse is propagated sequentially to adjacent mitochondria in a longitudinal axis within affected cardiomyocytes. Together, these data shed light onto the temporal and spatial induction of the MPT and also show that the MPT response is heterogeneous across distinct adjacent cells, despite putative maintenance of intracellular communication channels in the intact explanted heart. This heterogeneity in the response of adjacent cells under identical conditions is suggestive of variable cellular tolerance to oxidative stress and warrants further investigation. In addition, the demonstration that the MPT is evident during both ischemia and reperfusion supports the concept that the rescue of cardiomyocytes via therapeutic intervention at the time of reperfusion is a worthwhile goal to pursue.15
Although the introduction of TPLSM to the study of the intact explanted heart is a significant advance, a brief discussion of the limitations of the current technology is warranted. The main limitation in this study1 is that TPLSM currently can only be performed on noncontractile tissue. This limitation is especially significant in the study of the functioning of the mitochondria in the heart because of the exclusion of the contribution of the high-energy demand of cardiac contraction, which is probably central to the mitochondrial response to oxidative stress. An additional note of caution remains; Matsumoto-Ida et al1 investigated an extreme change in the mitochondrial membrane potential, and whether more modest perturbations in mitochondrial membrane potential, as evident in ischemic preconditioning5–7 and after the use of cardioprotective agents,16 can be measured with this technology is unknown. The limited sensitivity of this technique is illustrated when the measurement of autofluorescence of NADH in cardiomyocytes cannot be distinguished from background noise.17 Finally, the depth of tissue penetration used in this technology still limits the ability to explore the biology of ischemia affecting the mid- and endocardial regions of the heart.
Despite these shortcomings of this technology, its potential scientific applications are vast, based on the expanding fluorophore labeling technologies to tag cells, metabolites, organelle targeted proteins, small molecules, and genes (Figure). This "explosion" of labeled biological reagents, which can be introduced into the in vivo heart via genetic manipulation, direct injection, or intravascular administration should expand the potential for investigation into a myriad of biological pathways and programs using TPLSM. This technology therefore introduces an exciting new tool into the ever-expanding array of biomedical imaging devices to enhance our insight into the biochemical and molecular machinery involved in cardiac pathophysiology. The advances in medical imaging are further illustrated by the development of new probes to investigate real-time metabolic imaging with magnetic resonance imaging.18 Together, the improvements in biomedical imaging and the development of new labeled "biological probes" should propel scientific vision into the future to better determine the structure and functioning of the intracellular milieu. These in turn will improve our understanding of disease progression and will accelerate the development of novel therapies to alleviate and or prevent devastating diseases resulting from ischemia and reperfusion injury.
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Acknowledgments |
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Dr Sack is supported by the Division of Intramural Research of the National Heart, Lung, and Blood Institute at the National Institutes of Health.
Disclosures
None.
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Footnotes |
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The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
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References |
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