Dirksen, Robert T

Pugh, Edward N

Sheu, Shey-Shing

Jefferson Digital Commons

Thomas Jefferson UniversityJefferson Digital CommonsCenter for Translational Medicine Faculty Papers Center for Translational Medicine6-1-2012Perspectives on: SGP symposium onmitochondrial physiology and medicine:mitochondria take center stage.Shey-Shing SheuDepartment of Medicine, Center for Translational Medicine, Jefferson Medical College, Thomas Jefferson University, Shey-Shing.Sheu@jefferson.eduRobert T DirksenDepartment of Pharmacology and Physiology, University of Rochester Medical CenterEdward N PughCenter for Neuroscience, University of CaliforniaLet us know how access to this document benefits youFollow this and additional works at: http://jdc.jefferson.edu/transmedfpPart of the Other Medical Specialties CommonsThis Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of ThomasJefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarlypublications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers andinterested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion inCenter for Translational Medicine Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, pleasecontact: JeffersonDigitalCommons@jefferson.edu.Recommended CitationSheu, Shey-Shing; Dirksen, Robert T; and Pugh, Edward N, "Perspectives on: SGP symposium onmitochondrial physiology and medicine: mitochondria take center stage." (2012). Center forTranslational Medicine Faculty Papers. Paper 15.http://jdc.jefferson.edu/transmedfp/15The Rockefeller University PressJ. Gen. Physiol. Vol. 139 No. 6 391–393www.jgp.org/cgi/doi/10.1085/jgp.201210819 391Pe r spe c t i v eMitochondria are the ubiquitous power units of animal cells, responsible for “oxphos,” the aerobic synthesis of most of the ATP that serves as the common coin of cel-lular metabolism. To perform their primary task of oxi-dative phosphorylation and to manage many secondary tasks, mitochondria use more than 1,000 proteins, of which fewer than 100 are involved directly in oxidative phosphorylation. The “secondary” tasks mitochondria perform include their own maintenance, calcium regu-lation and signaling, lipid synthesis, and acting as gate-keepers for programmed cell death. Catalyzed by the advances in knowledge and technology that underlie great progress in all areas of cell biology, mitochondrial physiology has been undergoing a renaissance. In this special issue of the Journal we highlight some examples of this renaissance, offering a series of Perspectives on Mitochondria. The purpose of the Perspectives in Gen-eral Physiology is to provide a forum where scientific uncertainties or controversies are discussed in an au-thoritative, yet open manner. This series had its origin in the 65th Annual Symposium of the Society of Gen-eral Physiologists on “Mitochondrial Physiology and Medicine,” which took place in September 2011 in Woods Hole, MA. The meeting was organized by Shey-Shing Sheu (Thomas Jefferson University) along with a committee comprising leaders in mitochondrial research, including Robert Balaban (NHLBI), Paolo Bernardi (University of Padova), Robert Dirksen (Uni-versity of Rochester), Roberta Gottlieb (San Diego State University), Gyorgy Hajnóczky (Thomas Jefferson Uni-versity), and Brian O’Rourke (Johns Hopkins Univer-sity). A summary of the meeting was recently published (Sheu et al., 2011).Mitochondrial proteomesMitochondria contain more than 1,000 proteins, of which only 13 are encoded by mitochondrial DNA. In their Perspective, Zhang et al. summarize recent ad-vances in mitochondrial proteomics, including quanti-fying “subproteomes” of the mitochondria devoted to Correspondence to Edward N. Pugh: e n p u g h j r @ u c d a v i s . e d u different cellular functions. The proteomes of mito-chondria in various tissues (e.g., heart, muscle, brain) differ, and proteomic variation also causes or is associ-ated with disease. Substantial differences, for example, exist between heart and liver mitochondrial proteomes that can be directly related to tissue-specific needs. Of particular medical importance are the distinct pro-teomic features of mitochondria from healthy and dis-eased cardiac myocytes. Another important and rapidly developing aspect of mitochondrial proteomics is the characterization of posttranslational modifications, some of which are dynamic and may play critical roles in mod-ulating oxphos function on a subsecond scale (see next section). The key challenge, as articulated by Zhang et al., will be to translate the burgeoning mitochondrial proteomic databases into resources that can be readily tapped to advance our understanding of physiological function and pathophysiology.Metabolic homeostasis of the heartNothing better illustrates the amazing capacity of mito-chondria to meet the energy demands of tissue than their ability to power the cardiac contraction cycle throughout life, including workload increases of up to 10-fold during surges in sympathetic activity, such as those that accompany the “fight or flight response.” Mitochondria perform this feat with very little change in the steady-state concentrations of key metabolites such as ATP, ADP, Pi, and NADH. A.V. Hill (1950) first noted the challenge posed by these facts, which he called “metabolic homeostasis.” In his Perspective, Robert Balaban takes up Hill’s challenge. Balaban mar-shals evidence that posttranslational modifications of the F1F0 ATP synthase may underlie a 10-fold modula-tion of the maximum rate of ATP production (qATPmax), and that calcium in the mitochondrial matrix is a major regulator of qATPmax. These ideas comprise an impor-tant (although challenging to test) hypothesis that ties Perspectives on: SGP Symposium on Mitochondrial Physiology and MedicineMitochondria take center stageShey-Shing Sheu,1 Robert T. Dirksen,2 and Edward N. Pugh Jr.31Department of Medicine, Center for Translational Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 191072Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 146423Center for Neuroscience, University of California, Davis, Davis, CA 95616© 2012 Sheu et al. This article is distributed under the terms of an Attribution–Noncommercial– Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).The Journal of General Physiology392 SGP Symposium on Mitochondrial Physiology and Medicinewill be needed to resolve the relationship between pHm transients and superoxide flashes, and their role(s) in regulating mitochondrial energy and ROS generation, both signals hold much promise in revealing dynamic details of mitochondrial function in living cells.Mitochondrial calcium transportThe pivotal role of Ca2+ in regulating mitochondrial function and dysfunction is well recognized. The dogma for the mechanism of mitochondrial Ca2+ influx has been that it is determined primarily by the mitochon-drial Ca2+ uniporter (MCU), whose molecular identity was recently revealed (Baughman et al., 2011; De Stefani et al., 2011). Several studies have identified additional Ca2+ uptake pathways, including a rapid mode of up-take and the highly conserved mitochondrial protein leucine-zipper-EF-hand–containing transmembrane protein 1 (LETM1), which exhibit different Ca2+ affin-ity, kinetics, and pharmacology from the MCU. The Perspective by O-Uchi et al. examines recent studies that have attempted to uncover the molecular identities of mitochondrial Ca2+ influx mechanisms using genetic manipulations, including small interfering RNA. O-Uchi et al. argue that it is reasonable to expect that different tissues may regulate mitochondrial Ca2+ influx in differ-ent fashions, with different combinations and expression ratios of these channels and transporters, and point to studies that will clarify the diversity of Ca2+ influx mech-anisms, explain how these Ca2+ transporters are regu-lated by signaling molecules, and identify their distinct physiological functions and pathologies.A hotly debated issue is whether LETM1 acts as a high affinity mitochondrial Ca2+ influx mechanism. The Per-spective by Nowikovsky et al. summarizes current understanding of the molecular nature and of the physiology and pathophysiology of LETM1. LETM1 en-codes a mitochondrial protein conserved throughout eukaryotes, both animals and plants. Its deletion is em-bryonic lethal, and LETM1 mutations underlie the sei-zure disorder known as Wolf–Hirschhorn syndrome. The protein was identified first as a mitochondrial K+–H+ antiporter, but has since been argued to function as a Ca2+–H+ antiporter by Jiang et al. (2009). Nowikovsky et al. make the case that the normal electrochemical gradients for H+ and Ca2+ would not allow LETM1 to act as a Ca2+ influx mechanism, but rather that the driving forces under physiological conditions favor LETM1 serving as a Ca2+ efflux rather than influx mechanism.Shape changing: Role of morphology in  mitochondrial functionThe idea that mitochondrial morphology is also a key regulator of mitochondrial function has recently gained much attention. The Perspective by Galloway and Yoon summarizes the current understanding of the interplay together several key aspects of mitochondrial physiology, including the roles of variations in matrix calcium over time and of matrix proteins regulated by calcium.New insights from mitochondrial pH measurementsAs articulated in Mitchell’s chemiosmotic theory, ATP synthesis in mitochondria is driven by the proton mo-tive force, which consists of the chemical gradient for protons (pHm) and the mitochondrial membrane potential (m). Measurement of pHm has been a foundation of research in mitochondrial physiology. However, classical measurements were performed in bulk suspensions, disconnecting mitochondria from the milieu of the living cell, in which many natural and signal-dependent variations in pHi, Ca2+, and metabo-lites to which mitochondria are sensitive occur. Geneti-cally encoded (optogenetic) pH probes that promise to fill many gaps in the understanding of the chemios-motic mechanism in living cells have recently been developed. In their Perspective, Santo-Domingo and Demaurex summarize recent research with mitochon-drially targeted optogenetic pH probes, and, in particu-lar, work they have done with a very promising probe, mito-SypHer. Using this probe they have observed spon-taneous increases in pHm elevations that coincided with decreases in m in single mitochondria. Santo-Domingo and Demaurex propose that these spontaneous alkaliza-tions of the mitochondrial matrix reflect intrinsic properties of the mitochondrial proton circuit that function to stabilize the proton motive force with op-posing changes in m and pHm, and summarize the evidence that this stabilization process involves several distinct exchangers and symporters.Superoxide flashes: Quantal production of reactive oxygen species (ROS)Studying mitochondrial production of ROS has been challenging because of the limited methods capable of real-time monitoring of ROS levels in living cells and in situ. Recently, circularly permuted yellow fluorescent protein has been shown to specifically respond to super-oxide. When targeted to the mitochondrial matrix, this probe detects bursts of superoxide production, called mitochondrial superoxide flashes (mSOFs), in individ-ual mitochondria of intact quiescent cells. The Perspec-tive of Wei and Dirksen provides an updated assessment of the mechanisms underlying mSOF generation and raises several controversies regarding the generation of mSOFs, including the relationship between mSOFs and pHm transients, the molecular identity of the large pore channel that opens during a flash event, and the se-quence of events between pore opening and mSOF generation. Both mSOF and pHm signals require intact electron transport chain activities, because inhibition of any one of the electron transport complexes results in the abolition of the signals. Although future experiments  Sheu et al. 393Mitochondrial antioxidants and scavenging of ROSROS are normally generated at very low levels by the mitochondrial respiratory chain but increase under conditions of stress, and can ultimately lead to tissue damage. In this issue, Aon et al. performed experiments and theoretical analyses to assess the relative contribu-tion of glutathione (GSH) and thioredoxin (Trx), the two principal antioxidants in mitochondria, to the scav-enging of H2O2, a major ROS. Their results show that the reactions governing the concentrations GSH and Trx are essential to maintaining minimal emission of H2O2 during high level respiration, and that the reac-tions maintaining GSH and Trx work in close concert toward achieving this end.Letters to the editor related to these Perspectives will be published in the September 2012 issue of the Journal. Letters to the editor should be received no later than Monday, July 16, 2012. The letters may be no longer than two printed pages (approximately six double-spaced pages) and will be subject to editorial review. They may contain no more than one figure, no more than 15 references, and no significant references to un-published work (and may not include supplemental material). Letters should be prepared according to the Journal’s instructions and can be submitted electroni-cally at http://www.jgp.org. After the letters to the edi-tor have been published, further responses are limited to full manuscripts.R E F E R ENC E SBaughman, J.M., F. Perocchi, H.S. Girgis, M. Plovanich, C.A. Belcher-Timme, Y. Sancak, X.R. Bao, L. Strittmatter, O. Goldberger, R.L. Bogorad, et al. 2011. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature. 476:341–345. http://dx.doi.org/10.1038/nature10234De Stefani, D., A. Raffaello, E. Teardo, I. Szabò, and R. Rizzuto. 2011. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature. 476:336–340. http://dx.doi.org/10.1038/nature10230Hill, A.V. 1950. A challenge to biochemists. Biochim. Biophys. Acta. 4:4–11.Jiang, D., L. Zhao, and D.E. Clapham. 2009. Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ anti-porter. Science. 326:144–147. http://dx.doi.org/10.1126/science.1175145Seidlmayer, L.K., M.R. Gomez-Garcia, L.A. Blatter, E. Pavlov, and E.N. Dedkova. 2012. Inorganic polyphosphate is a potent acti-vator of the mitochondrial permeability transition pore in car-diac myocytes. J. Gen. Physiol. 139:321–331. http://dx.doi.org/10.1085/jgp.201210788Sheu, S.S., R.T. Dirksen, and E.N. Pugh Jr. 2011. The 65th Symposium of the Society for General Physiologists: Energizing research in mitochondrial physiology and medicine. J. Gen. Physiol. 138:563–567. http://dx.doi.org/10.1085/jgp.201110739between mitochondrial form and function, with a specific focus on how disturbances of this interaction lead to meta-bolic disease. This Perspective highlights the dynamic nature of the cross talk signaling between mitochon-drial shape and metabolic activity. Well-orchestrated mitochondrial fission, fusion, and molecular motor–based movement are necessary to ensure healthy bio-energetics homeostasis.Calcium buffering and calcium in cell deathThe JGP is also publishing several primary articles in-volving research on mitochondria presented at the 2011 SGP Symposium. Two of these deal with calcium influx into mitochondria and its role in triggering cell death. In work presented in this issue, Wei et al. thoroughly characterized two distinct modes of Ca2+ uptake in car-diac mitochondria, “MCUmode1” and “MCUmode2.” Mode 1 has a relatively low capacity, is engaged by small (0.1–0. 2-µM) increases in Ca2+, is completely inhibited by only relatively high levels (1 µM) of Ruthenium red (Ru360), and triggers relatively large changes in matrix free calcium. In contrast, mode 2 is responsible for the bulk of calcium transport for external calcium in the range of 2–10 µM, is inhibited by 0.1 µM Ru360, and is associated with very small changes in matrix free cal-cium owing to very high buffering. The data and analy-ses presented in this article support the conclusion that mode 1 calcium transport functions as a signal, perhaps to dynamically modulate oxidative phosphorylation (see Perspective by Balaban), whereas mode 2 is associated with a high capacity Ca2+-buffering function. The inves-tigation of Wei et al. also indicates that the trigger for activation of the mitochondrial permeability transition pore (mPTP) is unlikely to be free matrix Ca2+ itself, but could be a downstream byproduct of total mitochon-drial Ca2+ loading, a conclusion that dovetails nicely with work in another paper recently published in the JGP, by Seidlmayer et al. (2012).Seidlmayer et al. (2012) quantified the inorganic polyphosphate (polyP) content of mitochondria (300 pmol/mg of protein) in rabbit ventricular myocytes and manipulated polyP levels by means of a mitochondrially targeted polyphosphatase. Excess accumulation of Ca2+ in mitochondria is a key factor in triggering mPTP opening (and subsequent apoptosis) and consequent tissue damage such as occurs during cardiac ischemia reperfusion injury. Seidlmayer et al. (2012) found that depletion of polyP inhibited mPTP opening without affecting the matrix free calcium, and put forward the hypothesis that stable complexes of calcium with polyP act as triggers for mPTP opening. This is an important advance, which make a critical link between mito-chondrial calcium influx, calcium buffering by polyP, and activation of the mPTP.

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Perspectives on: SGP symposium on mitochondrial physiology and medicine: mitochondria take center stage.

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