Gyorgy Hajnoczky - US grants

DESCRIPTION (provided by applicant): Mitochondria sense and respond to calcium signals. The mitochondrial response is required for metabolism and other aspects of cell function and is likely to be central for several disease mechanisms activated by genetic impairments or environmental stress. During cytoplasmic [Ca2+] ([Ca2+]c) oscillations, mitochondrial Ca2+ sensing is ensured by local Ca2+ transfer from IP3 or ryanodine receptors to the mitochondrial Ca2+ uptake sites. The local communication has been proposed to occur at the sites where the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is closely associated with the mitochondria. In the past project period we and others have shown that the ER/SR-mitochondrial associations are supported by direct interorganellar physical links, referred as tethers. Furthermore, we and others have shown that at the sites of the ER-mitochondrial associations, mitochondria are exposed to at least 10-fold higher local [Ca2+] concentration than the global [Ca2+] c rise evoked by IP3-linked hormones. These results strengthen the concept that the ER-mitochondrial interface hosts a special structural arrangement. However, the miniature interface (<200nm diameter) is difficult to study with currently available approaches. Our first hypothesis is that mitochondrial Ca2+ sensing depends on IP3R localization, expression level, isoform variety, and activation-deactivation kinetics. We propose that IP3 receptors, SERCA pumps and VDACs are concentrated at the interface and form mutual interactions to present low background and high signal intensity for Ca2+ delivery to the mitochondria. To explore the molecular composition and function of the ER-mitochondrial contacts we have developed and will employ an array of novel molecular tools. Very recently, MICU1, and MCU have been identified as the first molecular components of the mitochondrial Ca2+ uniport but their function remains elusive. Our second hypothesis is that to control the Ca2+ transfer across the inner mitochondrial membrane, MICU1, an EF hand domain protein binds to MCU, the pore forming domain of the uniporter in a Ca2+ sensitive manner. Binding of MICU1 confers cooperativity to the Ca2+ uniport, a benefit of which is that at low [Ca2+] mitochondria excluded from Ca2+ handling and avoid Ca2+ overload. The third hypothesis is that the mitochondrial response to Ca2+ involves K+ influx and an ensuing increase in matrix volume. Mitochondrial matrix volume changes are central to the regulation of mitochondrial fusion, and provide a novel mechanism for the calcium signal to control mitochondrial fusion through. Fusion events, calcium and mitochondrial volume will be followed by fluorescence measurements of several novel reporters down to the level of individual organelles. Collectively, these studies will enhance the understanding of the fundamental mechanisms of mitochondrial calcium signaling and dynamics and will provide several molecular tools that will also facilitate the investigation of many other signaling paradigms.

DESCRIPTION (provided by applicant): Mitochondrial membrane permeabilization (MMP) is the step of commitment to cell death in a broad spectrum of cell injuries. MMP leads to the release of several factors activating the systematic execution of the cells and destroys the mitochondrial capacity to maintain ATP production. MMP is mediated by either pro-apoptotic Bcl-2 family proteins, Bax and Bak or by the permeability transition pore (PTP) that remains to be identified. In recent years, much attention has been focused on Bax that resides largely in the cytoplasm and upon to a variety of stress stimuli, gets inserted and activated in the outer mitochondrial membrane (OMM). By contrast, Bak is constitutively inserted to the OMM in most cell types and can respond to some activators faster than Bax. We and others have recently established that the targeting of Bak to the mitochondria is dependent on the presence of specifically the 2nd isoform of the Voltage Dependent Anion-selective Channel (VDAC2) in the OMM. We have also demonstrated that VDAC2 and the ensuing targeting of Bak to the mitochondria is required for the rapid response to insults that are relayed by Bid to the mitochondria. In our preliminary studies, we have shown that hepatocarcinoma mitochondria are far more sensitive to Bid than normal liver mitochondria and this difference is paralleled by more VDAC2 and Bak being present in the hepatocarcinoma. Our first hypothesis is that the differential sensitivity to Bid-induced cell killing in normal liver and liver tumor results from differential expression of VDAC2 and the ensuing differential recruitment of Bak to the mitochondria, offering a potential target for selective killing of hepatocarcinoma cells. We and others have provided evidence that both (a) Bid-induced mitochondrial membrane permeabilization and (b) IP3 receptor (IP3R)-mediated calcium signaling are effectively sensitized by reactive oxygen species (ROS). We propose that enhanced mitochondrial ROS production locally supports spreading of Bid-induced permeabilization among individual mitochondria and causes local enhancement of IP3R-mediated Ca2+ release and Ca2+ transfer to the mitochondria. The surplus Ca2+ further stimulates ROS production and facilitates opening of the PTP, initiating a vicious cycle that leads to cell death. To test this hypothesis, w have developed a novel molecular toolkit that allows us to directly monitor and perturb [ROS] and [Ca2+] dynamics at the mitochondrial surface or close associations of ER and mitochondria. Completion of this project will not only help to better understand the fundamental control of the mitochondrial membrane integrity but will also shed some light on the presently underappreciated role of Bak in cell killing and might uncover VDAC2 and Bak as candidates for selective targeting of human hepatocarcinomas. Tumors are commonly resistant to cell killing agents, and our work might show an Achilles' heel of hepatocarcinoma.

bioimaging /biomedical imaging; cell component structure /function; mitochondria; endoplasmic reticulum; biomedical resource;

DESCRIPTION (provided by applicant): In many apoptotic paradigms, a stress signal is delivered to the mitochondria (mitos) to induce release of apoptotic factors from the intermembrane space to the cytosol. These factors trigger execution of a program of cell death. In the cell, mitos often show coordinated activation of the individual organelles, giving rise to Ca 2+release waves and depolarization waves that depend on intermitochondrial communication. However, it remains elusive whether mitos interact with each other to establish coordinated release of apoptotic factors. Our principal hypothesis is that local intermitochondrial signaling is utilized to synchronize the response of individual organelles during apoptosis. We propose that several factors released by the mitos, such as pro-caspases, Ca 2+ and reactive oxygen species (ROS) may serve as a lateral signal that promotes recruitment of neighboring mitos to the apoptotic machinery. Furthermore, we propose that mitos release a novel factor, termed as mitochondrial permeabilization inducing factor (mPIF) that expands permeabilization to other mitos. Finally, we propose that intermitochondrial signaling may be particularly important in apoptosis of cells that are rich in mitos (e.g. muscle and liver). In the previous project period, we have demonstrated that calcium signal propagation to the mitos may trigger apoptosis. We have also shown that calcium release from mitos that undergo membrane permeabilization is important for the membrane permeabilization of the adjacent mitos, providing an example of the regenerative mechanisms that may underlie propagation of the mitochondrial death waves. Our preliminary studies have also shown coordinated recruitment of the mitos during truncated Bid (tBid)-induced mitochondrial permeabilization. Significantly, the tBid-lnduced wave of mitochondrial membrane permeabilization is not dependent on Ca. Furthermore, from mitos we have extracted a heat-sensitive and soluble factor that induces mitochondrial membrane permeabilization. To test our hypotheses, we will develop several high resolution fluorescence imaging methods to study mitochondrial function in single cells and will use single channel electrophysiology and a range of fluorometric, biochemical and molecular techniques. Dissection of the mechanisms of local intermitochondrial signaling is critical for understanding the operation of the apoptotic machinery and in turn, it is a key component in elucidating the regulation of a wide range of physiological and pathological processes that depend on apoptotic cell death.

DESCRIPTION (provided by applicant): Chronic alcohol abuse is associated with diseases of many organs, but the pathogenesis of these conditions still remains obscure. Studies on animal models have demonstrated that chronic ethanol feeding causes structural and functional derangements of mitochondria. Impairments of mitochondrial bioenergetics and the apoptosis-regulating function of mitochondria are considered to be central for the increased cell death in both alcoholic heart and liver disease. Emerging research indicates the interdependence between the factors that regulate mitochondrial morphology and function. Altered expression or mutation of the mitochondrial fusion- fission or motor proteins leads to cell/tissue injury. Our studies provide evidence that chronic ethanol exposure alters the mitochondrial morphology and fusion-fission dynamics. Our hypothesis is that the changes in mitochondrial morphology are crucial events in determining complex cellular and tissue responses to ethanol contributing to tissue injury. The research plan integrates genetics, advanced imaging and biochemistry approaches to unravel mechanisms, regulation and consequences of mitochondrial shape changes during ethanol-induced tissue injury. We have developed an array of live cell imaging approaches to visualize and quantitate both mitochondrial fusion-fission and motility, a toolkit that enables us to uncover the mechanisms, the regulation and impairment of mitochondrial dynamics upon alcohol exposure. The studies will focus on the tissue injury in heart and liver, where cell function relies on a high capacity of mitochondrial metabolism that can be regulated on demand. The experimental models will span the range from cell lines, through primary cultured cells to the intact organ. The experiments are organized into three aims as follows: (1) To determine the effects of chronic alcohol feeding on mitochondrial dynamics and analyze the underlying mechanisms; (2) To establish the relationship between mitochondrial bioenergetics and fusion-fission dynamics in control and alcohol-exposed tissue; (3) To determine whether the alterations in mitochondrial morphology and dynamics contribute to alcohol-induced cell injury. Understanding the role of mitochondrial dynamics in the chronic alcohol abuse-induced tissue injury will afford insights into the pathogenesis of alcoholic liver disease and cardiomyopathy and may open new avenues for developing diagnostic, prognostic and therapeutic tools. Narrative: Chronic alcoholism is associated with mitochondrial dysfunction and changes in mitochondrial morphology in multiple tissues. Mitochondrial dynamics is envisioned as a target of the alcohol's effect, which underlies changes in mitochondrial morphology which, in turn, lead to mitochondrial dysfunction. Recent progress in the study of mitochondrial structure and function in live cells will enable us to establish the mechanisms and significance of mitochondrial dynamics in the alcoholic liver and cardiac muscle injury.

DESCRIPTION (provided by applicant): This proposal brings together a unique multidisciplinary team of leading experts in alcohol studies and real time dynamic fluorescence imaging of cells and subcellular structures in intact tissues, high resolution electron tomography, sophisticated molecular biology and computational biology to address a fundamental question of how a deregulation of local interactions between organelles can result in global cell dysfunction, tissue damage and disease. The study addresses the regulation of the mitochondria-endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) interface and its relationship to localized Ca2+ signaling, formation of reactive oxygen species (ROS) and ER stress. The central hypothesis to be tested is that chronic alcohol exposure causes impaired ER/SR-mitochondrial structural and functional coupling and thereby increases the susceptibility to cell injury in liver and skeletal muscle. Our group has developed methods to manipulate the ER/SR-mitochondrial interface and has shown that this has consequences for localized Ca2+ signaling. Our first aim is to further develop these new experimental tools to be able a) to manipulate and measure the ER/SR-mitochondrial interface, b) to monitor the impact of controlled mitochondrial localization on local [Ca2+] signals and local ROS formation and c) to assess ER/SR stress responses in real time at the single cell level. This will help us analyze the relationship between ER/SR stress and changes in localized Ca2+ and ROS signaling. We will then test the concept that the effect of chronic alcohol exposure on ER/SR-mitochondrial interactions leads to impairments in ER/SR-mitochondrial structure, calcium signaling and induces ER/SR- mitochondrial stress responses in the liver and skeletal muscle. Finally, we will determine whether the alterations in ER-mitochondrial morphology and function contribute to alcohol-induced metabolic dysfunction and cell injury in vivo. With these approaches, we can answer fundamental questions about the subcellular organization of the ER-mitochondrial network and its disruption by ethanol. These studies will shed new light on the mechanisms by which alcohol abuse can cause tissue damage and will provide unique opportunities for the development of innovative treatment strategies. PUBLIC HEALTH RELEVANCE: Chronic alcoholism is associated with ER stress and mitochondrial dysfunction and changes in ER and mitochondrial morphology in multiple tissues. The ER/SR-mitochondrial junctions are important for the function and structure of both ER/SR and mitochondria and are envisioned as a target and an important mediator of the alcohol's effect on liver and skeletal muscle injury. A multidisciplinary approach utilizing expertise in alcohol studies and real time dynamic fluorescence imaging of cells and subcellular structures in intact tissues, high resolution electron tomography, sophisticated molecular biology and computational biology will help to address a fundamental question of how a deregulation of local interactions between organelles can result in global cell dysfunction, tissue damage and disease.

DESCRIPTION (provided by applicant): One third of alcohol abusers manifest decreased cardiac contractility and muscle strength termed respectively alcoholic cardiomyopathy and myopathy, accounting for about 6 million persons in the US. In fact, the most common cause of cardiomyopathy with congestive heart failure is chronic alcoholism. However, the pathogenesis of these conditions still remains obscure. Studies in human alcoholics and animal models have demonstrated that chronic ethanol consumption increases oxidative stress and other cellular stress factors and enhances the susceptibility to apoptosis. In addition to our demonstration of a marked increase in the rate of apoptosis in human skeletal muscle and heart from alcoholic patients, we have also shown decreased cardiac function in alcohol-fed animals. Our preliminary data indicate that ethanol also interferes with the proliferation and differentiation o skeletal muscle satellite cells and other stem cells in culture. These findings suggest that ethanol-induced sensitization to apoptosis/necrosis may alter the delicate cellular equilibrium between survival and cell death. We hypothesize that chronic alcohol abuse sensitizes heart and muscle to mitochondrial apoptosis elicited by Ca2+ overload and Bid at least in part by enhancing oxidative/nitrosative stress. We propose that chronic alcohol abuse also affects renewal mediated by progenitor cells in skeletal muscle, an organ that has a robust capacity for regeneration. Thus we propose that an impaired balance between cell death and renewal are central to the development of alcoholic cardiomyopathy and skeletal myopathy. The aims of this application are to i) evaluate stress response markers and apoptosis/necrosis in heart and skeletal muscle from human alcoholics and rats fed ethanol, with an emphasis on the mitochondrial stress pathways and ii) relate these parameters to the degree of alcoholic tissue injury, and iii) determine the effects of ethanol on progenitor cell number, proliferation and differentiation in skeletal muscle. We have available a collection of precisely annotated human heart and skeletal muscle tissues derived from patients on life support systems at the Hospital Clinic in Barcelona, Spain, and continue to obtain additional specimens. We have also established a productive collaboration with Dr. Pacher, the Chief of the Oxidative Stress and Tissue Injury Unit at NIAAA, who is a leader in research of oxidative/nitrosative stress in the cardiovascular system. Thus, the PIs' experience in calcium and Bcl-2 family protein-mediated mitochondrial stress and the pathologic aspects of human alcoholic cardiomyopathy and myopathy will be complemented by expertise in oxidative/nitrosative stress and by access to a unique human tissue resource. Furthermore, the studies will employ the recent advances in high resolution, high capacity and semi-automatic fluorescence imaging. We expect that the results of these studies will provide a unique bridge between the mechanisms underlying alcohol-induced tissue injury in animal models and in human alcoholics and will shed light on the pathogenesis of alcoholic cardiomyopathy and myopathy.

Mitochondrial Ca2+ uptake is central to energy metabolism, cell signaling and dynamics but exploration of the underlying molecular mechanisms and physiology has just started with the discovery of MCU, a pore-forming protein and MICU1, an EF-hand protein described as a critical regulator of the Ca2+ uniporter, which display striking co-evolution and co-expression. Recently, MICU2/3, paralogs of MICU1, MCUb, a dominant negative form of MCU, EMRE, an adaptor for MCU, and MCUR1, another regulator of the uniporter have also been described. Although a permanent and huge driving force supports mitochondrial Ca2+ uptake, there is a strict and complex regulation of the uniporter (mtCU) current by [Ca2+]. Cytoplasmic Ca2+ sensitivity to the MCU is conferred by MICUs via their EF hands. Work by us and others provided evidence that MICU1 is needed to keep the MCU closed at submicromolar cytoplasmic [Ca2+] ([Ca2+]c) and to promote co-operative MCU opening at higher [Ca2+]c. In the past <1.5 year project period, we have established the first mouse model for MICU1 and demonstrated that deletion of MICU1 increases the sensitivity to mitochondrial Ca2+ overload and cell death both in vivo and in vitro. Importantly, human disease associated with MICU1 loss of function has also been described in many patients. Thus, delineation of the mechanism relaying the effect of Ca2+ to the pore, and the functional significance and pharmacological targeting of mtCU are of vast significance. Moreover, because of complementing relevant skills in our group, and our previous success in and powerful toolkit for the study of the uniporter, we are uniquely qualified to move the stick in this area. Our hypotheses are that (1) MICU1 employs 3 distinct interaction sites for binding MCU, EMRE, and MICUs to relay the effect of Ca2+ to control the ion flux across the mtCU, (2) differences in the relative expression level of the mtCU components determine the tissue specific differential Ca2+ sensitivity and pharmacological properties of the mtCU, (3) in the liver and (4) the brain of MICU1-deficient mice, responses to physiological stimulation and responses to various stress conditions can be distinctively affected by impaired gatekeeping and cooperative activation of the mtCU, respectively. Testing of these ideas will provide clues to the fundamental mechanism controlling mtCU, to potential drug targets and to the pathogenesis initiated by perturbation of the mtCU constituents. The studies will utilize both novel human and murine genetic models and imaging methods.

? DESCRIPTION (provided by applicant): An elevation of cytosolic free calcium concentration is an integral component of the mechanism by which cells respond to hormones, growth factors and neurotransmitters. D-myo-inositol 1,4,5-trisphosphate ( IP3 ) is an intracellular messenger mediating the mobilization of Ca2+ from intracellular stores by interaction with an ubiquitous receptor ( IP3R ) that acts as a ligand-gated Ca2+ channel. IP3Rs are redox sensitive channels and are sensitized by oxidative stress. However, the molecular basis of this regulation is poorly understood. Ca2+ released from IP3Rs is locally transmitted to the mitochondria and can stimulate metabolism, and in higher amounts, can also initiate cell death. The overarching hypothesis of this study is that redox modulation of IP3Rs is an important component of the regulation of Ca2+ signals in cell death pathways. The proposal encompasses the following three specific aims: 1] To measure and map redox changes in IP3Rs. We have developed methods to determine the redox state of IP3Rs in vivo which will be used to quantitate the effects of exogenous and endogenous agents causing oxidative stress. Preliminary studies using mass-spectroscopy identify a subset of 11 cysteines that become oxidized in IP3R-1. The type of oxidative modifications occurring will be identified. Redox-sensitive thiols will be mutate and the functional sensitivity to oxidative stress will be assessed. 2] To measure IP3R redox state at the ER/mitochondrial junction. We will test the hypothesis that the pool of IP3Rs located at the ER/mito junction is particularly prone to ROS modifications. We will employ subcellular fractionation and imaging methods utilizing targeted IP3Rs, ROS-sensitive fluorescent proteins, ROS-producing photosensitive probes and targeted catalases. 3] To investigate the role of IP3R redox changes in models of ER stress/apoptosis. We will test the hypothesis that ER-resident NADPH oxidases play an important role in IP3R redox regulation. Liver will be used as an experimental model to induce ER stress. The role of IP3R redox regulation in ER stress pathways activated by fructose will be examined. The long-term goal of the proposal is to obtain a detailed understanding of how oxidative stress impacts intracellular Ca2+ signaling under normal and disease conditions.

? DESCRIPTION (provided by applicant): A range of environmental agents causes tissue injury that has been attributed to reactive oxygen species (ROS) produced by mitochondria. However, the causative pathways remain largely unknown because it has been difficult to directly monitor or specifically perturb ROS. In the R21 phase, this proposal brings together efforts to develop a new, genetically-targeted toolkit to perturb and measure ROS and calcium (Ca2+) signals in a sensitive and specific manner. Furthermore, this toolkit will allow recording of ROS and Ca2+ down to the level of specific subcompartments of the mitochondria, which likely make differential contributions in ROS dysregulation. The R33 phase will use adeno-associated viruses and transgene expression to bring the novel toolkit into mice to enable study of the effect of various environmental agents on ROS and Ca2+ signals in situ in the liver, heart, and skeletal muscle. Within phases one and two, the project will study the specific involvement of ROS and Ca2+ in the stress pathways triggered by arsenic (As), cadmium (Cd), and dioxin. The investigators will specifically test the novel hypothesis that environmental stress induced by these agents causes impaired mitochondria- endoplasmic/sarcoplasmic reticulum (ER/SR) functional and structural coupling, providing an important mechanism underlying cell injury in various tissues, including the liver, cardiac and skeletal muscle. This team has developed methods to manipulate the mitochondrial-ER/SR interface and has shown that this has consequences for localized Ca2+ signaling. These studies will allow for a paradigm shift in the way mitochondrial pathogenesis of environmental stress is studied, and will shed new light on the mechanisms by which environmental agents can cause tissue damage, leading to unique opportunities for the development of innovative treatment strategies.

PROJECT SUMMARY/ABSTRACT Liver cancers with increasing frequency in the world have become the second cause of cancer related death; however an important gap in current knowledge remains the molecular fingerprint and organization of hepatocarcinoma/hepatoma cells which might allow their specific targeting. We found that the expression level of two proteins, VDAC2 and Bak, that are central to a mitochondrial apoptosis pathway, is low in normal hepatocytes and is increased during hepatic tumor progression. Furthermore, we also found that the pro- apoptotic BH3-only Bcl-2 family protein, Bid induces mitochondrial apoptosis more efficiently in hepatocarcinoma than in normal liver. This difference can be eliminated by VDAC2 silencing in hepatocarcinoma cells, and by VDAC2 expression in normal hepatocytes but only if Bak isn?t knocked out. The potential medical significance of the VDAC2-Bak heterogeneity is supported by human databases that show upregulation of VDAC2 and Bak proteins in liver cancer. We have also documented both subcellular and cell- to-cell differences in the tBid-Bak pathway. Finally, we have shown at least in cell culture, that hepatocarcinoma cells can be selectively killed through the tBid-Bak pathway (using its activators and suppressors of its inhibitor, Mcl-1 while normal hepatocytes are spared. We here, postulate that the heterogeneity in VDAC2 and/or Bak abundance in the liver are important for hepatoma/ hepatocarcinoma (1) growth and (2) targeting by the combination of an Mcl-1 inhibitor drug and a cell permeable hydrocarbon stapled Bid BH3 peptide. Heterogeneity is considered (1) within the cells among individual mitochondria, (2) in each cell type among single cells, and (3) among the different cell types. To test our hypothesis we have established a combination of genetic targeting, tumorigenesis in mouse, and biochemical and microscopic imaging approaches. In the study we will focus on (1) assessing heterogeneity of VDAC2, Bak and Bak-mediated OMM permeabilization in hepatocarcinoma cells and normal hepatocytes and their dependence on VDAC2 expression and mitochondrial fusion; (2) determining whether increased expression of VDAC2 through upregulation of Bak affects hepatoma/hepatocarcinoma progression; (3) testing if activation of Bak by treatment with Mcl-1 inhibitor (S-63845) and a cell permeable hydrocarbon stapled Bid peptide can be used to kill hepatocyte-derived tumors without damaging normal hepatocytes; and (4) determining additional VDAC2-dependent proteins in hepatic tumor cells and to evaluate their heterogeneity in the liver and their relevance for hepatoma/hepatocarcinoma growth. By addressing these points, our study will provide clues to the contribution of mitochondrial heterogeneity to hepatic tumorigenesis and test a novel tumor-selective targeting approach.

Abstract Early studies of mitochondria isolated from various tissues have demonstrated that Ca2+ uptake is driven by the membrane potential (??m), and is mediated by a ruthenium red-sensitive electrogenic uniport, referred to as ?Ca2+ uniporter? (mtCU). Electrophysiological recording of mtCU documented a similar inwardly rectifying Ca2+ current in mitoplasts derived from different tissues but great differences appeared in the current density, which was particularly low in cardiac mitochondria. Recently, the major mtCU forming proteins have been identified, including the pore, MCU, its dominant-negative form, MCUb, a scaffold, EMRE, and Ca2+-sensitive regulators, MICU1 and MICU2. To date, a MICU complex (a hetero/homo-dimer of MICU1 and MICU2) appears to determine both the threshold and cooperative activation of the mtCU by Ca2+, thus providing a mechanism for the sigmoidal [Ca2+] dependence of the mtCU. MICU1 deletion in mouse is perinatal lethal, likely because of mitochondrial Ca2+ overload-induced cell death. mtCU components show tissue-specific expression and MICU1 is expressed at a particularly low level in cardiac muscle. The tissue specific differences in the mtCU current and molecular composition of the mtCU -shown by our initial studies- are particularly interesting in the context of the distinct calcium signaling patterns that mitochondria from cardiac muscle and other tissues such as the liver have to cope with. Our preliminary results also show that heart failure patients have elevated cardiac MICU1 and MICU1-to-MCU ratio. We put forward the hypothesis that meeting the heart-specific calcium signaling needs depends on the relative abundance of the mtCU components, and adaptations to physiologic and pathogenic stress in the heart are associated with and determined by plasticity in the mtCU molecular composition. We also propose that the composition of the mtCU determines how mitochondrial Ca2+ uptake influences oxidative metabolism and mitochondrial fusion dynamics, which is central to keeping mitochondria in shape. The proposed studies will depend on an array of sophisticated live imaging techniques, novel genetic mouse models and animal models for heart failure. The hypothesis will be tested through the following specific aims. Aim1 will determine the impact of the protein levels and stoichiometry of the mtCU components on the Ca2+-dependent regulation of mitochondrial Ca2+ uptake, ATP production and fusion dynamics in cardiac muscle. Aim2 will determine the relevance of the mtCU protein profiles and the Ca2+ dependence of mitochondrial Ca2+ uptake on contractile function, including in the context of physiological and pathological stress. Collectively, these studies will be useful to establish how mtCU composition at the tissue level, namely the extent by which the population of MCUs is regulated by MICU1 and MICU2, and the resulting tissue-level pattern of mitochondrial Ca2+ uptake, determine the cardiac specific pattern of contractility, bioenergetics and mitochondrial quality control, under unchallenged conditions as well as under conditions of increased physiological or pathological cardiac workload.

Project Summary/Abstract This proposal requests partial support for a brand-new Gordon Research Conference (GRC) entitled ?Mitochondria in Health and Disease?, which is scheduled for March 2019. This conference is brand new to the GRC, an organization known world-wide because of the high-quality, cutting-edge nature of its meetings. We anticipate 200 participants, including a large contingent of graduate students, postdocs and early career independent scientists. The program will provide unique opportunities for the many female scientists and other under-represented minorities working on the field by scheduling 10+ female speakers and discussion leaders, a power hour, a meeting with NIH program directors, and fellowships and short talks reserved for minorities. Mitochondria were traditionally thought to act only as cellular powerplants. Recent progress in genetics, proteomics and imaging approaches to track mitochondria in vivo have provided evidence that mitochondria are central to cell signaling and dynamics. However, we still know very little about how they behave in the cell, and what influences them biochemically and physically. The proposed conference will focus on these aspects of mitochondrial existence both in model systems and cells with specialized structural and functional arrangements like neurons, cardiomyocytes and muscle fibers. The program will have an emphasis on the physiological situations but will also cover mitochondrial stress responses elicited by various conditions, including environmental exposures which initiate or augment cell injury, and the balance between homeostasis and apoptosis. Considering the dominance of cardiac, neuronal, and muscular impairments in mitochondrial diseases, the program of the new conference will focus on these tissues. The following subject areas will be discussed in 9 separate platform sessions: Construction of mitochondria, biosynthesis of lipids; Mitochondrial transfer and positioning; Contact formation and function; Membrane fusion-fission and content exchange; Signaling on the mitochondrial surface (BCL-2 family complexes and other signaling complexes) and in the intermembrane space; Calcium and ROS signaling; Mitochondrial stress responses; Diseases originated from or progressed by mitochondrial pathobiology; New technologies to facilitate mitochondrial biology research. Thus, we will address the unmet needs for comprehensively support of the broad and rapidly emerging mitochondrial biology research and the wealth of scientists who have been working on this field, including junior investigators who seek training. We intend to capture the full diversity of global mitochondrial biology and medicine research with our conference, which should set us apart from other meetings and should result in a deep and lasting impact on the field. Overall, supporting this conference is of multifold relevance for the mission of NIH: (1) will accelerate research by providing a new forum for a robustly developing field, (2) will foster a cross-disciplinary perspective, (3) will educate and facilitate networking and career opportunities for junior scientists, (4) will create opportunities for under-represented minorities.