Peipei Ping - US grants

DESCRIPTION (provided by applicant): The central hypothesis of this competitive renewal is formulated on the basis of two key developments in the field of ischemia biology and cardioprotection: (i) the identification of multiprotein complex formation as a means for cardioprotective signaling and (ii) the establishment of the mitochondria as a critical organelle for cell death and survival. The functional link between these two developments is mitochondrial permeability transition (MPT) in ischemic injury. MPT involves impaired mitochondrial function leading to cell death and is carried out by a multiprotein complex, the MPT pore. Deciphering the proteomic basis of the MPT pore and elucidating its role in cardioprotection constitute the major goals of this application. The proposed studies will focus on nitric oxide (NO)-induced late preconditioning (PC), a well-characterized pharmacological means of cardioprotection with a poorly-defined proteomic basis for its underlying cellular mechanisms. To achieve our goals, the application directs its effort at two targets: the Oracle isozymes, a novel family of scaffolding proteins critical to NO protective signaling, and the subproteome of ANT1, a repertoire of ANT1-associating proteins that are essential to MPT pore function. We hypothesize that attenuated propensity for mitochondrial permeability transition is an essential signaling event in NO-mediated cardioprotection and that the Oracle family of proteins plays a critical role in this process by modulating the ANT1 subproteome and contributing to the protection of mitochondrial function. Three specific aims, with tightly related experimental protocols are proposed. Aim 1 will establish the essential role of Oracle, a molecular backbone of signaling complexes, in NO-induced preconditioning. Aim 2 will build upon knowledge gained in Aim 1 to elucidate the function of the MPT pore in NO-mediated cardioprotection and determine the role of Oracle in this process. Finally, Aim 3 will define a subproteome, composed of ANT1-associating proteins, which is critical to the regulation of MPT pore function in cardiac mitochondria. Importantly, for the first time, the proteomic basis of the participation of the MPT pore in NO protection will be determined and the role of Oracle in the assembly of this subproteome will be fully characterized. A comprehensive strategy encompassing pharmacological methods, transgenic models, and proteomic characterization is proposed. These studies will elucidate the proteomic basis of NO preconditioning, define mechanisms underlying ANT1 regulation, and identify novel molecular targets that are critical to the modulation of MPT pore function. Thus, the findings will have broad implications for future investigations concerning pharmacological cardioprotective interventions.

2010-2014 has been a productive period. Our success in devising novel methods to study the molecular mechanisms underlying cardioprotection has exceeded expectations. We achieved four major accomplishments, all of which were driven by technological innovations. Briefly, we created novel platforms to analyze quantitative protein phosphorylation and protein turnover rates; we expanded the reach of 'omics techniques to a widening field of novel proteome parameters; we built novel computational tools and data-to- knowledge workflows (Cardiac Organellar Protein Atlas Knowledgebase (COPaKB) and ProTurn) and integrated them into our experimental designs and thought processes. These endeavors are highly significant because (1) through them we gained an elevated understanding into the many aspects of cardiac mitochondrial functions within the purview of cardioprotection; and (ii) these new tools pave the way for future investigations and the testing of new hypotheses in multifarious directions. The major scientific outputs supported by this Award are highlighted by a series of 11 peer-reviewed publications in journals such as Circ Res, J Clin Invest, and J Proteome Res (the Ping laboratory has contributed 36 publications from 2011- 2014). Furthermore, the MERIT Award supported 24 trainees. The trainees collectively received 11 academic honors and recognitions. The PI also received 3 international awards, participated in 6 NIH workshops, co- authored 4 white papers/ reviews, and succeeded in 1 patent application (UC 2013-137-0). In the extension period, we will examine the spatiotemporal dynamics of mitochondrial cardioprotective signaling pathways. Innovation will maintain priority in the MERIT Award, as we will continue to create new in- house technologies and apply them directly to elucidate the regulatory mechanisms of cardiac disease and protection. Three Specific Aims are proposed in this renewal. First, we will elucidate the spatial distribution of mitochondrial protein complex assembly and interaction in the setting of cardioprotection and/or elevated oxidative stress. Second, we will examine the effect of cardioprotection and/or oxidative stress on protein temporal dynamics. Third, we will design and construct an 'omics-based screening strategy to select human induced-pluripotent stem cell (iPSC)-derived cardiomyocytes that are resistant to oxidative stress and injury. We will validate molecular markers and evaluate their informativeness in predicting stress-resistant phenotypes in subsequent lines. We are convinced that these studies will propel our knowledge on cardioprotective environments and also avail the use of iPSC-derived cardiomyocyte experimental models in future discoveries and therapies. RELEVANCE (See instructions): Myocardial ischemic injury affects millions of people worldwide and is a leading cause of death. The proposed studies will examine the mitochondrial signaling mechanisms of NO-donor or PKCe-mediated cardioprotection against myocardial ischemic injury. Understanding the pathogenic mechanisms of and protection from ischemic injury has major implications to improve human health, and will create numerous opportunities to make key contributions to the fundamental knowledge of mitochondria and survival signaling using new technologies.

DESCRIPTION (provided by applicant): The cardiac mitochondria are critical regulators of cell survival during ischemic injury. Considerable research has been directed at interrogating molecular mechanisms that impact their normal and pathological function in the heart. Despite these efforts, to date, very limited information is known regarding the proteomic basis of this vital organelle in the normal and diseased myocardium. Consequently, a fundamental knowledge base to advance our understanding of the role of mitochondria in cardiac disease pathogenesis is lacking. At least two proteomic approaches may be utilized to map the mitochondrial sub-proteome. One focuses on broad-scale annotation of proteins (i.e., expression proteomics) and the second undertakes not only the identification but also the functional characterization of a sub-group of proteins (i.e., functional proteomics). We reason that a simple catalog of proteins without a functional correlate, or a partial catalog of proteins without information regarding the manner in which the mitochondrial proteins are organized to perform their cellular tasks, will be insufficient to generate a thorough understanding of the true proteomic basis of cardiac mitochondrial function. Thus, the application of functional proteomics is more appropriate and critically important to investigate the cardiac mitochondrial sub-proteome, because it gains proteomic information that is linked to the essential biological function of mitochondria. Recent studies have demonstrated that proteins are organized as multiprotein complexes within functional sub- 3roteomes. These multiprotein complexes are assembled to facilitate signal transduction in biological systems. Accordingly, characterization of multiprotein complexes enables not only identification of proteins in sub-proteomes but also characterization of the biological functions that they support. Nevertheless, a versatile functional proteomic Platform specifically designed to target characterization of native mitochondrial multiprotein complexes in the heart is absent. Such a platform would serve as an essential tool to delineate the proteomic basis of mitochondrial function in health and disease, and to decipher the role of multiprotein signaling complexes in this organelle. The central goal of this application is to develop a state-of-the-art proteomic platform with high-sensitivity and high-speed that is tailored for analysis of mitochondrial multiprotein complexes, to gain insights on mitochondrial protein function at a large-scale, and to explore the dynamic modulation of the mitochondrial subproteome during cardiac ischemia. The specific aims are: Aim 1--To develop and optimize a functional proteomic platform to analyze cardiac mitochondrial multiprotein complexes to discern: what proteins make up these complexes, specific protein-protein interactions among proteins within the complexes, and post-translational modifications of these proteins. Aim 2-- To elucidate the effect of myocardial ischemia on mitochondrial multiprotein complexes, protein-protein interactions, protein function, and post-translational modifications using the platform optimized in Aim 1.

ISCHEMIC INJURY AND PROTECTION Program Project Grant Departments of Physiology, Medicine & Anesthesiology David Geffen School of Medicine University of California, Los Angeles UCLA Principal Investigator/Program Director (Last, First, Middle): Ping, PROJECT 2: PKCe-DEPENDENT MODULATION OF VDAC AND ANT IN ISCHEMIC INJURY AND PROTECTION: IMPACT ON THE MPT PORE CLA Project Leader: Peipei Ping, Ph.D. Co-Project Leader: Ligia Toro, Ph.D. PHS 398 (Rev. 05/01) Page 121 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Ping, Project 2) Project 2 is designed to address the central theme of the PPG by examining kinase-dependent regulation of the mitochondrial permeability transition (MPT) pore in ischemic injury and protection. Project 2 complements the effort of Project 1 in characterizing signaling events that modulate mitochondrial permeability transition (MPT). Project 2 will delineate the molecular mechanism by which PKCe, a well-established cardioprotective kinase, interacts with and modifies key components of the MPT pore. The present studies are built upon our previous findings that the two core elements of the MPT pore, the outer membrane protein VDAC and the inner membrane protein ANT, are members of the PKCe sub-proteome at the mitochondria. These findings have been confirmed by the observation of strong localization of VDAC and ANT to PKCe immuno-complexes. Accordingly, Project 2 hypothesizes that PKCe interacts with, and modulates, VDAC and ANT via phosphorylation and that these post- translational modifications of VDAC and ANT impact their pore-forming abilities in the setting of ischemic injury and cardiac protection. Specifically, in collaboration with the Heart Biology Core, Project 2 will determine subcellular colocalization of PKCe with VDAC and ANT (using high resolution confocal imaging techniques) and will examine distribution of this kinase at the outer and inner mitochondrial membranes (by EM analyses). In collaboration with the Proteomic Core, Project 2 will determine the phosphorylation sites on endogenous VDAC and ANT in the in vivo setting. This knowledge in hand, Project 2 will take advantage of the strong expertise of the investigator team with membrane proteins to perform liposome reconstitution assays to conclusively examine the pore-forming abilities of wild type VDAC and ANT as well as their mutants either devoid of phosphorylation or constitutively activated. Furthermore, in collaboration with Project 4, Project 2 will determine how cell death pathway regulatory proteins (e.g., Bcl-2 and Bax) regulate the pore-forming abilities of VDAC and ANT and will examine whether the interactions of VDAC and ANT with these proteins are affected by PKCe-dependent phosphorylation. The proposed investigations will integrate information gained through membrane biophysics and proteomics studies with that from functional analysis at the organelle and system level. These studies will define, for the first time, the precise manner in which a cardioprotective kinase interacts with the MPT pore components; will map endogenous phosphorylation sites on VDAC and ANT in the normal myocardium and examine changes in these modifications that occur in the setting of cardioprotection; and will provide novel insights regarding how PKCe-VDAC-ANT complexes may be regulated by the Bcl-2 family of proteins.

DESCRIPTION (provided by applicant): Investigations of ischemic injury and cardiac protection over the past two decades have greatly advanced our understanding of the cellular events that mediate these biological processes. However, significant controversies exist with regard to the specific molecules and signaling mechanisms that underlie cardiac cell death and survival. We believe that the use of divergent models in isolation and the analysis of individual proteins-in the absence of thorough investigation of the associated molecules and their respective interactions at the subproteomic level-has hampered accurate characterization of how proteins contribute to a phenotype in vivo. These issues have in turn negatively impacted the translation of basic science advances of the past 20 years to improved treatment of ischemic heart disease. This Program Project takes a novel approach to address the aforementioned limitations and therefore affords great opportunities for conceptual and mechanistic advancement of the field of myocardial ischemia and cardiac protection. We feel that a significant paradigm shift is required to accelerate translational research in the area of myocardial ischemia. Accordingly, instead of relying on one particular experimental model, our unique team of investigators enables the characterization of the role of specific proteins using an array of models at the system, organ, cell and organelle levels. Instead of focusing the study on one pathway and/or a single molecule in isolation, the novel proteomic technology platform provided by the Proteomic Core enables systematic examination of a protein and its associating partners, i.e., the subproteome in which the protein functions in the setting of ischemic injury and cardiac protection. Moreover, subsequent to subproteome mapping studies, our investigator team will engage in a rigorous target validation process. This validation process will be facilitated by the state-of-the-art techniques assembled in individual Projects as well as the Heart Biology Core. These techniques include cardiac-targeted inducible transgenesis, high resolution confocal microscopy, and integrative physiology, which in combination will allow for comprehensive and unequivocal investigation of the mechanistic nature of ischemic injury and protection. Importantly, these studies will target subproteomes involved in physiologic phenotypes and therefore the findings will facilitate an understanding of the discrete molecular context in which specific molecules elicit their cellular effects. These investigations will elucidate more accurate targets for therapeutic intervention and thus will be inherently more translatable to the pre-clinical arena.

[unreadable] DESCRIPTION (provided by applicant): Despite many investigations establishing the role of mitochondrial permeability transition (MPT)-an opening of non-selective pores spanning the matrix to the cytosol that leads to mitochondrial dysfunction and cell death-in ischemic injury, the discrete mechanisms governing modulation of a proposed multiprotein complex that regulates MPT (i.e. the MPT pore), remain poorly defined. Two proteins, the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have been implicated as the core pore-forming units of the MPT pore by virtue of their independent abilities to form non-selective channels in response to stresses associated with ischemia/reperfusion injury. The focus of this application is to define the fundamental roles of these molecules in protection against myocardial ischemic insult. [unreadable] [unreadable] Previous studies have mapped both VDAC and ANT localization to contact sites between outer and inner mitochondrial membranes. Pharmacological interventions targeting VDAC and ANT show effective modulation of MPT in cell and organelle settings. Despite this, the sub-organelle distribution and isoform-specific function(s) of distinct VDAC and ANT isoforms-critical to understand the regulatory mechanisms of the MPT-have been virtually unexplored, especially in cardiac research. Our recent preliminary findings suggest that targeted inhibition of VDAC is sufficient to protect the heart against ischemic insult and that induction of pore-formation by ANT blocks the protective effects of pharmacologic and genetic cardioprotective regiments. Furthermore, our preliminary data demonstrate for the first time that multiple isoforms of VDAC (VDAC1-3) and ANT (ANT1-2) are expressed in the adult mouse heart as detected by both immunoblotting (using antibodies we developed since the original submission) and LC/MS/MS. These findings are very exciting, as they dispel the widely-held belief that all actions performed by myocardial VDAC and ANT can be attributed to VDAC1 and ANT1. [unreadable] [unreadable] This A1 application has been completely revised in response to critiques by the review panel. Stimulated by our new data as well as other developments in the field, the revised proposal addresses a number of key questions that are critical to the role of MPT in myocardial ischemic injury. Our central hypothesis is that isoforms of VDAC and ANT participate in distinct manners in the phenomenon of MPT, in part owing to their differential suborganellar localization, pore-forming properties, and interactions with regulatory proteins (e.g., PKC(, Bcl-2, and Bax). We will define the sub-organelle distribution of distinct isoforms of VDAC and ANT in the normal heart and during cardioprotection and will rigorously elucidate the pore-forming properties of these molecules in the reconstituted liposome format in response to known injurious factors present during ischemia/reperfusion injury (including ROS and Ca2+). We will examine the expression, localization and interactions among critical components of the MPT pore subproteome using confocal and electron microscopy. Lastly, we will delineate post-translational modification of MPT pore components using mass spectrometry and interrogate the role of these modifications to regulate MPT. These studies will provide novel and mechanistic information to aid our understanding of mitochondrial function in myocardial ischemic injury. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Investigations in the past decades have significantly advanced our understanding of signaling mechanisms underlying the protection and pathogenesis of myocardial ischemic injury. It is increasingly recognized that preservation of mitochondrial function plays a pivotal role in cardioprotection against ischemia reperfusion injury (I/R). However, it remains virtually unknown as to who the molecular targets of cardioprotection are in the mitochondria; what specific molecular events led to the protection of mitochondria; and whether there is a systems integration of cardioprotective signaling at the mitochondria to support the manifestation of a protected phenotype. Using a murine model of nitric oxide (NO) induced late phase of cardioprotection, we elect to examine the plausible intrinsic signaling properties of mitochondria using a novel experimental strategy enabling a parallel examination of mitochondrial signaling, mitochondrial proteomes, and mitochondrial behavior by computational modeling. The proposed studies are based upon preliminary evidence by others and our own demonstrating that activation of PKC?-Src module occurs in the NO donor treated mice and that both are localized to mitochondrial membranes. In this proposal we will test the innovative hypothesis that the PKC?-Src module interacts with the brief mPTP openings to protect cardiomyocytes from Ca++ overload induced jury. The working hypothesis is that NO activates PKC?-Src module, leading to brief mPTP openings which results in transients Ca++ releases and reactive oxygen species (ROS) bursts, and consequently inactivates Ca++ reuptake and further activates PKC?-Src to form a feed- forward loop. When the homeostasis is interrupted (e.g., calcium overload or elevated ROS), brief mPTP openings transits into irreversible, long-lasting mPTP openings, which instead induce cardiac injury. In this application we propose to delineate the functional effects of brief openings of mPTP on Ca++ handling and ROS production; and to elucidate mPTP regulation by the mitochondrial PKC?-Src module in the setting of NO-induced late phase of cardioprotection (Aim 1). In the specific Aim 2 we will conclusively establish the activation of a PKC?-Src signaling module in the mitochondria as a mandatory signaling element of NO-induced cardioprotection against myocardial ischemic injury. At last we will systematically define the molecular targets of mitochondrial Src-kinase in NO- induced late phase of cardioprotection (Aim 3). The proposed studies will advance our understanding of cardiac biology by providing novel mechanistic insights into how interactions of brief mPTP openings with mitochondrial PKC?-Src module can be beneficial in mediating NO-induced late phase of cardioprotection. (End of Abstract)

The overall goal of the UCLA Proteomics Center is to gain mechanistic and functional insights into cardiovascular disease through characterization of the core organelle constituents of the mitochondria and proteasome in normal and diseases myocardium. Additionally, molecular pathways that regulate these organelles will be delineated with the ultimate goal of identifying novel therapeutic targets.

DESCRIPTION (provided by applicant): Oxidative stress has been increasingly recognized as a common feature among different forms of heart disease. Elevated reactive oxygen species (ROS) has been shown as a convergent signaling messenger leading to failing heart either of ischemic or non-ischemic origin. Despite significant progress in many areas of ROS related investigations, two fundamental issues remain unresolved and will constitute the center of this proposed investigation. The first question is who are the regulators of ROS in the diseased myocardium? The second question is what are the molecular targets of ROS and how do ROS-induced molecular modifications result in cardiac dysfunction? This application is inspired by our exciting data identifying PP2Cm as a novel regulator of ROS;and by the intriguing preliminary evidence that the cardiac proteasome complexes are a new class of molecular targets for ROS. Accordingly, the proposed investigation will address a novel aspect of ROS signaling: its modulation by PP2Cm, and it will embark on a largely unexplored area of research: the functional consequences of ROS elevation--its impact on the proteasome systems and their substrates. The application will determine the emerging role of PP2Cm in ROS biology;it will establish proteasome subunits as a new set of molecular targets for the elevated ROS;and it will systematically characterize perturbed protein degradation pathways in the normal and stressed myocardium. Furthermore, the application will identify potential therapeutic windows whereby disrupted protein quality control may be rescued. To accomplish our goals, two related models of cardiac stress--pressure overload by transverse-aortic constriction (TAC) and myocardial ischemic injury (I/R)-are employed. Three specific aims are proposed: Aim 1 will elucidate mechanisms underlying PP2Cm mediated protection of the heart;it will determine its role in governing ROS regulation and examine the impact of genetic perturbations of PP2Cm in TAC and I/R using the newly established PP2Cm genetic models in-house (the null/LacZ knock-in KO and the cardiac conditional inducible Tet-Off). Aim 2 will establish roles of the 20S and 26S proteasomes in the two stress models with respect to proteasome complex assembly, function, and degradation capacity;it will decipher molecular events underlying ROS damaged 20S and 26S proteasomes. It will apply a targeted proteomic approach to delineate the molecular modification of proteasome subunits;and it will define the functional significance of such modifications. Aim 3 will define functional consequences of ROS-injured 20S and 26S proteasomes in the two pathological models;it will characterize the substrate repertoire of 20S and 26S proteasomes in the normal and stressed myocardium. Our research plan is supported by "a technology tool box" combining established methods and innovative approaches assembled by the investigator team. It encompasses genetic models, proteasome biology, ROS biology, disease models, quantitative proteomics, and high-resolution imaging. Collectively, the proposed studies will conclusively characterize PP2Cm regulation of ROS in the stress myocardium;it will establish proteasome subunits as novel targets of ROS;and it will provide mechanistic insights into protein homeostasis in the two stress models. PUBLIC HEALTH RELEVANCE: Our proposal addresses a novel hypothesis that oxidative stress in the diseased myocardium is regulated in part by a mitochondria specific protein phosphatase;and one important functional impact of the elevated levels of reactive oxygen species is the damage of cardiac proteasomes. The proposed investigation will gain insights on these diseases and will help to develop new therapeutic strategies to prevent or reverse pathological remodeling in the diseased myocardium. Therefore, our proposal has significant clinical relevance as well as potential key contributions to the fundamental knowledge of mitochondrial biology, ROS biology, proteasome biology, and their functional impact on the cardiovascular systems.

DESCRIPTION (provided by applicant): Ischemic and pharmacologic preconditioning (PC) constitute the most powerful protection of the heart from ischemia/reperfusion (I/R) injury; however, the detailed molecular mechanisms underlying cardioprotection are still being defined. There is a general consensus that mitochondria are the final effectors of cardioprotective signaling regimes, and hexokinase (HK) has been suggested by multiple groups to regulate the mitochondrial permeability transition (MPT). Though the association of HK with voltage-dependent anion channels (VDAC) was elucidated over 10 years ago, two fundamental questions regarding the physiologic consequences of this interaction have remained unanswered, and consequently, have stalled the progression of the cardioprotection field. First, is the dissociation of HK from cardiac mitochondria a molecular trigger of cell death? That is, does HK dissociation from mitochondria precede all other cell death events [e.g., MPT, DY loss, and cytochrome C (cyto C) release]? Second, what are the unknown molecular players that stabilize the HK-VDAC interaction and impart its unique cardioprotective properties? Unequivocal answers to these questions have been unattainable due to the lack of technologies for (i) temporal profiling of the spatial distribution of HK in relation to MPT, ?? loss, and cyto release in live cardiomyocytes, and (ii) quantifying the molecular constituents of the HK-VDAC complex and deciphering their stoichiometry. In view of these challenges, our program has tailored state-of-the-art live-cell imaging and quantitative proteomic innovations to comprehensively delineate the dynamics of HK-induced cardioprotection on a biological timescale. We hypothesize that HK is a core regulator of cardioprotection, common to multiple models of injury and preconditioning. We will employ real-time imaging in live myocytes to define the temporal profile of the molecular events during injury (Aim 1); we will use an extensive biochemical and genetic toolbox to delineate the molecular paradigm of HK interaction with mitochondria as well as its physiological consequences mediating cardioprotection (Aim 2); we will quantitatively define the proteome dynamics and molecular stoichiometry of HK interaction with VDAC; characterize isoform-selective changes in assembly of the HK- VDAC interactome; and identify candidate proteins essential to stabilize the HK interaction with VDAC during cardioprotection (Aim 3); and we will use cardiac gene delivery of HK constructs or other molecular candidates identified in Aims 1-3, to test an in vivo gene therapy strategy to protect adult rats from I/R injury (Aim 4). The proposed investigations promise conceptual, technological, and methodological innovations. We will leverage close collaborations with the UCLA NHLBI Proteomics Center for immediate and efficient translation of knowledge obtained in cell/animal models to clinical studies. The success of the proposed investigations will undoubtedly propel the field of cardioprotection forward.

DESCRIPTION (provided by applicant): The inception of the BD2K Initiative is a testament to the foresight of NIH and our community. Clearly, the future of biomedicine rests on our collective ability to transform Big Data into intelligible scientific facts. In line with the BD2K objectives,our goal is to revolutionize how we address the universal challenge to discern meaning from unruly data. Capitalizing on our investigators' complementary strengths in computational biology and cardiovascular medicine, we will present a fusion of cutting-edge innovations that are grounded in a cardiovascular research focus, encompassing: (i) on-the-cloud data processing, (ii) crowd sourcing and text-mining data annotation, (iii) protein spatiotemporal dynamics, (iv) multi-omic integration, and (v) multiscale clinical data modeling. Drawing from our decade of experience in creating and refining bioinformatics tools, we propose to amalgamate established Big Data resources into a generalizable model for data annotation and collaborative research, through a new query system and cloud infrastructure for accessing multiple omics repositories, and through computational-supported crowdsourcing initiatives for mining the biomedical literature. We propose to interweave diverse data types for revealing biological networks that coalesce from molecular entities at multiple scales, through machine learning methods for structuring molecular data and defining relationships with drugs and diseases, and through novel algorithms for on-the-cloud integration and pathway visualization of multi-dimensional molecular data. Moreover, we propose to innovate advanced modeling tools to resolve protein dynamics and spatiotemporal molecular mechanisms, through mechanistic modeling of protein properties and 3D protein expression maps, and through Bayesian algorithms that correlate patient phenotypes, health histories, and multi-scale molecular profiles. The utility and customizability o our tools to the broader research population is clearly demonstrated using three archetypical workflows that enable annotations of large lists of genes, transcripts, proteins, or metabolites; powerful analysis of complex protein datasets acquired over time; and seamless aQoregation of diverse molecular, textual and literature data. These workflows will be rigorously validated using data from two significant clinical cohorts, the Jackson Heart Study and the Healthy Elderly Longevity (Wellderly). In parallel, a multifaceted strategy will be implemented to educate and train biomedical investigators, and to engage the public for promoting the overall BD2K initiative. We are convinced that a community-driven BD2K initiative will best realize its scientific potential and transform the research culture in a sustainable manner, exhibiting lasting success beyond the current funding period.

DESCRIPTION (provided by applicant): Mitochondrial dysfunctions are commonly observed in cardiac injury and heart failure. Despite significant progress in past decades, the molecular sequence of events that transition the cardiac mitochondrial proteome from a healthy to dysfunctional state is not known. Current technologies that measure static protein abundance are insufficient to fully understand the time-dimensional features of this process, necessitating new conceptual and technical approaches to promote a more comprehensive understanding and to drive discovery of therapies. In this proposal, we will use alterations in mitochondrial protein turnover to mechanistically define cardiac remodeling, and we will investigate the differential role of intramitochondrial and extramitochondrial protein degradation pathways in transitioning the mitochondrial proteome from a healthy to diseased state. We hypothesize that ROS-induced damage to the mitochondrial proteome triggers alterations in protein half-lives-the functional window-of proteins comprising key functional components of mitochondria (e.g., ETC), which induces mitochondrial malfunction and propels cardiac remodeling. We postulate that protein turnover rates will unveil time-dependent molecular features of pathology that are otherwise masked in steady-state protein measurements, thus providing novel mechanistic insights into cardiac hypertrophy. The rationale for these studies is supported by recent discoveries on the importance of protein quality control and mitochondrial dynamics in cardiac phenotypes, and by our preliminary investigations regarding the effects of ROS on intra-mitochondrial protease activities. The proposed experiments will examine (i): protein turnover during cardiac mitochondrial remodeling, and (ii) the manner in which protein degradation pathways affect cardiac phenotypes. Aim 1 capitalizes on a novel heavy water (D2O) labeling strategy recently developed in our lab, which examines the temporal dynamics of the mitochondrial proteome in three settings: (i) isoproterenol (ISO)-induced remodeling; (ii) injury by elevated ROS; and (iii) cardioprotection by active PKC?. We anticipate that our findings will identify the protein turnover perturbations during remodeling and will elucidate their functional consequences. Aim 2 will determine proteolytic activities of individual protein degradation pathways targeting cardiac mitochondrial proteins. We expect these studies to determine how each proteolytic pathway changes during ISO treatment, ROS elevation, and PKC? cardioprotection to regulate mitochondrial dynamics. Finally, Aim 3 will integrate the knowledge gained from Aim 1 and Aim 2 to create and refine a translational model for measuring protein turnover in clinical studies. We expect these pilot studies to lay the foundation for comprehensive investigations of protein quality control in human heart diseases. Together, we expect these aims to reveal new insights on the nature of mitochondrial dynamics at the individual protein level in the heart.

A critical challenge in Big Data science is the overall lack of data ahalysis platforms available for transforming Big Data into biological knowledge. To address this challenge, we propose a set of interconnected computational tools capable of organizing and analyzing heterogeneous data to support combined inquiries and to de-convolute complex relationships embedded within large-scale data. We demonstrate its utility with a cardiovascular-centric platform that is easily generalizable to similar efforts in other disciplines. Our Center has designed a federated data architecture of existing resources substantiated by a solid and growing user base, and innovations to elevate functionality. Novel crowdsourcing and text-mining methods will extract the wealth of untapped knowledge embedded in biomedical literature, and novel in-depth proteomics analytical tools will unprecedentedly elucidate dynamic protein features. A key strength of our platform will be the rigorous validation using clinical data from Jackson Heart Study and the Healthy Elderly Active Longevity (HEAL; Wellderly) cohorts. Our proposal includes nine scientific aims that address three main focus areas: (i) we will build a new model platform that amalgamates community-supported Big Data resources, enabling data annotations and collaborative analyses; (ii) we will integrate molecular data with drug and disease information, both structured and unstructured, for knowledge aggregation, and (iii) we will create on-the-cloud analytical and modeling tools to power in-depth protein discoveries. Specifically, we will create a novel distributed query system and cloud-based infrastructure that is capable of providing unified access to multi-omics datasets; we will develop computational and crowdsourcing methods to systematically define relationships between genes, proteins, diseases, and drugs from the literature, emphasizing cardiovascular medicine; we will rally community participation and promote awareness of collaborative research through outreach and educational games; we will create a platform to analyze and visualize multi-scale pathway models of genes, proteins, and metabolites; we will develop tools and algorithms to mechanistically model spatiotemporal protein networks in organelles and to. predict higher physiological phenotypes; and we will correlate individual phenotypes, health histories, and multi-scale molecular profiles to examine cardiovascular disease mechanisms. These tools will be implemented, delivered, and executed on the cloud infrastructure to minimize the computational power required of users.

The ultimate goal of the BD2K Initiative and therefore, of each BD2K Center, is to enable the biomedical research community to use the various types of Big Data for research. Inherent in the success of each BD2K Center is not only the engineering of novel tools and platforms for handling and analyzing biomedical Big Data, but also the utilization of the diverse expertise and specialties of each Center, thereby connecting with the scientific community as well as the general public to disseminate and build enthusiasm for Big Data research. This concept underscores the supreme necessity for a BD2K Center Consortium that is united under one mission with global influence. The joint-efforts by all the NIH BD2K Centers as a Consortium will synergistically empower the entire community. We envision that these efforts may be collaboratively organized to gain both a broad spectrum of contemporary data science software tools for addressing targeted challenges in biomedical research, and a collection of training resources for fulfilling the educational needs at multiple levels. Our Center will completely serve and support the NIH BD2K Initiative. Accordingly, we will structure our BD2K Center Consortium Activities to achieve four specific aims: 1) Our Center will fully abide the governance of the BD2K Center Consortium through the leadership of Steering Committee (SC), the NIH BD2K Project Team (BPT), and recommendations from the Independent Experts Committee (lEC). We will actively collaborate, organize and participate in all BD2K Center Consortium meetings, SC meetings, and visiting other Centers as instructed by NIH; 2) Our Center will serve the BD2K Center Consortium by assisting the NIH BPT in establishing policies/guidelines to transform the current research culture, and in encouraging Big Data standardization to facilitate data sharing and interoperability in biomedical research; 3) Our Center will proactively collaborate with other NIH BD2K Centers by synergizing workforces and resources, supporting the development of DSR and Training components in the broad BD2K Consortium; and 4) Our Center will unreservedly commit to foster a continuous public recognition and endowment in data science, ensuring an exuberant vitality of data science in biomedical research, rendering the BD2K Initiative a sustainable life beyond the proposed NIH funding period.

Highly synergistic collaborative efforts among determined, skilled, and professional investigators often produce novel insights greater than those that could have been generated by a single effort. It is in this regard that the Administration Component will work fervently to foster collaboration and promote multi-institutional productivity through providing integral organization, management, and support of the proposed multilayered BD2K Initiative Center. Specifically, the Administration Component serves a fundamental role in the success of the three main Components of the Center, namely. Data Science Research (DSR), Training, and Consortium Activities, as well as in collaboration among all BD2K Centers, the NIH, and the greater biomedical scientific community. The Administration Component aims to support the mission of the NIH BD2K Initiative by providing administrative assistance with the following Specific Aims; In our capacity within our own Center, we plan: 1a. To support the Center and the mission of the NIH BD2K Initiative in providing innovative research and effective technological engineering of software and computational tools, with a central theme for the organization, management, and processing of Big Data for biomedical research. 1b. To coordinate meetings, workshops, and seminars in an effort to promote collaboration among the various components of the Center and to nurture productivity across Components. 1c. To manage the daily activities of the Center including budgeting, travel reservations, website maintenance, and other critical administrative tasks. In our activities beyond our own Center, we aim: 2a. To promote the dissemination of software, tools, and knowledge by generating a more profound interest in Big Data analysis, modeling, and literacy thereby eliciting a substantial impact on the scientific community. 2b. To foster a working relationship between the community and scientific researchers with bioinformatics knowledge and tools through the effective dissemination of data and products generated by the Center, thus propelling innovations and advancement in Big Data research. Under the leadership of N|H, we aim to support the scientific and educational goals of the NIH BD2K Initiative in overcoming challenges and revitalizing the integration of Big Data Science and biomedical research.

Mounting amounts of diverse biomedical data have been generated. Extracting meaningful information from these datasets has relied on the efforts of informaticians, who are extensively trained in the computer science realm, with little to no training in biology. Similarly, biologists in general are not proficient to analyze, annotate, and translate their large datasets into valuable biomedical insights. In addition, there has been an overall lack of public understanding for the importance of Big Data science, hindering the enthusiasm to advance data science in the biomedical field. To bridge the gaps that exist among data generation, interpretation and awareness, our training program will provide critical data science education to current biomedical researchers, expand the data science workforce in the biomedical field, and elicit a broad public recognition of data science. Accordingly, we have engineered an integrated training program with four specific aims: 1) To empower current biomedical researchers with the ability to manage and interpret Big Data by gaining proficiency in utilizing data science software tools; 2) To utilize the training component as an interactive testing field for software packages developed by the Data Science Research (DSR) component. User critiques/feedback will refine and transform software tools to a professional grade, facilitating the community to capture the full value of Big Data; 3) To cultivate a new generation of developers with transdisciplinary expertise in both computational biology and biomedical informatics; and 4) To heighten public awareness of and enthusiasm for the substantial opportunities embedded within computational biology, which has the potential to transform biomedical research and medicine. To achieve these aims, we have constructed three trainee-oriented modules: Biomedical Researcher /User-Oriented Module, Big Data Science Researcher-Oriented Module, and General Public-Oriented Module. A trans-institutional collaboration has been organized (i.e., UCLA, TSRI, UMMC, and EMBL-EBI), and all components have demonstrated outstanding track records in education. This collaboration will ensure successful execution of the training component substantiated by distinguished experts and meritorious educators from a wide breadth of disciplines, spanning -omics, bioinformatics, and computational science.

? DESCRIPTION (provided by applicant): During the cardiac remodeling that precedes heart failure (HF), multiple biomolecules (e.g., nucleic acids and proteins) are simultaneously shifting towards new steady state as the heart undergoes massive and progression changes in cell state. Systems technology now allows the molecular profiles of multiple biomolecules to be simultaneously measured, but results are often difficult to interpret due to the poor correlation between mRNA and protein abundance, in part because the synthesis and degradation rates of protein molecules are unaccounted for. Recent findings from our group suggest that cardiac remodeling is characterized by widespread remodeling in protein turnover dynamics, especially in nuclear proteins. Protein turnover rate correlates well with phenotypic changes whilst being largely orthogonal from protein abundance, highlighting that it is a missing dimension to our understanding of biological regulation of cardiac remodeling. We postulate that a class of heretofore unexplored disease drivers exists in the cardiac nuclei whose decreased turnover due to impaired proteolysis drives the pathogenic process of cardiac remodeling. To test this hypothesis, we designed three specific aims. Aim 1 will utilize a technological platform we recently developed to measure RNA abundance, protein abundance, and protein turnover in combination in vivo. We will search for candidate protein drivers that exhibit decreased/unchanged mRNA expression, decreased/unchanged protein turnover, but increased abundance, suggesting impaired proteolysis. Furthermore, we will (i) utilize a systems genetic model to contrast mouse strains that are susceptible vs. resistant to a well-characterized model of cardiac remodeling (isoproterenol challenge); and (ii) prioritize molecular features that are restored during reverse remodeling following isoproterenol withdrawal. Aim 2 will validate the candidate drivers by examining their mechanism of proteolysis and susceptibility to proteasomal degradation in vitro. Aim 3 will validate the disease proteins using in vitro models and in human NYHA Class IV HF patients and HF patients with LVAD-mediated reverse remodeling to ensure the discovered protein drivers are translationally relevant. We expect the experiments to shed light on the role of proteolysis in cardiac remodeling and disease susceptibility.

PROJECT SUMMARY/ABSTRACT The inception of the R35 mechanism is a testament to the foresight and vision of NHLBI leadership. Clearly, this will provide unparalleled opportunities for driving discovery and enhancing human health. Capitalizing on a 22-year track record of scientific innovation, training and service to the community, as well as unique abilities in leveraging the technical foundation built by the NIH BD2K Center of Excellence at UCLA, this application presents a multi-pronged strategy for identifying molecular signatures that drive cardiac phenotypes. This application addresses two critical biomedical challenges. Firstly, there is a knowledge gap in how we conceptualize proteins, including how they interplay with other omes, and how their dynamics contribute to functional phenotypes. Secondly, there is an inadequacy of computational tools for systematically linking phenotypic and molecular data, and the cardiovascular community lacks a shared informatics management environment where both datasets and resources are accessible and interoperable for integrative analyses. Accordingly, this R35 proposes two areas of focus for breaking new ground. The first focus area will be to unveil how cardiac mitochondrial spatio-temporal proteomes and their interplay with metabolomic and genomic information drive cardiac phenotypes. This involves the advancement of technological platforms to characterize global spatio-temporal dynamics of cardiac proteins, metabolites, and pathways, producing both valuable molecular datasets from model systems and human cohorts and optimized kinetic models for enabling global dynamic analyses. The second focus area will be to build analytical tools for integrating molecular and phenotypic data, and to construct a prototypic cardiovascular data commons for supporting on-demand interactions among data, tools, resources, and users. These efforts will enable the elucidation of interconnected biological networks from proteomic, metabolomic, genetic variation, and clinical data types through a novel mixed model regression algorithm. Moreover, this will result in novel components for supporting a specialized data commons in cardiovascular medicine, including new APIs, graphical user interface, cloud-computing infrastructure, and data management pipeline. In summary, this R35 proposes to build a translational data ecosystem of seamless data acquisition and informatics platforms for enabling a new model of data-driven knowledge production. Discoveries will propel the NHLBI mission forward, including unveiling molecular signatures of disease in model systems and humans, fostering the training of future biomedical professionals, and disseminating advances to the scientific community and public via a specialized cardiovascular commons, all in the realization of precision medicine.

Abstract: This new T32 proposal will support graduate students and postdoctoral fellows pursuing integrated data science training in cardiovascular (CV) medicine at UCLA. Integrated data science training programs are very limited in today?s CV biomedical community. Data science challenges facing CV medicine are in many ways unique and have a long historic track record of information and data. CV diseases are chronic, heterogeneous disorders that exhibit distinct temporal profiles combined with multi-organ alterations, necessitating novel analysis platforms that integrate findings across expanded continuums of diverse information (e.g., text, imaging, omics). The complexity and size of CV datasets have pushed computational approaches to their limits, thus attenuating the rate for adding value in CV medicine. To this end, there is broad consensus that we must merge data science with CV medicine. The creation of a next-generation workforce having more advanced understanding of data science tactics for addressing real-world CV problems will ultimately realize precision CV medicine. Our T32 fills a unique niche in CV data science that is currently missing, both at UCLA and nationally. The UCLA Integrated Data Science Training in CV Medicine (iDISCOVER) Program draws upon faculty from the UCLA Schools of Medicine and Engineering, to establish a program for trainees committed to intensive data science applications in CV medicine. We have a substantiated track record in establishing training programs, as evidenced by our NIH Big Data to Knowledge (BD2K) Initiative, Heart BD2K Center at UCLA. Experience has enabled us to construct a T32 research program targeting the most pressing data science questions in CV medicine. Our two-year program will accept qualified students who have completed 1st year PhD training from Computer Science (CS), Bioinformatics (BI) or Bioengineering (BE); and eligible postdoc fellows from elite CV programs. We will train predoctoral students during their second and third year of PhD training, and postdoctoral fellows during their first and second year of their fellowship. Trainees will engage in advanced coursework within the specific focus areas: (i) omics phenotyping-supported outcome studies; (ii) machine learning-supported approaches in CV medicine; and (iii) information indexing and knowledgebase construction. Trainees will engage in CV clinical rotations to give them exposure to pressing CV data science questions. Trainees will be guided by a co-mentoring arrangement (1 CV mentor and 1 data science mentor). We have an outstanding group of 14 core faculty and 6 clinical supporting faculty members in the UCLA Schools of Medicine, Engineering and Life Sciences. Our faculty have established, vibrant and well-funded research programs with strong histories of guiding students to successful careers. Our program promotes the training of underrepresented minority groups, as demonstrated by training records of all faculty. Our iDISCOVER program has the institutional backing from Schools of Medicine, Engineering, Grad Division, Depts. of CS, BI, and BE, as well as Cardiology and Physiology. These elements ensure solid support for program implementation, advancement and success.

This proposal requests NHLBI?s support for the 38th Annual Conference of the International Society for Heart Research (ISHR)?North American Section (NAS) in 2020. As the President of ISHR NAS, it is my great honor to serve as the PI of this R13 application and I hope to continue the success in ISHR NAS meetings. The Scientific Program will be co-chaired by Dr. Timothy McKinsey and Dr. Kika Sucharov, and supported by Ms. Kelly Brodsky, the chair of the Local Organizing Committee; all three are located at the University of Colorado- Denver. They will serve as co-PIs and collaborator. We have been working together since July 2018 to plan a first-class conference with broad impact in cardiovascular biology and medicine. The meeting organizers have extensive experience in implementing conferences, and their scientific expertise covers a broad range of disciplines, e.g. molecular mechanisms of heart failure, epigenetics, proteomics and big data. We are confident based on the steady growth of ISHR NAS membership over past two years that this 2020 meeting will attract approximately 300-400 attendees. Three Specific Aims of the conference are proposed. Aim 1 is to elevate and enrich the careers of early- and middle-career investigators (ECIs and MCIs); this conference will provide them both with hands-on execution of every phase of conference operation, including reviewing abstracts, selecting presentations, organizing and implementing topic-specific scientific sessions. Importantly, an educational panel discussaion on scientific rigor and integrity has been carefully planned to guide the next generation of investigators in ethical scientific conduct; Aim 2 is to illuminate cardiovascular drug targets and drug development through the marriage of basic science, clinical science and industrial participation through four specific scientific goals; and Aim 3 is to present state-of-the-art cardiovascular science through interactive domains (workshops, ?how-to? sessions, and panel discussions), where candid, direct debates on challenges and important issues are emphasized. Individuals with dichotomous viewpoints will be invited to present in these discussions. This ISHR NAS meeting carries the tradition with a vibrant program featuring special sessions for ECIs and MCIs, 20 scientific symposia, 2 young investigator competitions, 6 workshops, 2 featured lectures, and several community-building networking events. At the time of submission, 92 speakers from academic institutions and industries have been invited (80% confirmed, 40% are female); they are from 43 cities across 29 US states and 3 cities in Canada. Furthermore, 8 travel awards are dedicated to support participation of underrepresented minority groups. Every aspect of the meeting will go to support the NHLBI mission of promoting the prevention and treatment of heart diseases and enhancing the health of individuals, as well as elucidating mechanistic insights on disease causes, translating basic discoveries into clinical practice, fostering training of emerging scientists and physicians, and communicating research advances to the public.