David A. Liem

Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS‐based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism, and biogenesis. In addition, there are several other clusters, including proteolysis, protein folding, and reduction/oxidation signaling, which ostensibly represent previously under‐appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue‐ and species‐specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles.

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (2H2O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with 2H2O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level 2H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d−1 and 0.163 d−1, respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (∼60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.

Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.

Differential Regulation of Proteasome Function in Isoproterenol-Induced Cardiac Hypertrophy

Rationale: Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation. Objective: The study aimed at identification of molecular mechanisms changing proteasome function in the hypertrophic heart. Method and Results: Proteasome function, expression, and assembly were analyzed during the development of cardiac hypertrophy induced by &bgr;-adrenergic stimulation. The analysis revealed, for the first time, divergent regulation of proteasome function in cardiac hypertrophy. Proteasome complexes have 3 different proteolytic activities, which are ATP-dependent for 26S complexes (19S assembled with 20S) and ATP-independent for 20S core particles. The 26S activities were enhanced in hypertrophic hearts, partially because of increased expression and assembly of 19S subunits with 20S core complexes. In contrast, caspase- and trypsin-like 20S activities were significantly decreased. Activation of endogenous cAMP-dependent protein kinase (PKA) rescued the depressed 20S functions, supporting the notion that PKA signaling is a positive regulator of protein degradation in the heart. Chymotrypsin-like 20S activity was stably maintained during cardiac remodeling, indicating a switch in proteasome subpopulations, which was supported by altered expression and incorporation of inducible &bgr; subunits. Conclusions: Three novel mechanisms for the regulation of proteasome activities were discovered in the development of cardiac hypertrophy: (1) increased incorporation of inducible subunits in 20S proteasomes; (2) enhanced 20S sensitivity to PKA activation; and (3) increased 26S assembly. PKA modulation of proteasome complexes may provide a novel therapeutic avenue for restoration of cardiac function in the diseased myocardium.

PROTEOMIC AND METABOLOMIC ANALYSIS OF CARDIOPROTECTION: INTERPLAY BETWEEN PROTEIN KINASE C EPSILON AND DELTA IN REGULATING GLUCOSE METABOLISM OF MURINE HEARTS

We applied a combined proteomic and metabolomic approach to obtain novel mechanistic insights in PKCvarepsilon-mediated cardioprotection. Mitochondrial and cytosolic proteins from control and transgenic hearts with constitutively active or dominant negative PKCvarepsilon were analyzed using difference in-gel electrophoresis (DIGE). Among the differentially expressed proteins were creatine kinase, pyruvate kinase, lactate dehydrogenase, and the cytosolic isoforms of aspartate amino transferase and malate dehydrogenase, the two enzymatic components of the malate aspartate shuttle, which are required for the import of reducing equivalents from glycolysis across the inner mitochondrial membrane. These enzymatic changes appeared to be dependent on PKCvarepsilon activity, as they were not observed in mice expressing inactive PKCvarepsilon. High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy confirmed a pronounced effect of PKCvarepsilon activity on cardiac glucose and energy metabolism: normoxic hearts with constitutively active PKCvarepsilon had significantly lower concentrations of glucose, lactate, glutamine and creatine, but higher levels of choline, glutamate and total adenosine nucleotides. Moreover, the depletion of cardiac energy metabolites was slower during ischemia/reperfusion injury and glucose metabolism recovered faster upon reperfusion in transgenic hearts with active PKCvarepsilon. Notably, inhibition of PKCvarepsilon resulted in compensatory phosphorylation and mitochondrial translocation of PKCdelta. Taken together, our findings are the first evidence that PKCvarepsilon activity modulates cardiac glucose metabolism and provide a possible explanation for the synergistic effect of PKCdelta and PKCvarepsilon in cardioprotection.

Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.

Divergent Mitochondrial Biogenesis Responses in Human Cardiomyopathy

Background— Mitochondria are key players in the development and progression of heart failure (HF). Mitochondrial (mt) dysfunction leads to diminished energy production and increased cell death contributing to the progression of left ventricular failure. The fundamental mechanisms that underlie mt dysfunction in HF have not been fully elucidated. Methods and Results— To characterize mt morphology, biogenesis, and genomic integrity in human HF, we investigated left ventricular tissue from nonfailing hearts and end-stage ischemic (ICM) or dilated (DCM) cardiomyopathic hearts. Although mt dysfunction was present in both types of cardiomyopathy, mt were smaller and increased in number in DCM compared with ICM or nonfailing hearts. mt volume density and mtDNA copy number was increased by ≈2-fold (P<0.001) in DCM hearts in comparison with ICM hearts. These changes were accompanied by an increase in the expression of mtDNA-encoded genes in DCM versus no change in ICM. mtDNA repair and antioxidant genes were reduced in failing hearts, suggestive of a defective repair and protection system, which may account for the 4.1-fold increase in mtDNA deletion mutations in DCM (P<0.05 versus nonfailing hearts, P<0.05 versus ICM). Conclusions— In DCM, mt dysfunction is associated with mtDNA damage and deletions, which could be a consequence of mutating stress coupled with a peroxisome proliferator-activated receptor &ggr; coactivator 1&agr;–dependent stimulus for mt biogenesis. However, this maladaptive compensatory response contributes to additional oxidative damage. Thus, our findings support further investigations into novel mechanisms and therapeutic strategies for mt dysfunction in DCM.

Integration of Cardiac Proteome Biology and Medicine by a Specialized Knowledgebase

Rationale: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. Objective: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. Methods and Results: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10 924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. Conclusions: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.

Protein kinetic signatures of the remodeling heart following isoproterenol stimulation

Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (2H2O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of β-adrenergic-induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.

THY-1 Receptor Expression Differentiates Cardiosphere-Derived Cells with Divergent Cardiogenic Differentiation Potential

Summary Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation.