RESUMEN
Hydrogen sulfide, an endogenous signaling molecule, plays an important role in the physiology and pathophysiology of the cardiovascular system. Using a mouse model of myocardial infarction, we investigated the anti-inflammatory and anti-apoptotic effects of the H2S donor sodium hydrosulfide (NaHS). The results demonstrated that the administration of NaHS improved survival, preserved left ventricular function, limited infarct size, and improved H2S levels in cardiac tissue to attenuate the recruitment of CD11b(+)Gr-1(+) myeloid cells and to regulate the Bax/Bcl-2 pathway. Furthermore, the cardioprotective effects of NaHS were enhanced by inhibiting the migration of CD11b(+)Gr-1(+) myeloid cells from the spleen into the blood and by attenuating post-infarction inflammation. These observations suggest that the novel mechanism underlying the cardioprotective function of H2S is secondary to a combination of attenuation the recruitment of CD11b(+)Gr-1(+) myeloid cells and regulation of the Bax/Bcl-2 apoptotic signaling.
Asunto(s)
Sulfuro de Hidrógeno/farmacología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Isquemia Miocárdica/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Antígeno CD11b/metabolismo , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Sulfuro de Hidrógeno/administración & dosificación , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Células Mieloides/patología , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/mortalidad , Infarto del Miocardio/patología , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/mortalidad , Isquemia Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Receptores de Quimiocina/metabolismo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Mitochondria are a common energy source for organs and organisms; their diverse functions are specialized according to the unique phenotypes of their hosting environment. Perturbation of mitochondrial homeostasis accompanies significant pathological phenotypes. However, the connections between mitochondrial proteome properties and function remain to be experimentally established on a systematic level. This uncertainty impedes the contextualization and translation of proteomic data to the molecular derivations of mitochondrial diseases. We present a collection of mitochondrial features and functions from four model systems, including two cardiac mitochondrial proteomes from distinct genomes (human and mouse), two unique organ mitochondrial proteomes from identical genetic codons (mouse heart and mouse liver), as well as a relevant metazoan out-group (drosophila). The data, composed of mitochondrial protein abundance and their biochemical activities, capture the core functionalities of these mitochondria. This investigation allowed us to redefine the core mitochondrial proteome from organs and organisms, as well as the relevant contributions from genetic information and hosting milieu. Our study has identified significant enrichment of disease-associated genes and their products. Furthermore, correlational analyses suggest that mitochondrial proteome design is primarily driven by cellular environment. Taken together, these results connect proteome feature with mitochondrial function, providing a prospective resource for mitochondrial pathophysiology and developing novel therapeutic targets in medicine.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Proteoma , Animales , Cromatografía Liquida , Drosophila melanogaster , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones , Espectrometría de Masas en TándemRESUMEN
Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.
Asunto(s)
Ventrículos Cardíacos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Ácidos Hidroxámicos/farmacología , Daño por Reperfusión Miocárdica/enzimología , Miocardio/enzimología , Ácido Valproico/farmacología , Acetilación/efectos de los fármacos , Animales , Cromatografía Liquida , Regulación de la Expresión Génica , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/patología , Histona Desacetilasas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Ratones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Transducción de Señal , Espectrometría de Masas en Tándem , VorinostatRESUMEN
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.
Asunto(s)
Bases de Datos de Proteínas , Bases del Conocimiento , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Proteómica/métodos , Biología de Sistemas , Integración de Sistemas , Acceso a la Información , Animales , Caenorhabditis elegans , Difusión de Innovaciones , Drosophila , Humanos , Ratones , Diseño de Software , Flujo de TrabajoRESUMEN
The innovations in mass spectrometry-based investigations in proteome biology enable systematic characterization of molecular details in pathophysiological phenotypes. However, the process of delineating large-scale raw proteomic datasets into a biological context requires high-throughput data acquisition and processing. A spectral library search engine makes use of previously annotated experimental spectra as references for subsequent spectral analyses. This workflow delivers many advantages, including elevated analytical efficiency and specificity as well as reduced demands in computational capacity. In this study, we created a spectral matching engine to address challenges commonly associated with a library search workflow. Particularly, an improved sliding dot product algorithm, that is robust to systematic drifts of mass measurement in spectra, is introduced. Furthermore, a noise management protocol distinguishes spectra correlation attributed from noise and peptide fragments. It enables elevated separation between target spectral matches and false matches, thereby suppressing the possibility of propagating inaccurate peptide annotations from library spectra to query spectra. Moreover, preservation of original spectra also accommodates user contributions to further enhance the quality of the library. Collectively, this search engine supports reproducible data analyses using curated references, thereby broadening the accessibility of proteomics resources to biomedical investigators. This article is part of a Special Issue entitled: From protein structures to clinical applications.
Asunto(s)
Algoritmos , Bases de Datos de Proteínas , Espectrometría de Masas/métodos , Biblioteca de Péptidos , Animales , RatonesRESUMEN
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 ((2)H(2)O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with (2)H(2)O 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 (2)H 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.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Proteolisis , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Óxido de Deuterio , Semivida , Marcaje Isotópico , RatonesRESUMEN
Proteasomes are ubiquitously expressed multicatalytic complexes that serve as key regulators of protein homeostasis. There are several lines of evidence indicating that proteasomes exist in heterogeneous subpopulations in cardiac muscle, differentiated, in part, by post-translational modifications (PTMs). PTMs regulate numerous facets of proteasome function, including catalytic activities, complex assembly, interactions with associating partners, subcellular localization, substrate preference, and complex turnover. Classical technologies used to identify PTMs on proteasomes have lacked the ability to determine site specificity, quantify stoichiometry, and perform large-scale, multi-PTM analysis. Recent advancements in proteomic technologies have largely overcome these limitations. We present here a discussion on the importance of PTMs in modulating proteasome function in cardiac physiology and pathophysiology, followed by the presentation of a state-of-the-art proteomic workflow for identifying and quantifying PTMs of cardiac proteasomes.
Asunto(s)
Corazón/fisiología , Miocardio/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Proteómica , Animales , Biología Computacional , Humanos , Datos de Secuencia Molecular , Miocitos Cardíacos/fisiología , Transducción de Señal/fisiología , Fracciones Subcelulares/metabolismoRESUMEN
Aldehydes are common reactive constituents of food, water and air. Several food aldehydes are potentially carcinogenic and toxic; however, the direct effects of dietary aldehydes on cardiac ischemia-reperfusion (IR) injury are unknown. We tested the hypothesis that dietary consumption of aldehydes modulates myocardial IR injury and preconditioning. Mice were gavage-fed the alpha, beta-unsaturated aldehyde acrolein (5mg/kg) or water (vehicle) 24h prior to a 30-min coronary artery occlusion and 24-hour reperfusion. Myocardial infarct size was significantly increased in acrolein-treated mice, demonstrating that acute acrolein exposure worsens cardiac IR injury. Furthermore, late cardioprotection afforded by the nitric oxide (NO) donor diethylenetriamine/NO (DETA/NO; dose: 0.1mg/kg x 4, i.v.) was abrogated by the administration of acrolein 2h prior to DETA/NO treatment, indicating that oral acrolein impairs NO donor-induced late preconditioning. To examine potential intracellular targets of aldehydes, we investigated the impact of acrolein on mitochondrial PKCepsilon signaling in the heart. Acrolein-protein adducts were formed in a dose-dependent manner in isolated cardiac mitochondria in vitro and specific acrolein-PKCepsilon adducts were present in cardiac mitochondrial fractions following acrolein exposure in vivo, demonstrating that mitochondria are major targets of aldehyde toxicity. Furthermore, DETA/NO preconditioning induced both PKCepsilon translocation and increased mitochondrial PKCepsilon localization. Both of these responses were blocked by acrolein pretreatment, providing evidence that aldehydes disrupt cardioprotective signaling events involving PKCepsilon. Consumption of an aldehyde-rich diet could exacerbate cardiac IR injury and block NO donor-induced cardioprotection via mechanisms that disrupt PKCepsilon signaling.
Asunto(s)
Acroleína/toxicidad , Mitocondrias Cardíacas/enzimología , Infarto del Miocardio/enzimología , Daño por Reperfusión Miocárdica/enzimología , Proteína Quinasa C-epsilon/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Cardiotónicos/farmacología , Dieta/efectos adversos , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos ICR , Mitocondrias Cardíacas/patología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Miocardio/enzimología , Miocardio/patología , Transporte de Proteínas/efectos de los fármacos , Triazenos/farmacologíaRESUMEN
Aldehydes are ubiquitous pollutants with well-indicated but ill-defined cardiovascular toxicity. To investigate the direct toxic effects of environmental aldehyde exposure on the myocardium, 8-wk-old male ICR (Institute of Cancer Research) strain mice were gavage fed trans-2-hexenal (0.1, 1, 10, or 50 mg/kg/wk) or corn oil (vehicle) for 4 wk, during which cardiac function, myocardial morphology, cardiomyocyte apoptosis, and the cytochrome cmediated caspase activation apoptotic pathway were determined. Quantification by enzyme-linked immunosorbent assay (ELISA) revealed that aldehyde- protein adducts increase in mouse hearts following hexenal treatment, whereas echocardiographic analysis displayed a significant impairment of basal left-ventricular contractile function. Both histological analysis and TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) staining indicated condensed nuclei and a significant increase in cardiomyocyte apoptosis in these mice, but immunohistochemistry-based confocal microscope revealed no marked myofibril disarray. Release of cytochrome c from mitochondria into the cytosol, concomitant with activation of caspase-3 and -9, was also found in hexenal-treated groups. In addition, isolated cardiac mitochondria formed hexenal-protein adducts when treated with hexenal, providing indirect evidence that the cardiac mitochondrion is one of primary subcellular targets of aldehyde toxins. These findings suggest that trans-2-hexenal exposure results in direct cardiac toxicity through, at least in part, induction of mitochondrial cytochrome c release-mediated apoptosis in cardiomyocytes, indicating that the cardiac mitochondrion is one of principal subcellular targets of aldehyde toxins.