Study on diverse pathological characteristics of heart failure in different stages based on proteomics

Heart failure is a process characterized by significant disturbance of protein turnover. To elucidate the alterations in cardiac protein expression during the various phases of heart failure and to understand the nature of the processes involved, we analysed the proteome in an established heart failure model at different time points to monitor thousands of different proteins simultaneously. Here, heart failure was induced by transverse aortic constriction (TAC) in KM mice. At 2, 4 and 12 weeks after operation, protein expression profiles were determined in sham‐operated (controls) and TAC mice, using label‐free quantitative proteomics, leading to identification and quantification of almost 4000 proteins. The results of the KEGG pathway enrichment analysis and GO function annotation revealed critical pathways associated with the transition from cardiac hypertrophy to heart failure, such as energy pathways and matrix reorganization. Our study suggests that in the pathophysiology of heart failure, alterations of protein groups related to cardiac energy substrate metabolism and cytoskeleton remodelling could play the more dominant roles for the signalling that eventually results in contractile dysfunction and heart failure.     

Region-resolved proteomics profiling of monkey heart

Nonhuman primates (NHPs) play an indispensable role in biomedical research because of their similarities in genetics, physiological, and neurological function to humans. Proteomics profiling of monkey heart could reveal significant cardiac biomarkers and help us to gain a better understanding of the pathogenesis of heart disease. However, the proteomic study of monkey heart is relatively lacking. Here, we performed the proteomics profiling of the normal monkey heart by measuring three major anatomical regions (vessels, valves, and chambers) based on iTRAQ-coupled LC-MS/MS analysis. Over 3,200 proteins were identified and quantified from three heart tissue samples. Furthermore, multiple bioinformatics analyses such as gene ontology analysis, protein–protein interaction analysis, and gene-diseases association were used to investigate biological network of those proteins from each area. More than 60 genes in three heart regions are implicated with heart diseases such as hypertrophic cardiomyopathy, heart failure, and myocardial infarction. These genes associated with heart disease are mainly enriched in citrate cycle, amino acid degradation, and glycolysis pathway. At the anatomical level, the revelation of molecular characteristics of the healthy monkey heart would be an important starting point to investigate heart disease. As a unique resource, this study can serve as a reference map for future in-depth research on cardiac disease-related NHP model and novel biomarkers of cardiac injury.     

Alteration of collagenous protein profile in congestive heart failure secondary to myocardial infarction

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.     

Proteomic analysis of metabolic, cytoskeletal and stress response proteins in human heart failure

Human heart failure is a complex syndrome and a primary cause of morbidity and mortality in the world. However, the molecular pathways involved in the remodelling process are poorly understood. In this study, we performed exhaustive global proteomic surveys of cardiac ventricle isolated from failing and non-failing human hearts, and determined the regulatory pathway to uncover the mechanism underlying heart failure. Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was used to identify differentially expressed proteins in specimens from failing (n = 9) and non-failing (n = 6) human hearts. A total of 25 proteins with at least 1.5-fold change in the failing heart were identified; 15 proteins were up-regulated and 10 proteins were down-regulated. The altered proteins belong to three broad functional categories: (i) metabolic [e.g. NADH dehydrogenase (ubiquinone), dihydrolipoamide dehydrogenase, and the cytochrome c oxidase subunit]; (ii) cytoskeletal (e.g. myosin light chain proteins, troponin I type 3 and transthyretin) and (iii) stress response (e.g. αB-crystallin, HSP27 and HSP20). The marked differences in the expression of selected proteins, including HSP27 and HSP20, were further confirmed by Western blot. Thus, we carried out full-scale screening of the protein changes in human heart failure and profiled proteins that may be critical in cardiac dysfunction for future mapping.     

Tissue-specific expression and post-translational modification of histone H3 variants

Analyses of histone H3 from 10 rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissociation showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addition, a novel rat testes-specific H3 protein was identified with this approach.     

Heterotrimeric G proteins in heart disease

Guanine nucleotide binding proteins (G proteins) are largely grouped into three classes: heterotrimeric G proteins, ras-like or small molecular weight GTP binding proteins, and others like Gh. In the heart G proteins transduce signals from a variety of membrane receptors to generate diverse effects on contractility, heart rate, and myocyte growth. This central position of G proteins forming a switchboard between extracellular signals and intracellular effectors makes them candidates possibly involved in the pathogenesis of cardiac hypertrophy, heart failure, and arrhythmia. This review focuses primarily on discoveries of heterotrimeric G protein alterations in heart diseases that help us to understand the pathogenesis and pathophysiology. We also discuss the underlying molecular mechanisms of heterotrimeric G protein signalling.     

Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

Introduction: Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood.

Areas covered: Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart.

Expert commentary: Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets.     

Regulation of the (pro)renin-renin receptor in cardiac remodelling

The (pro)renin-renin receptor [(P)RR] was discovered as an important novel component of the renin-angiotensin system (RAS). The functional significance of (P)RR is widely studied in renal and vascular pathologies and has sparked interest for a potential role in cardiovascular disease. To investigate the role of (P)RR in cardiac pathophysiology, we aimed to assess (P)RR regulation in adverse cardiac remodelling of the failing heart. In particular, we evaluated the expression of (P)RR in different models of heart failure and across different species. Significantly increased levels of (P)RR mRNA were found in post-myocardial infarcted (MI) hearts of rats (1.6-fold, P < 0.05) and mice (5-fold, P < 0.01), as well as in transgenic rats with overexpression of the mouse renin gene (Ren2) (2.2-fold, P < 0.01). Moreover, we observed a strong increase of (P)RR expression in hearts of dilated cardiomyopathy (DCM) patients (5.3-fold, P < 0.001). Because none of the tested commercially available antibodies appeared to detect endogenous (P)RR, a (P)RR-specific polyclonal antibody was generated to study (P)RR protein levels. (P)RR protein levels were significantly increased in the post-MI rat heart (1.4-fold, P < 0.05) as compared to controls. Most interestingly in DCM patients, a significant 8.7-fold (P < 0.05) increase was observed. Thus, protein expression paralleled gene expression. These results demonstrate that (P)RR expression is strongly up-regulated both in rodent models of heart failure and in the failing human heart, hinting to a potential role for (P)RR in cardiac pathophysiology.     

An overview of human protein databases and their application to functional proteomics in health and disease

Functional proteomics can be defined as a strategy to couple proteomic information with biochemical and physiological analyses with the aim of understanding better the functions of proteins in normal and diseased organs. In recent years, a variety of publicly available bioinformatics databases have been developed to support protein-related information management and biological knowledge discovery. In addition to being used to annotate the proteome, these resources also offer the opportunity to develop global approaches to the study of the functional role of proteins both in health and disease. Here, we present a comprehensive review of the major human protein bioinformatics databases. We conclude this review by discussing a few examples that illustrate the importance of these databases in functional proteomics research.     

心脏疾病中G蛋白的变化

G蛋白是一类重要的信号转导分子,其生理功能是将细胞膜受体所识别的各种细胞外信号同细胞内一系列效应分子偶联起来,引起核基因转录及蛋白质结构和功能的变化。G蛋白在心脏表达的亚型有Gs、Gi/o、Gq／11、G12／13,参与心肌收缩力、心率、心律和心肌细胞生长的调节。本文着重讨论了心脏G蛋白的分类、结构和功能,以及在心肌肥大、心力衰竭、急性心肌缺血和心律失常等心脏疾病中的改变,以加深对这些疾病的发病机制和病理生理过程的认识。     

Cardiac neural crest

Neural crest cells (NCCs) contribute to many organs and tissues during embryonic development. Amongst these, the cardiovascular system represents a fascinating example. In this review, recent advances in our understanding of the developmental biology and molecular genetics regulating cardiac NCC maturation will be summarized. While the existence of a significant neural crest (NC) contribution to the developing heart has been appreciated for more than 20 years, only in the last few years have molecular pathways regulating this process been elucidated and the significant contribution of these mechanisms to the etiology of congenital heart disease in man become apparent. Emerging data suggest that ongoing studies will reveal complex inductive interactions between cardiac NC and a series of other cell types contributing to the developing cardiovascular system.     

Proteomics landscape of radiation-induced cardiovascular disease: somewhere over the paradigm

Introduction: Epidemiological studies clearly show that thoracic or whole body exposure to ionizing radiation increases the risk of cardiac morbidity and mortality. Radiation-induced cardiovascular disease (CVD) has been intensively studied during the last ten years but the underlying molecular mechanisms are still poorly understood.

Areas covered: Heart proteomics is a powerful tool holding promise for the future research. The central focus of this review is to compare proteomics data on radiation-induced CVD with data arising from proteomics of healthy and diseased cardiac tissue in general. In this context we highlight common and unique features of radiation-related and other heart pathologies. Future prospects and challenges of the field are discussed.

Expert commentary: Data from comprehensive cardiac proteomics have deepened the knowledge of molecular mechanisms involved in radiation-induced cardiac dysfunction. State-of-the-art proteomics has the potential to identify novel diagnostic and therapeutic markers of this disease.     

Challenges in mass spectrometry-based proteomics

During the last decade, protein analysis and proteomics have been established as new tools for understanding various biological problems. As the identification of proteins after classical separation techniques, such as two-dimensional gel electrophoresis, have become standard methods, new challenges arise in the field of proteomics. The development of "functional proteomics" combines functional characterization, like regulation, localization and modification, with the identification of proteins for deeper insight into cellular functions. Therefore, different mass spectrometric techniques for the analysis of post-translational modifications, such as phosphorylation and glycosylation, have been established as well as isolation and separation methods for the analysis of highly complex samples, e.g. protein complexes or cell organelles. Furthermore, quantification of protein levels within cells is becoming a focus of interest as mass spectrometric methods for relative or even absolute quantification have currently not been available. Protein or genome databases have been an essential part of protein identification up to now. Thus, de novo sequencing offers new possibilities in protein analytical studies of organisms not yet completely sequenced. The intention of this review is to provide a short overview about the current capabilities of protein analysis when addressing various biological problems.     

Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle

Trichinella spiralis is an intracellular nematode parasite of mammalian skeletal muscle. Infection of the muscle cell leads to the formation of a host-parasite complex that results in profound alterations to the host cell and a re-alignment of muscle-specific gene expression. The role of parasite excretory-secretory (ES) proteins in mediating these effects is currently unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, a global proteomics approach was used to analyse the ES proteins from T. spiralis muscle larvae. Following 2-DE of ES proteins,MALDI-TOF-MS and LC-MS/MS were used to identify the peptide spots. Specific Trichinella EST databases were assembled and used to analyse the data. Despite the current absence of a Trichinella genome-sequencing project, 43 out of 52 protein spots analysed were identified and included the major secreted glycoproteins. Other novel proteins were identified from matches with sequences in the T. spiralis database. Our results demonstrate the value of proteomics as a tool for the identification of Trichinella ES proteins and in the study of the molecular mechanism underpinning the formation of the host-parasite complex during Trichinella infections.     

利用EST序列构建Mascot本地数据库

对蛋白质质谱数据进行数据库比对和鉴定是蛋白质组学研究技术中的一个重要步骤。由于公共数据库蛋白质数据信息不全,有些蛋白质质谱数据无法得到有效的鉴定。而利用相关物种的EST序列构建专门的质谱数据库则可以增加鉴定未知蛋白的几率。本文介绍了利用EST序列构建Mascot本地数据库的具体方法和步骤,扩展了Mascot检索引擎对蛋白质质谱数据的鉴定范围,从数据库层面提高了对未知蛋白的鉴别几率,为蛋白质组学研究提供了一种较为实用的生物信息学分析技术。     

A Draft Map of Rhesus Monkey Tissue Proteome for Biomedical Research

Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research.     

Cardiac amyloidosis: the need for early diagnosis

Amyloidosis is a collection of systemic diseases characterised by misfolding of previously soluble precursor proteins that become infiltrative depositions, thereby disrupting normal organ structure and function. In the heart, accumulating amyloid fibrils lead to progressive ventricular wall thickening and stiffness, resulting in diastolic dysfunction gradually progressing to a restrictive cardiomyopathy. The main types of cardiac amyloidosis are amyloid light chain (AL) amyloidosis caused by an underlying plasma cell dyscrasia, amyloid transthyretin (TTR) amyloidosis of wild-type (normal) TTR at older age (ATTRwt) and hereditary or mutant amyloid TTR (ATTRm) in which a genetic mutation leads to an unstable TTR protein. Overall survival is poor once heart failure develops, underlining the need for early referral and diagnosis. Treatment for AL amyloidosis has improved markedly over the last decades, and TTR amyloidosis gene silencers and orally available transthyretin stabilisers are ready to enter the clinical arena after recent positive outcome trials. Novel therapies aiming at fibril degradation with monoclonal antibodies are under investigation. In this review, we focus on ‘red flag’ signs and symptoms, diagnosis and management of cardiac amyloidosis which differs considerably from the general management of heart failure. Only by increasing awareness, prognosis for patients with this devastating disease can be improved.

     

A comprehensive dictionary of protein accession codes for complete protein accession identifier alias resolving

In mass spectrometry-based proteomics, protein identification results usually consist of peptide sequences and database-dependent accession identifiers of the matching proteins. Often certain annotations are only available in particular databases that in turn must be queried by a certain identifier. In order to simplify and unify the tracing of identified proteins back to their original annotation information, a system capable of set-oriented mapping the different accession identifiers of proteins derived from multiple sequence database sources has been developed. This allows unification of the access to protein information and tracing to other online resources providing additional information as well as resolving cross-references of protein identifications. The interface of seqDB is available via http://www.protein-ms.de following the link to seqDB.     

Proteomics technologies and challenges

Proteomics is the study of proteins and their interactions in a cell. With the completion of the Human Genome Project, the emphasis is shifting to the protein compliment of the human organism. Because proteome reflects more accurately on the dynamic state of a cell, tissue, or organism, much is expected from proteomics to yield better disease markers for diagnosis and therapy monitoring. The advent of proteomics technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of diseases. High-throughput proteomics technologies combining with advanced bioinformatics are extensively used to identify molecular signatures of diseases based on protein pathways and signaling cascades. Mass spectrometry plays a vital role in proteomics and has become an indispensable tool for molecular and cellular biology. While the potential is great, many challenges and issues remain to be solved, such as mining low abundant proteins and integration of proteomics with genomics and metabolomics data. Nevertheless, proteomics is the foundation for constructing and extracting useful knowledge to biomedical research. In this review, a snapshot of contemporary issues in proteomics technologies is discussed.