Left ventricular hypertrophy induced by abdominal aortic banding and its prevention by angiotensin receptor blocker telmisartan—a proteomic analysis

Cardiac hypertrophy is frequently caused by pressure overload (i.e., high blood pressure or hypertension) and can lead to heart failure. The major objective of the present study was to investigate the proteomic changes in response to the development of left ventricular hypertrophy (LVH) induced by abdominal aortic banding (AB) and its prevention by antihypertensive treatment with angiotensin II receptor blocker (ARB) telmisartan. One week after AB and Sham surgery, rats were assigned into three groups: SHAM–control, aortic banding without treatment (AB–Ctrl) and aortic banding with telmisartan treatment (AB–Telmi; 5mg/kg/day for 8 weeks). Echocardiography, hemodynamics, and pathology were performed to assess LVH. Left ventricular myocardium was sampled. The analysis of proteomic proteins from myocardium was performed by two-dimensional gel electrophoresis and MALDI–TOF–MS. In AB–Ctrl, heart rate, systolic arterial blood pressure, diastolic blood pressure, left ventricular end systolic pressure, interventricular septal thickness at diastole, posterior wall thickness in diastole, heart weight (HW) and HW/body weight (BW) were increased, indicating that both hypertension and LVH developed. Telmisartan prevented hypertension and LVH. Concurrently, among numerous proteins, there were 17 that were differentially expressed among hypertrophic hearts, normal hearts, and the hearts where hypertrophic response was suppressed by ARB treatment. Primarily, proteins involved in cell structure, metabolism, stress and signal transduction exhibited up-regulations in LVH, providing cellular and molecular mechanism for hypertrophic development. These changes were prevented or greatly attenuated by telmisartan regimen. Interestingly, antioxidative-related heat shock protein 2 was detected neither in SHAM–Ctrl nor in AB–Ctrl, but in AB–Telmi. LVH is accompanied by series changes of protein expression. Both LVH and proteomic changes can be prevented by blockade of renin–angiotensin system with telmisartan. These protein alterations may constitute mechanistic pathways leading to hypertrophy development and experimental targets for novel therapeutic strategy.     

Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome

We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.     

γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l ‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.     

Proteomic analysis of the mosquito Aedes aegypti midgut brush border membrane vesicles

We analyzed brush border membrane vesicle proteins from isolated midguts of the mosquito Aedes aegypti, by two proteomic methods: two-dimensional gel electrophoresis (isoelectric focusing and SDS-PAGE) and a shotgun two-dimensional liquid chromatographic (LS/LS) approach based on multidimensional protein identification technology (MudPIT). We were interested in the most abundant proteins of the apical brush border midgut membrane. About 400 spots were detected on 2D gels and 39 spots were cored and identified by mass spectrometry. 86 proteins were identified by MudPIT. Three proteins, arginine kinase, putative allergen and actin are shown to be the most predominant proteins in the sample. The total number of 36 proteins detected by both methods represents the most abundant proteins in the BBMV.     

Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation.     

Multidimensional analysis of the insoluble sub-proteome of Oceanobacillus iheyensis HTE831, an alkaliphilic and halotolerant deep-sea bacterium isolated from the Iheya ridge

We report the first proteomic analysis of the insoluble sub-proteome of the alkaliphilic and halotolerant deep-sea bacterium Oceanobacillus iheyensis HTE831. A multidimensional gel-based and gel-free analysis was utilised and a total of 4352 peptides were initially identified by automated MS/MS identification software. Automated curation of this list using PROVALT reduced our peptide list to 467 uniquely identified peptides that resulted in the positive identification of 153 proteins. These identified proteins were functionally classified and physiochemically characterised. Of 26 proteins identified as hypothetical conserved, we have assigned function to all but four. A total of 41 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify three of the five bacterial classes of signal peptide, namely: (i) twin-arginine translocation; (ii) Sec-type and (iii) lipoprotein transport. Our proteomic strategy has also allowed us to identify, at neutral pH, a number of proteins described previously as belonging to two putative transport systems believed to be of importance in the alkaliphilic adaptation of O. iheyensis HTE831.     

Identification of additional proteins in differential proteomics using protein interaction networks

In this study, we developed a novel computational approach based on protein–protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein–protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest‐ranked proteins, beta‐arrestin 1, and beta‐arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost‐effective means to identify additional proteins that remain elusive for current 2D gel‐based proteomic profiling techniques.     

Pilot study identifying myosin heavy chain 7, desmin,insulin‐like growth factor 7, and annexin A2 as circulating biomarkers of human heart failure

In‐depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac‐specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria, including myosin heavy chain 7, insulin‐like growth factor‐binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all four proteins. We verified antibody cross‐reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from four unaffected and four heart failure patients and demonstrated that all four proteins increased between twofold and 150‐fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human.     

Comparison of the protein profile of established and regressed hypertension-induced left ventricular hypertrophy

Established left ventricular hypertrophy (LVH) showed a significant alteration in the cardiac protein profile compared with normal heart. The main finding of this work was to identify proteins differently expressed in hypertension-induced LVH and the fact that after regression of LVH (histologically determined), the proteome still maintains a number of expressed proteins characteristic of the hypertrophied heart. These unrecovered proteins play an essential role in the energy production pathway, in cellular stress defense and also in hypertrophy regulation.     

Establishment of a 2-D human urinary proteomic map in IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is the most common form of immune complex-mediated glomerulonephritis worldwide. Although chronic renal failure develops in considerable numbers of IgAN patients, the exact etiology has not yet been clearly elucidated. To establish the urinary protein map of IgAN, we performed a urinary proteomic analysis. Thirteen patients with IgAN and 12 normal controls were recruited. Morning midstream spot urine samples were used with Centriprep ultrafiltration for concentration and desalting. 2-DE was performed and compared between IgAN and normal control, and urinary proteins were identified by MALDI-TOF MS. A large number of protein spots were identified in IgAN and normal control samples, with means of 311 spots and 174 spots, respectively. Approximately 216 protein spots were detected as differentially expressed in IgAN. Among these, 82 spots were over-expressed, and 134 spots were under-expressed compared to normal controls. A total of 84 differentially expressed spots, representing 59 different proteins, were finally identified in IgAN. We have established a urinary proteomic map of IgAN and this result helps in the identification. Further study is needed to determine the potential pathogenic role of these proteins.     

C-terminal antibodies (CTAbs): a simple and broadly applicable approach for the rapid generation of protein-specific antibodies with predefined specificity

Recent advances in proteomic techniques have resulted in an ever-increasing need to produce antibodies. Here, to address this problem, a technically simple approach of targeting the extreme C-termini of proteins with antibodies (CTAbs) was investigated in proteins secreted by the human pathogen Streptococcus pyogenes. Target proteins were identified by a conventional proteomic approach and CTAbs produced against synthetic five amino acid peptides representing the C-terminus of each target protein. In every case where protein secretion was demonstrated (n = 20), CTAbs were successfully produced and bound specifically to the target protein (100% success rate). The apparent specificity was consistent with the structural heterogeneity of the C-termini of S. pyogenes proteins. The global specificity of CTAb binding was defined using a combinatorial library of synthetic peptides representing structural variants of the original synthetic immunogen. This is a systematic and comprehensive approach for the development of antibodies with defined specificity that can be used in a range of applications.     

Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT)

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.     

Sequential alterations in Akt, GSK3β, and calcineurin signalling in the mouse left ventricle after thoracic aortic constriction

Left ventricular hypertrophy (LVH) is an adaptive response to chronic biomechanical stress that generally progresses to maladaptive hypertrophy and heart failure (HF). We studied the activation of protein kinase B (Akt/PKB), glycogen synthase kinase 3 beta (GSK3β), and calcineurin (Cn) at 3, 7, 15, 30, and 60 days following transverse aortic constriction (TAC) in 4-week-old mice. Following TAC, GSK3β inactivation at day 3 was associated with Akt activation, whereas at days 15 and 30, it appeared to be controlled by other kinases. Moderate nonsignificant Cn activation occurred at the early stages, and peak activation at day 30, concomitant with GSK3β inactivation and overt LVH and HF. At the latest stage (day 60), despite further progression of LVH and HF, Cn activation appeared attenuated. Early stages of LVH were associated with Ca2+-handling protein upregulation, whereas major Cn activation, associated with GSK3β inactivation, appeared to engage maladaptive hypertrophy and progression to HF associated with Ca2+-handling protein downregulation.     

Dynamic microRNA expression during the transition from right ventricular hypertrophy to failure

MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there are no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC vs. control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: four miRs (34a, 28, 148a, and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, extracellular matrix turnover, and impaired proteosomal function. The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload-stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.     

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.     

Increased expression of cardiotrophin-1 during ventricular remodeling in hypertensive rats

Cardiotrophin-1 (CT-1) stimulates longitudinal myocardial cell hypertrophy. We examined the expression of CT-1, leukemia inhibitory factor (LIF), and gp130 by competitive RT-PCR and Western blotting in Dahl salt-sensitive (DS) rats with a high-salt diet, which showed a distinct transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF). The expression levels of CT-1 mRNA and protein were significantly increased at the CHF stage compared with the LVH stage and age-matched Dahl salt-resistant (DR) rats (n = 6 for each group). mRNA expression of LIF was not changed in the left ventricle at any stage by RT-PCR. gp130 mRNA and protein levels of DS rats at 11 and 17 wk were significantly increased compared with age-matched DR rats. The isolated myocyte length of DS rats at 17 wk was the longest among the four groups of rats. The LV end-diastolic dimension (LVDd) of DS rats, determined by echocardiography, was significantly increased at the CHF stage. There was a significant correlation between the CT-1 protein level and LVDd. CT-1 may play a role in ventricular remodeling during transition from LVH to CHF in the rat hypertensive model.