A gut microbial metabolite of dietary polyphenols reverses obesity-driven hepatic steatosis.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.

Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes.

Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure, and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study, we used a multiomics approach to vertically integrate unbiased data generated using an assay for transposase-accessible chromatin with high-throughput sequencing, RNA-Seq, and proteomics. We then horizontally integrated these data across different models of hyperammonemia, including myotubes and mouse and human muscle tissues. Changes in chromatin accessibility and/or expression of genes resulted in distinct clusters of temporal molecular changes including transient, persistent, and delayed responses during hyperammonemia in myotubes. Known responses to hyperammonemia, including mitochondrial and oxidative dysfunction, protein homeostasis disruption, and oxidative stress pathway activation, were enriched in our datasets. During hyperammonemia, pathways that impact skeletal muscle structure and function that were consistently enriched were those that contribute to mitochondrial dysfunction, oxidative stress, and senescence. We made several novel observations, including an enrichment in antiapoptotic B-cell leukemia/lymphoma 2 family protein expression, increased calcium flux, and increased protein glycosylation in myotubes and muscle tissue upon hyperammonemia. Critical molecules in these pathways were validated experimentally. Human skeletal muscle from patients with cirrhosis displayed similar responses, establishing translational relevance. These data demonstrate complex molecular interactions during adaptive and maladaptive responses during the cellular stress response to hyperammonemia.

Comparative proteomics reveals elevated CCN2 in NGLY1-deficient cells.

N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-ß signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-ß pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-ß signaling to microscopic liver fibrosis in NGLY1 patients.

Atrial fibrillation rhythm is associated with marked changes in metabolic and myofibrillar protein expression in left atrial appendage.

Atrial fibrillation (AF) is strongly associated with risk of stroke and heart failure. AF promotes atrial remodeling that increases risk of stroke due to left atrial thrombogenesis, and increases energy demand to support high rate electrical activity and muscle contraction. While many transcriptomic studies have assessed AF-related changes in mRNA abundance, fewer studies have assessed proteomic changes. We performed a proteomic analysis on left atrial appendage (LAA) tissues from 12 patients with a history of AF undergoing elective surgery; atrial rhythm was documented at time of surgery. Proteomic analysis was performed using liquid chromatography with mass spectrometry (LC/MS-MS). Data-dependent analysis identified 3090 unique proteins, with 408 differentially expressed between sinus rhythm and AF. Ingenuity Pathway Analysis of differentially expressed proteins identified mitochondrial dysfunction, oxidative phosphorylation, and sirtuin signaling among the most affected pathways. Increased abundance of electron transport chain (ETC) proteins in AF was accompanied by decreased expression of ETC complex assembly factors, tricarboxylic acid cycle proteins, and other key metabolic modulators. Discordant changes were also evident in the contractile unit with both up and downregulation of key components. Similar pathways were affected in a comparison of patients with a history of persistent vs. paroxysmal AF, presenting for surgery in sinus rhythm. Together, these data suggest that while the LAA attempts to meet the energetic demands of AF, an uncoordinated response may reduce ATP availability, contribute to tissue contractile and electrophysiologic heterogeneity, and promote a progression of AF from paroxysmal episodes to development of a substrate amenable to persistent arrhythmia.

A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesis and turnover.

The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary cilium during mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few known interacting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminal Gon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase binding assays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact. ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining after blockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in both molecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networks formed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike WT cells. Targeted LC-MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semitryptic peptide arising from cleavage at Gly2196-Leu2197 We noted that this scissile bond is in the linker between fibronectin modules III17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previously observed in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.

Phosphoproteomics identifies pathways underlying the role of receptor-interaction protein kinase 3 in alcohol-associated liver disease and uncovers apoptosis signal-regulating kinase 1 as a target.

Receptor-interaction protein kinase 3 (RIP3), a critical determinant of the necroptotic pathway of programmed cell death, contributes to injury in murine models of alcohol-associated liver disease (ALD); however, the underlying mechanisms are unknown. We investigated the effect of chronic ethanol feeding on the hepatic phosphoproteome in C57BL/6 and RIP3-deficient (Rip3-/- ) mice, focusing on death receptor (DR) signaling pathways. C57BL/6 and Rip3-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet. A label-free mass spectrometry-based approach identified differentially phosphorylated proteins that were mapped to pathways affected by ethanol and Rip3 genotype. Identified targets were validated in both the murine model of ALD and in liver tissue from patients with alcohol-associated hepatitis (AH) and healthy controls. Chronic ethanol dysregulated hepatic tumor necrosis factor-induced DR signaling pathways. Of particular importance, chronic ethanol feeding to C57BL/6 mice decreased the phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) at serine (S)1036/S1040 (S1029/S1033 human), sites linked with the inhibition of ASK1 death-promoting activity. This decrease in phosphorylation of inhibitory sites was muted in Rip3-/- mice. Decreased phosphorylation at S1033 was also lower in liver of patients with severe AH compared to healthy controls, and phosphorylation at the ASK1 activation site (threonine [Thr]-838) was increased in patients with AH. The net impact of these changes in phosphorylation of ASK1 was associated with increased phosphorylation of p38, a downstream target of ASK1, in patients with AH and C57BL/6 but not Rip3-/- mice. Similarly, chronic ethanol feeding affected the c-Jun N-terminal kinase pathway in C57BL/6 but not Rip3-/- mice. Taken together, our data indicate that changes in inhibitory phosphorylation of ASK1 are an important target in ALD and suggest the involvement of noncanonical functions of Rip3 in ALD.

Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C.

Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262.

Tyrosine 656 in topoisomerase IIß is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and ß are phosphorylated, site-specific phosphorylation of topo IIß is poorly characterized. Using LC-MS/MS analysis of topo IIß, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIß. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIß activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIß, underscore the importance of careful interpretation of modifications having the same nominal mass.

Excessive Inorganic Phosphate Burden Perturbed Intracellular Signaling: Quantitative Proteomics and Phosphoproteomics Analyses.

Inorganic phosphate (Pi) is an essential nutrient for the human body which exerts adverse health effects in excess and deficit. High Pi-mediated cytotoxicity has been shown to induce systemic organ damage, though the underlying molecular mechanisms are poorly understood. In this study, we employed proteomics and phosphoproteomics to analyze Pi-mediated changes in protein abundance and phosphorylation. Bioinformatic analyses and literature review revealed that the altered proteins and phosphorylation were enriched in signaling pathways and diverse biological processes. Western blot analysis confirms the extensive change in protein level and phosphorylation in key effectors that modulate pre-mRNA alternative splicing. Global proteome and phospho-profiling provide a bird-eye view of excessive Pi-rewired cell signaling networks, which deepens our understanding of the molecular mechanisms of phosphate toxicity.

A Single Human-Relevant Fast Food Meal Rapidly Reorganizes Metabolomic and Transcriptomic Signatures in a Gut Microbiota-Dependent Manner.

BACKGROUND: A major contributor to cardiometabolic disease is caloric excess, often a result of consuming low cost, high calorie fast food. Studies have demonstrated the pivotal role of gut microbes contributing to cardiovascular disease in a diet-dependent manner. Given the central contributions of diet and gut microbiota to cardiometabolic disease, we hypothesized that microbial metabolites originating after fast food consumption can elicit acute metabolic responses in the liver. METHODS: We gave conventionally raised mice or mice that had their microbiomes depleted with antibiotics a single oral gavage of a liquified fast food meal or liquified control rodent chow meal. After four hours, mice were sacrificed and we used untargeted metabolomics of portal and peripheral blood, 16S rRNA gene sequencing, targeted liver metabolomics, and host liver RNA sequencing to identify novel fast food-derived microbial metabolites and their acute effects on liver function. RESULTS: Several candidate microbial metabolites were enriched in portal blood upon fast food feeding, and were essentially absent in antibiotic-treated mice. Strikingly, at four hours post-gavage, fast food consumption resulted in rapid reorganization of the gut microbial community and drastically altered hepatic gene expression. Importantly, diet-driven reshaping of the microbiome and liver transcriptome was dependent on an intact microbial community and not observed in antibiotic ablated animals. CONCLUSIONS: Collectively, these data suggest a single fast food meal is sufficient to reshape the gut microbial community in mice, yielding a unique signature of food-derived microbial metabolites. Future studies are in progress to determine the contribution of select metabolites to cardiometabolic disease progression and the translational relevance of these animal studies.

Proteomics identifies a convergent innate response to infective endocarditis and extensive proteolysis in vegetation components.

Infective endocarditis is a life-threatening infection of heart valves and adjacent structures characterized by vegetations on valves and other endocardial surfaces, with tissue destruction and risk of embolization. We used high-resolution mass spectrometry to define the proteome of staphylococcal and non-staphylococcal vegetations and Terminal Amine Isotopic Labeling of Substrates (TAILS) to define their proteolytic landscapes. These approaches identified over 2000 human proteins in staphylococcal and non-staphylococcal vegetations. Individual vegetation proteomes demonstrated comparable profiles of quantitatively major constituents that overlapped with serum, platelet, and neutrophil proteomes. Staphylococcal vegetation proteomes resembled one another more than the proteomes of non-staphylococcal vegetations. TAILS demonstrated extensive proteolysis within vegetations, with numerous previously undescribed cleavages. Several proteases and pathogen-specific proteins, including virulence factors, were identified in most vegetations. Proteolytic peptides in fibronectin and complement C3 were identified as potential infective endocarditis biomarkers. Overlap of staphylococcal and non-staphylococcal vegetation proteomes suggests a convergent thrombotic and immune response to endocardial infection by diverse pathogens. However, the differences between staphylococcal and non-staphylococcal vegetations and internal variance within the non-staphylococcal group indicate that additional pathogen- or patient-specific effects exist. Pervasive proteolysis of vegetation components may arise from vegetation-intrinsic proteases and destabilize vegetations, contributing to embolism.

Epithelial-derived gasdermin D mediates nonlytic IL-1ß release during experimental colitis.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1ß. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1ß-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1ß, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1ß colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1ß, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1ß-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1ß sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

Integrative proteomics and phosphoproteomics in pulmonary arterial hypertension.

Pulmonary arterial endothelial cells (PAEC) are mechanistically linked to origins of pulmonary arterial hypertension (PAH). Here, global proteomics and phosphoproteomics of PAEC from PAH (n = 4) and healthy lungs (n = 5) were performed using LC-MS/MS to confirm known pathways and identify new areas of investigation in PAH. Among PAH and control cells, 170 proteins and 240 phosphopeptides were differentially expressed; of these, 45 proteins and 18 phosphopeptides were located in the mitochondria. Pathologic pathways were identified with integrative bioinformatics and human protein-protein interactome network analyses, then confirmed with targeted proteomics in PAH PAEC and non-targeted metabolomics and targeted high-performance liquid chromatography of metabolites in plasma from PAH patients (n = 30) and healthy controls (n = 12). Dysregulated pathways in PAH include accelerated one carbon metabolism, abnormal tricarboxylic acid (TCA) cycle flux and glutamate metabolism, dysfunctional arginine and nitric oxide pathways, and increased oxidative stress. Functional studies in cells confirmed abnormalities in glucose metabolism, mitochondrial oxygen consumption, and production of reactive oxygen species in PAH. Altogether, the findings indicate that PAH is typified by changes in metabolic pathways that are primarily found in mitochondria.

Massive aggrecan and versican accumulation in thoracic aortic aneurysm and dissection.

Proteoglycan accumulation is a hallmark of medial degeneration in thoracic aortic aneurysm and dissection (TAAD). Here, we defined the aortic proteoglycanome using mass spectrometry, and based on the findings, investigated the large aggregating proteoglycans aggrecan and versican in human ascending TAAD and a mouse model of severe Marfan syndrome. The aortic proteoglycanome comprises 20 proteoglycans including aggrecan and versican. Antibodies against these proteoglycans intensely stained medial degeneration lesions in TAAD, contrasting with modest intralamellar staining in controls. Aggrecan, but not versican, was increased in longitudinal analysis of Fbn1mgR/mgR aortas. TAAD and Fbn1mgR/mgR aortas had increased aggrecan and versican mRNAs, and reduced expression of a key proteoglycanase gene, ADAMTS5, was seen in TAAD. Fbn1mgR/mgR mice with ascending aortic dissection and/or rupture had dramatically increased aggrecan staining compared with mice without these complications. Thus, aggrecan and versican accumulation in ascending TAAD occurs via increased synthesis and/or reduced proteolytic turnover, and correlates with aortic dissection/rupture in Fbn1mgR/mgR mice. Tissue swelling imposed by aggrecan and versican is proposed to be profoundly deleterious to aortic wall mechanics and smooth muscle cell homeostasis, predisposing to type-A dissections. These proteoglycans provide potential biomarkers for refined risk stratification and timing of elective aortic aneurysm repair.

Site-specific quantitation of protein nitration using liquid chromatography/tandem mass spectrometry.

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, Y162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162YnLYEIAR168 and the corresponding decrease in the 162YLYEIAR168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK75, also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.

The 36K protein of zebrafish CNS myelin is a short-chain dehydrogenase.

Previous studies identified homologues to mammalian myelin genes expressed in the teleost central nervous system (CNS), including myelin basic protein (MBP), protein zero (P0), and a member of the proteolipid protein family, DM20. In addition, an uncharacterized 36-kDa (36K) protein is a major component of teleost myelin, but is not a major component of myelin in other species. In the present study, we sought to better understand myelin proteins and myelination in one teleost, zebrafish, by molecular characterization of the zebrafish 36K protein. Purified zebrafish CNS myelin was isolated and the amino acid sequences of peptides present in the 36-kDa band were determined by mass spectrometry. These sequences matched a previously uncharacterized EST in The Institute for Genome Research (TIGR) zebrafish database that is related to the short-chain dehydrogenase/reductase (SDR) protein family. In vitro expression of the zebrafish 36K cDNA in Neuro 2a cells resulted in a protein product that was recognized by a 36K polyclonal antibody. The zebrafish 36K mRNA and protein expression patterns were determined and correlated to other known myelin gene expression profiles. In addition, we determined by in situ hybridization that a human 36K homologue (FLJ13639) is expressed in oligodendrocytes and neurons in the adult human cortex. This study identified a major myelin protein in zebrafish, 36K, as a member of the SDR superfamily; an expression pattern similar to other myelin genes was demonstrated.

Phosphorylation of serine 1106 in the catalytic domain of topoisomerase II alpha regulates enzymatic activity and drug sensitivity.

Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II alpha. This effect can be mimicked in sensitive cells treated with the intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II alpha. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II alpha containing a S1106A substitution is 4-fold less active than wild type topo II alpha in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposide-stabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II alpha protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca(2+)-regulated phosphorylation of Ser-1106 in the catalytic domain of topo II alpha modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.

Increased beta-myosin heavy chain in acute cellular rejection following human heart transplantation.

BACKGROUND: Increased expression of smooth muscle and nonmuscle myosin heavy chains has been previously reported in animal models of cardiac allograft rejection. However, altered expression of beta-myosin heavy chain in human cardiac rejection has not been determined. METHODS: Two-dimensional (2D)-gel electrophoresis of endomyocardial biopsies taken from patients with (Grade 3A, n = 6) and without (Grade 0, n = 6) acute rejection were analyzed. Increased expression of two protein spots (MW approximately 12 kDa) were identified in the presence of acute rejection and were further characterized by mass spectrometry analysis. In patients who had acute rejection, protein expression was subsequently analyzed by immunoblotting on biopsies preceding, during, and following treatment of rejection. RESULTS: Mass spectrometric analysis of the protein spots detected 6 and 22 tryptic peptides, respectively. Protein sequence database search analysis identified the first protein as beta-myosin heavy chain and the second spot consisted of proteins of unidentified nature that may represent novel proteins. Immunoblotting analysis showed 1.4 x fold increase (p < 0.01) of protein expression of beta-myosin heavy chain expression in the presence of acute rejection. CONCLUSIONS: To our knowledge, this is the first 2D-gel study to describe increased expression of beta-myosin heavy chain and other proteins of unidentified nature in association with human cardiac allograft rejection.