Justyna Fert-Bober - Academia.edu (original) (raw)

Papers by Justyna Fert-Bober

Circulation-cardiovascular Genetics, Feb 1, 2014

V ascular Ehlers-Danlos syndrome (VEDS), formerly known as type IV EDS, leads to reduced life exp... more V ascular Ehlers-Danlos syndrome (VEDS), formerly known as type IV EDS, leads to reduced life expectancy because of arterial dissections and rupture, as well as hollow organ rupture, with life-threatening complications occurring as early as the second decade of life. 1 Despite the identification of the causative gene, COL3A1, >2 decades ago, 2-4 there has been limited progress on understanding the disease mechanism beyond that of connective tissue weakness because of structural defects or reduced amounts of type III procollagen. The only human treatment intervention trial to date was modestly beneficial to arterial complications using celiprolol, a mixed β(1)-adrenoceptor antagonist and β(2)-adrenoceptor agonist that is not available in the United States. 5 Although the drug mechanism remains unclear, a protective role for transforming growth factor-β (TGF-β) has been suggested. 5 In a COL3A1 haploinsufficiency mouse model study, doxycycline, a matrix metalloproteinase inhibitor, reduced arterial lesions. 6 Whether this will be applicable to other mutations that disrupt the collagen triple helix is not known nor have the results been validated in humans. Editorial see p 5 Clinical Perspective on p 88 The COL3A1 gene product, procollagen III, is processed to form the mature collagen III homotrimer. The most common COL3A1 mutations in VEDS are missense resulting in the substitution of other amino acids for glycine located throughout the triple-helical domain, with the next most common being splice site mutations that lead to single exon skipping (ES). 1,7 These mutation classes result in a dilated endoplasmic reticulum in skin fibroblasts, reduced type III procollagen secretion, elastic fiber abnormalities, and alterations in the size and distribution of the major collagen fibrils Background-Vascular Ehlers-Danlos syndrome (VEDS) causes reduced life expectancy because of arterial dissections/ rupture and hollow organ rupture. Although the causative gene, COL3A1, was identified >20 years ago, there has been limited progress in understanding the disease mechanisms or identifying treatments. Methods and Results-We studied inflammatory and transforming growth factor-β (TGF-β) signaling biomarkers in plasma and from dermal fibroblasts from patients with VEDS. Analyses were done in terms of clinical disease severity, genotype-phenotype correlations, and body composition and fat deposition alterations. VEDS subjects had increased circulating TGF-β1, TGF-β2, monocyte chemotactic protein-1, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and leptin and decreased interleukin-8 versus controls. VEDS dermal fibroblasts secreted more TGF-β2, whereas downstream canonical/noncanonical TGF-β signaling was not different. Patients with COL3A1 exon skipping mutations had higher plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and VEDS probands had abnormally high plasma C-reactive protein versus affected patients identified through family members before any disease manifestations. Patients with VEDS had higher mean platelet volumes, suggesting increased platelet turnover because of ongoing vascular damage, as well as increased regional truncal adiposity. Conclusions-These findings suggest that VEDS is a systemic disease with a major inflammatory component. C-reactive protein is linked to disease state and may be a disease activity marker. No changes in downstream TGF-β signaling and increased platelet turnover suggest that chronic vascular damage may partially explain increased plasma TGF-β1. Finally, we found a novel role for dysregulated TGF-β2, as well as adipocyte dysfunction, as demonstrated through reduced interleukin-8 and elevated leptin in VEDS.

Methods in molecular biology, Dec 15, 2021

Citrullination, the Ca2+-driven enzymatic conversion of arginine residues to citrulline, is a pos... more Citrullination, the Ca2+-driven enzymatic conversion of arginine residues to citrulline, is a posttranslational modification, implicated in several physiological and pathological processes. Several methods to detect citrullinated proteins have been developed, including color development reagent, fluorescence, phenylglyoxal, and antibody-based methods. These methods yet suffer from limitations in sensitivity, specificity, or citrullinated site determination. Mass spectrometry (MS)-based proteomic analysis has emerged as a promising method to resolve these problems. However, due to low abundance of citrullinated proteins and similar MS features to deamidation of asparagine and glutamine, confident identification of citrullinated proteome is challenging. Here, we present a systematic approach to identify a compendium of steps to enhance the number of detected citrullinated residue and implement diagnostic MS feature that allow the confidence of MS-based identifications. Our method is based on the concept of generation of hyper-citrullinated library with high-pH reversed-phase peptide fractionation that allows to enrich in low abundance citrullinated peptides and amplify the effect of charge loss upon citrullination. Application of our approach to complex global citrullino-proteome datasets demonstrates the confident assessment of citrullinated peptides, thereby enhancing the size and functional interpretation of citrullinated proteomes.

Expert Review of Proteomics, Dec 2, 2021

ABSTRACT Introduction Arginine deimination (citrullination) is a post-translational modification ... more ABSTRACT Introduction Arginine deimination (citrullination) is a post-translational modification catalyzed by a family of peptidyl arginine deiminase (PAD) enzymes. Cell-based functional studies and animal models have manifested the key role of PADs in various cardiovascular diseases (CVDs). Area Covered This review summarizes the past 10 years of knowledge on the role of PADs in CVD pathogenesis. It focuses on the PAD functions and diverse citrullinated proteins in cardiovascular conditions like deep vein thrombosis, ischemia/reperfusion, and atherosclerosis. Identification of PAD isoforms and citrullinated targets are essential for directing diagnosis and clinical intervention. Finally, anti-citrullinated protein antibodies (ACPAs) are addressed as an independent risk factor for cardiovascular events. Expert Opinion PAD is an unique family of enzymes that permanently converts amino acid arginine to amino acid citrulline in protein . Overexpression or increased activity of PAD has been observed in various CVDs with acute and chronic inflammation as the background. Importantly, far beyond being simply involved in forming neutrophil extracellular traps (NETs), accumulating evidence indicated PAD activation as a trigger for numerous processes, such as transcriptional regulation, endothelial dysfunction, and thrombus formation. In summary, the findings so far have testified the important role of deimination in cardiovascular biology, while more basic and translational studies are essential for further exploration.

Journal of Proteome Research, Jan 25, 2023

medRxiv (Cold Spring Harbor Laboratory), Jul 19, 2021

Background. Amidst the millions of individuals affected directly by the pandemic, pronounced sex ... more Background. Amidst the millions of individuals affected directly by the pandemic, pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating not only the acute response but also recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, we deliberately examined sexspecific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. Methods. We used a bead-based array containing over 90 autoantigens previously linked to a range of classic autoimmune diseases to assess autoantibody (AAB) titers in 177 participants. All participants had confirmed evidence of prior SARS-CoV-2 infection based on presence of positive anti-nucleocapsid IgG serology results (Abbott Diagnostics, Abbott Park, Illinois). We used multivariate analysis to determine whether sex-bias was associated with increased rates of AABs reactivity and symptom burden after SARS-CoV2 infection. Results. 82.4% of AABs reactivity was associated with being male compared to 17.6% with female. We found a diversity of AABs responses that exhibited sex-specific patterns of frequency distribution as well as associations with symptomatology and symptom burden. Conclusion. Our results reveal a remarkable sex-specific prevalence and selectivity of AAB responses to SARS-CoV-2. Further understanding of the nature of triggered and persistent AAB activation among men and women exposed to SARS-CoV-2 will be essential for developing effective interventions against immune-mediated sequelae of COVID-19.

Current Neurology and Neuroscience Reports, Oct 1, 2022

Rheumatology, Dec 19, 2016

Objective. High levels of ACPAs in RA are associated with more severe arthritis and worse prognos... more Objective. High levels of ACPAs in RA are associated with more severe arthritis and worse prognosis. However, the role of ACPAs in mediating the increased risk of heart failure in RA remains undefined. We examined whether specific ACPAs were associated with subclinical left ventricular (LV) phenotypes that presage heart failure. Methods. Sera from RA patients without clinical cardiovascular disease were assayed for specific ACPAs using a custom Bio-Plex bead assay, and their cross-sectional associations with cardiac magnetic resonance-derived LV measures were evaluated. High ACPA level was defined as 5 75th percentile. Findings were assessed in a second independent RA cohort with an expanded panel of ACPAs and LV measures assessed by 3D-echocardiography. Results. In cohort 1 (n = 76), higher levels of anti-citrullinated fibrinogen 4160 and anti-citrullinated vimentin antibodies were associated with a 10 and 6% higher adjusted mean LV mass index (LVMI), respectively, compared with lower antibody levels (P < 0.05). In contrast, higher levels of anticitrullinated biglycan 247266 were associated with a 13% lower adjusted mean LVMI compared with lower levels (P < 0.001). In cohort 2 (n = 74), the association between ACPAs targeting citrullinated fibrinogen and citrullinated vimentin peptides or protein and LVMI was confirmed: higher anticitrullinated fibrinogen 556575 and anti-citrullinated vimentin 5877 antibody levels were associated with a higher adjusted mean LVMI (19 and 15%, respectively; P < 0.05), but no association with biglycan was found. Conclusion. Higher levels of antibodies targeting citrullinated fibrinogen and vimentin peptides or protein were associated with a higher mean LVMI in both RA cohorts, potentially implicating autoimmune targeting of citrullinated proteins in myocardial remodelling in RA.

Journal of Translational Medicine, Dec 1, 2021

Background: Pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection... more Background: Pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating the acute response and recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, the aim of the study was to examine sex-specific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. Methods: In this study, we assessed autoantibody (AAB) reactivity to 91 autoantigens previously linked to a range of classic autoimmune diseases in a cohort of 177 participants (65% women, 35% men, mean age of 35) with confirmed evidence of prior SARS-CoV-2 infection based on presence of antibody to the nucleocapsid protein of SARS-CoV-2. Data were compared to 53 pre-pandemic healthy controls (49% women, 51% men). For each participant, sociodemographic data, serological analyses, SARS-CoV-2 infection status and COVID-19 related symptoms were collected by an electronic survey of questions. The symptoms burden score was constructed based on the total number of reported symptoms (N = 21) experienced within 6 months prior to the blood draw, wherein a greater number of symptoms corresponded to a higher score and assigned as more severe burden. Results: In multivariable analyses, we observed sex-specific patterns of autoreactivity associated with the presence or absence (as well as timing and clustering of symptoms) associated with prior COVID-19 illness. Whereas the overall AAB response was more prominent in women following asymptomatic infection, the breadth and extent of AAB reactivity was more prominent in men following at least mildly symptomatic infection. Notably, the observed reactivity included distinct antigens with molecular homology with SARS-CoV-2. Conclusion: Our results reveal that prior SARS-CoV-2 infection, even in the absence of severe clinical disease, can lead to a broad AAB response that exhibits sex-specific patterns of prevalence and antigen selectivity. Further

Immunological Reviews, Dec 25, 2019

The presence of autoantibodies and autoreactive T cells to citrullinated proteins and citrullinat... more The presence of autoantibodies and autoreactive T cells to citrullinated proteins and citrullinating enzymes in patients with rheumatoid arthritis (RA), together with the accumulation of citrullinated proteins in rheumatoid joints, provides substantial evidence that dysregulated citrullination is a hallmark feature of RA. However, understanding mechanisms that dysregulate citrullination in RA has important challenges. Citrullination is a normal process in immune and non-immune cells which is likely activated by different conditions (e.g. inflammation) with no pathogenic consequences. In a complex inflammatory environment such as the RA joint, unique strategies are therefore required to dissect specific mechanisms involved in the abnormal production of citrullinated proteins. Here, we will review current models of citrullination in RA and discuss critical components that, in our view, are relevant to understanding the accumulation of citrullinated proteins in the RA joint, collectively referred to as the RA citrullinome. In particular, we will focus on potential caveats in the study of citrullination in RA, and will highlight methods to precisely detect citrullinated proteins in complex biological samples, which is a confirmatory approach to mechanistically link the RA citrullinome with unique pathogenic pathways in RA.

Circulation Research, Apr 27, 2018

There is an exponential increase in biological complexity as initial gene transcripts are spliced... more There is an exponential increase in biological complexity as initial gene transcripts are spliced, translated into amino acid sequence, and post-translationally modified. Each protein can exist as multiple chemical or sequencespecific proteoforms, and each has the potential to be a critical mediator of a physiological or pathophysiological signaling cascade. Here, we provide an overview of how different proteoforms come about in biological systems and how they are most commonly measured using mass spectrometry-based proteomics and bioinformatics. Our goal is to present this information at a level accessible to every scientist interested in mass spectrometry and its application to proteome profiling. We will specifically discuss recent data linking various protein post-translational modifications to cardiovascular disease and conclude with a discussion for enablement and democratization of proteomics across the cardiovascular and scientific community. The aim is to inform and inspire the readership to explore a larger breadth of proteoform, particularity post-translational modifications, related to their particular areas of expertise in cardiovascular physiology.

Circulation, Nov 16, 2021

Introduction: Recently, an irreversible enzymatic post-translational modification, citrullination... more Introduction: Recently, an irreversible enzymatic post-translational modification, citrullination, has been shown to be present in the cardiovascular system (e.g. aorta). Citrullination, in which arginine is converted to citrulline, can alter protein structure and function, and produce autoantigens Hypothesis: Emerging evidence suggest a potential role for autoimmunity in the pathogenesis of atherosclerosis. However, it remains unknown whether of post-translational citrullination of arterial proteins contributes to local inflammatory processes that initiate or accelerate the atherosclerotic process. The aim of this study was to investigate changes in protein citrullination in human left anterior descending (LAD) coronary artery specimens with and without early atherosclerosis obtained from autopsied young adults with no clinical manifestations of coronary disease. Methods: Segments of the mid-LAD from 74 autopsied individuals (age range:15-55 yrs., 75% male, 67% White) were graded by a pathologist for percent of surface area involved with fatty streaks (FS) or fibrous plaque (FP). The frozen samples were homogenized and digested with Lys-C for subsequent proteomic mass-spectrometry using a data-independent acquisition (DIA) proteomic strategy with a hyper-citrullinated spectral library approach and our published CitFinder software. Results: We found 133 differentially expressed proteins (FDR&amp;lt;0.05) between control, FS and FP groups. Among them, 19 citrullinated proteins with 26 modified amino acid residues were identified. Citrullinated peptides from fibrinogen alpha chain (R84) and transgelin (R146) were upregulated in FP samples in comparison to FS and normal samples. Similarly, the citrullination site (R269) on alpha-enolase was significantly increased in the FP samples compared to normal. Interestingly, citrullination (R110) on myosin light polypeptide 6 was downregulated in FP samples. Conclusions: These data represent the most comprehensive interrogation of citrullinated proteins in human arterial samples to-date and suggest a possible association between this modification of several key proteins involved in tight junction, vascular smooth muscle contraction and inflammation and early atherosclerosis.

Journal of Inflammation, Nov 18, 2022

Background: Macrophages are effector cells of the innate immune system that undergo phenotypical ... more Background: Macrophages are effector cells of the innate immune system that undergo phenotypical changes in response to organ injury and repair. These cells are most often classified as proinflammatory M1 and anti-inflammatory M2 macrophages. Protein arginine deiminase (PAD), which catalyses the irreversible conversion of protein-bound arginine into citrulline, is expressed in macrophages. However, the substrates of PAD and its role in immune cells remain unclear. This study aimed to investigate the role of PAD in THP-1 macrophage polarization to the M1 and M2 phenotypes and identify the citrullinated proteins and modified arginines that are associated with this biological switch using mass spectrometry. Results: Our study showed that PAD2 and, to a lesser extent, PAD1 and PAD4 were predominantly expressed in M1 macrophages. We showed that inhibiting PAD expression with BB-Cl-amidine decreased macrophage polarization to the M1 phenotype (TNF-α, IL-6) and increased macrophage polarization to the M2 phenotype (MRC1, ALOX15). This process was mediated by the downregulation of proteins involved in the NF-κβ pathway. Silencing PAD2 confirmed the activation of M2 macrophages by increasing the antiviral innate immune response and interferon signalling. A total of 192 novel citrullination sites associated with inflammation, cell death and DNA/RNA processing pathways were identified in M1 and M2 macrophages. Conclusions: We showed that inhibiting PAD activity using a pharmacological inhibitor or silencing PAD2 with PAD2 siRNA shifted the activation of macrophages towards the M2 phenotype, which can be crucial for designing novel macrophage-mediated therapeutic strategies. We revealed a major citrullinated proteome and its rearrangement following macrophage polarization, which after further validation could lead to significant clinical benefits for the treatment of inflammation and autoimmune diseases.

Journal of Proteome Research, Apr 16, 2019

Proteome Science, Jan 29, 2010

Background: Although mechanical ventilation (MV) is a major supportive therapy for patients with ... more Background: Although mechanical ventilation (MV) is a major supportive therapy for patients with acute respiratory distress syndrome, it may result in side effects including lung injury. In this study we hypothesize that MMP-9 inhibition by doxycycline might reduce MV-related lung damage. Using a proteomic approach we identified the pulmonary proteins altered in high volume ventilation-induced lung injury (VILI). Forty Wistar rats were randomized to an orally pretreated with doxycycline group (n = 20) or to a placebo group (n = 20) each of which was followed by instrumentation prior to either low or high tidal volume mechanical ventilation. Afterwards, animals were euthanized and lungs were harvested for subsequent analyses. Results: Mechanical function and gas exchange parameters improved following treatment with doxycycline in the high volume ventilated group as compared to the placebo group. Nine pulmonary proteins have shown significant changes between the two biochemically analysed (high volume ventilated) groups. Treatment with doxycycline resulted in a decrease of pulmonary MMP-9 activity as well as in an increase in the levels of soluble receptor for advanced glycation endproduct, apoliporotein A-I, peroxiredoxin II, four molecular forms of albumin and two unnamed proteins. Using the pharmacoproteomic approach we have shown that treatment with doxycycline leads to an increase in levels of several proteins, which could potentially be part of a defense mechanism. Conclusion: Administration of doxycycline might be a significant supportive therapeutic strategy in prevention of VILI.

Arthritis & rheumatology, Oct 28, 2015

Objective. Anti-citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthrit... more Objective. Anti-citrullinated protein antibodies (ACPAs) are characteristic of rheumatoid arthritis (RA). However, their presence years before the onset of clinical RA is perplexing. Although multiple putative citrullinated antigens have been identified, no studies have demonstrated the specific capacity of these antigens to initiate inflammatory arthritis. This study was undertaken to recapitulate the transition from preclinical to clinical RA and to demonstrate the capacity of local citrullination to facilitate this transition. Methods. We performed proteomic analysis of activated human neutrophils to identify citrullinated proteins, including those targeted as part of the RA immune response. Using enzyme-linked immunosorbent assay, we compared RA and osteoarthritis synovial fluid for levels of citrullinated histone H2B and its immune complex. Using macrophage activation assays, we assessed the effect of histone citrullination on immunostimulatory capacity and evaluated the stimulatory capacity of native and citrullin-ated H2B immune complexes. Finally, we assessed the potential for anti-citrullinated H2B antibodies to mediate arthritis in vivo. Results. We identified robust targeting of neutrophilderived citrullinated histones by the ACPA immune response. More than 90% of the RA patients had anticitrullinated H2B antibodies. Histone citrullination increased innate immunostimulatory capacity, and immune complexes containing citrullinated histones activated macrophage cytokine production and propagated neutrophil activation. Finally, we demonstrated that immunization with H2B was arthritogenic, but only in the setting of underlying articular inflammation. Conclusion. Our findings indicate that citrullinated histones, specifically citrullinated H2B, are an antigenic target of the ACPA immune response. Furthermore, local generation of citrullinated antigen during low-grade articular inflammation provides a mechanistic model for the conversion from preclinical autoimmunity to inflammatory arthritis. Rheumatoid arthritis (RA) is associated with antibodies targeting proteins which have undergone posttranslational modification of arginine to citrulline by a family of enzymes known as peptidylarginine deiminase (PAD) (1-3). Although several lines of evidence implicate these antibodies in RA pathogenesis, the presence of these anti-citrullinated protein antibodies (ACPAs) up to several years before the onset of disease development (4-6) has called into question their direct role in the mediation of synovial inflammation. Similarly, there are multiple citrullinated protein targets of the ACPA immune response with no dominant immunopathogenic antigen identified. Recent studies suggest that proteins generated in multiple pathways of neutrophil activation may serve as antigenic

Methods in molecular biology, 2017

Data independent acquisition mass spectrometry (DIA-MS) strategies and applications provides uniq... more Data independent acquisition mass spectrometry (DIA-MS) strategies and applications provides unique advantages for qualitative and quantitative proteome probing of a biological sample allowing constant sensitivity and reproducibility across large sample sets. These advantages in LC-MS/MS are being realized in fundamental research laboratories and are quickly transitioning into clinical research applications. However, the ability to translate raw LC-MS/MS proteomics data (high-throughput) into biological knowledge is a complex and difficult task requiring the use of many algorithms and tools for which there is no widely accepted standard or recognized best practice available. In fact many of the tools used for the biological interpretations of proteomic data were developed for use with RNA and genomic analyzes. Use of these tools inherently fail to capture the full interpretation that proteomics uniquely supplies including the dynamics of quickly reversible chemically modified states of proteins, irreversible amino acid modifications, signaling truncation events and finally, the determining the presence of protein from allele specific transcripts. This Chapter highlights key steps and publicly available algorithms required to translate DIA-MS data into knowledge.

The Journal of Allergy and Clinical Immunology, Feb 1, 2014

RATIONALE: ST2 (IL1RL1), in its soluble form (sST2) neutralizes its ligand, IL-33, by acting as a... more RATIONALE: ST2 (IL1RL1), in its soluble form (sST2) neutralizes its ligand, IL-33, by acting as a decoy receptor. Serum sST2 has been used as a biomarker for disease severity and outcome for multiple inflammatory and lung diseases, including atopic asthma. We previously showed a 10-SNP haplotype as a key determinant of serum sST2 levels in targeted deep resequencing studies of ST2 in 241 samples of African ancestry (p50.0002). METHODS: Serum sST2 was measured by ELISA on an additional 440 African American and 196 European American samples. Four SNPs selected from the 10-SNP haplotype were genotyped using the ABI Taqman assay. Single-variant tests for the common variants were performed separately by race using the linear regression models assuming additive effects of alleles on log serum total ST2 adjusted for age, gender, asthma, batch, study and principal components to adjust for admixture. RESULTS: All four SNPs were significantly replicated (p<0.05) in the independent African American sample. Three of the four SNPs were strongly associated with sST2 levels in the European American samples (p<10-8). Importantly, predicted allelic effects were almost identical between the racial groups. CONCLUSIONS: We confirmed association between sST2 levels and SNPs in a previously identified ST2 haplotype in an independent sample of 440 African Americans, and replicated this association in a European American population with identical allelic effects. Two SNPs were predicted to be in a region with minimal binding evidence for Polymerase (RNA) II (DNA Directed) Polypeptide A (POLR2A) which is a DNAdependent RNA polymerase catalyzing transcription of DNA into RNA.

Proteomics, Jun 1, 2008

Ischemia/reperfusion (I/R) injury is a serious problem resulting from clinical setting of coronar... more Ischemia/reperfusion (I/R) injury is a serious problem resulting from clinical setting of coronary revascularization. Despite extensive studies on I/R injury, the molecular bases of cardiac dysfunction caused by I/R are still unknown, but are likely to result from alterations in protein expression. Isolated rat hearts were subjected to 15-30 min of no-flow ischemia without (Ischemia protocol) or with 30 min of reperfusion (I/R protocol). 2-DE analysis of heart proteins from both experimental protocols showed wide-ranging changes in protein levels. In the Ischemia protocol, 39 protein spots were changed in ischemic groups and those changes correlated with duration of ischemia. Ninety percent of the affected proteins were increased. In contrast to increased protein levels, the total messenger RNA (mRNA) level decreased approximately two fold. Compared to the Ischemia protocol, changes in protein levels in the I/R protocol did not correlate with the duration of ischemia and the degree of recovery of mechanical function. The decrease of affected protein from I/R protocol was associated with the increase in total protein level in reperfusate. Our studies show that the protein increase is correlated with the mechanical function of the I/R hearts and the increase is not likely associated with an increase in protein synthesis.