Martin Cadeiras

Protein kinetic signatures of the remodeling heart following isoproterenol stimulation

Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (2H2O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of β-adrenergic-induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.

Regulation of Acetylation Restores Proteolytic Function of Diseased Myocardium in Mouse and Human

Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

New-onset graft dysfunction after heart transplantation—incidence and mechanism-related outcomes

BACKGROUND Graft dysfunction (GD) after heart transplantation (HTx) is a major cause of morbidity and mortality. The impact of different pathophysiologic mechanisms on outcome is unknown. In this large, single-center study we aimed to assess the incidence of GD and compare the outcomes with different histopathologic mechanisms of rejection. METHODS We analyzed a data set of 1,099 consecutive patients after their HTx at Columbia University Medical Center between January 1994 and March 2008, and identified all patients hospitalized with new-onset GD. Based on the histopathologic data, patients were divided into GD-unexplained (Group-GD-U), GD-antibody-mediated rejection (Group-GD-AMR), GD-cardiac allograft vasculopathy (Group-GD-CAV) and GD-acute cellular rejection (Group-GD-ACR) groups. We compared the in-hospital and 3-, 6- and 12-month mortality across these groups using the chi-square test. We also compared the 3-, 6- and 12-month survival curves across groups using the log-rank test. RESULTS Of 126 patients (12%) identified with GD, complete histology data were available for 100 patients. There were 21, 20, 27 and 32 patients identified in Group-GD-U, Group-GD-AMR, Group-GD-CAV and Group-GD-ACR, respectively. The in-hospital mortality rates were 52%, 20%, 15% and 6%, respectively. The in-hospital mortality rate was significantly higher in Group-GD-U compared with all other groups (p = 0.0006). The 3-, 6- and 12-month survival rate was also significantly lower in Group-GD-U compared with all other groups. CONCLUSION A significant proportion of patients presenting with new-onset GD have unexplained histopathology. Unexplained GD is associated with a significantly higher mortality rate. New diagnostic tools are necessary to better understand and detect/predict this malignant phenotype.

Characterization of human plasma proteome dynamics using deuterium oxide

High‐throughput quantification of human protein turnover via in vivo administration of deuterium oxide (2H2O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of 2H2O to human subjects.

Gene Expression Profiles of Patients With Antibody-Mediated Rejection After Cardiac Transplantation

ntibody-mediated rejection (AMR) is characterized y interstitial edema, prominent endothelial cell damge, occasional inflammatory cells, donor-specific ntibodies and C4d deposition, and may cause acute raft loss after heart transplantation. Unfortuately, there is no non-invasive method to accurately redict or diagnose AMR. Peripheral blood mononuclear cell (PBMC) gene ignatures allow for identification of patients at risk of ejection. We conducted a pilot study to test the ypothesis that patients with AMR show specific PBMC ene expression profiles. We included all patients at our center who were part f the Cardiac Allograft Rejection Gene expression bservational (CARGO) study and evaluated with gene icroarrays. Gene probes with expression values resent in 70% of the samples were filtered retaining ,688 probes of the original 7,370. AMR was defined as ew-onset graft dysfunction in the absence of cellular ejection, with light-microscopic criteria of endothelial welling, requiring specific treatment according to our nstitutional practice. Repeat samples from the same atients were averaged. Candidate genes were identied by Significance Analysis of Microarrays (SAM). unctional analysis was performed with High Throughut GOminer (HTGM) and Gene Set Enrichment Analsis (GSEA). Clinical variables were compared using a -test or chi-square test when appropriate.

Drawing networks of rejection - a systems biological approach to the identification of candidate genes in heart transplantation

Technological development led to an increased interest in systems biological approaches to characterize disease mechanisms and candidate genes relevant to specific diseases. We suggested that the human peripheral blood mononuclear cells (PBMC) network can be delineated by cellular reconstruction to guide identification of candidate genes. Based on 285 microarrays (7370 genes) from 98 heart transplant patients enrolled in the Cardiac Allograft Rejection Gene Expression Observational study, we used an information‐theoretic, reverse‐engineering algorithm called ARACNe (algorithm for the reconstruction of accurate cellular networks) and chromatin immunoprecipitation assay to reconstruct and validate a putative gene PBMC interaction network. We focused our analysis on transcription factor (TF) genes and developed a priority score to incorporate aspects of network dynamics and information from published literature to supervise gene discovery. ARACNe generated a cellular network and predicted interactions for each TF during rejection and quiescence. Genes ranked highest by priority score included those related to apoptosis, humoural and cellular immune response such as GA binding protein transcription factor (GABP), nuclear factor of κ light polypeptide gene enhancer in B‐cells (NFκB), Fas (TNFRSF6)‐associated via death domain (FADD) and c‐AMP response element binding protein. We used the TF CREB to validate our network. ARACNe predicted 29 putative first‐neighbour genes of CREB. Eleven of these (37%) were previously reported. Out of the 18 unknown predicted interactions, 14 primers were identified and 11 could be immunoprecipitated (78.6%). Overall, 75% (n= 22) inferred CREB targets were validated, a significantly higher fraction than randomly expected (P < 0.001, Fisher’s exact test). Our results confirm the accuracy of ARACNe to reconstruct the PBMC transcriptional network and show the utility of systems biological approaches to identify possible molecular targets and biomarkers.

Low Variability of Intraindividual Longitudinal Leukocyte Gene Expression Profiling Cardiac Allograft Rejection Scores

with ET-Kyoto solution before pancreas preservation significantly improved islet yield from both deceased donor and autologous live pancreata (4, 9). Both collagenase infusion and DI require a pancreatic duct, therefore, an opened main pancreatic duct as created by the Puestow procedure is a major problem. To overcome this issue, we reconstructed the pancreatic duct and jejunum conduit using autosuture. This simple procedure made it possible for us to reconstruct a watertight conduit for collagenase infusion and DI. This is the first report to show that the reconstruction of the pancreatic duct resulted in successful islet isolation and, in turn, insulin independence. In addition, duct reconstruction could be applicable to posttraumatic pancreatectomy cases in which a laceration or damage to the duct could be present (10). Bacteria contamination is a concern using this technique. In this case, bacteremia was not observed; however, we recommend careful observation for bacteremia. Even if pancreatitis is in its advanced phase, TP with AIT could effectively reduce abdominal pain and maintain excellent glycemic control as long as patients receive a sufficient quantity of islets (11). This reconstruction method should be useful to promote TP with AIT after Puestow procedure for patients who have advanced pancreatitis.

Reduced HLA Class II antibody response to proteasome inhibition in heart transplantation

Antibody-mediated rejection (AMR) has become an increasingly recognized entity in heart transplantation linked to poor outcomes. Treatment algorithms for AMR vary from institution to institution, and therapeutic options remain limited. Responses to standard therapies, such as plasmapheresis, intravenous immunoglobulin, corticosteroids, and rituximab, are not always apparent. Bortezomib, a first-in-class 26S proteasome inhibitor that was approved by the U.S. Food and Drug Administration for multiple myeloma, is emerging as an alternative therapy option for AMR. Proteasome inhibition results in depletion of plasma cells, a primary source of antibody production. Additionally, a major function of the 26S proteasome is antigen processing for HLA Class I presentation. We postulated that compared with Class II responses, bortezomib would be more effective in preventing de novo Class I antibody production. To date, there have not been published reports of the variable immunologic responses of specific HLA antibody types to bortezomib in heart transplantation. Here we report the first case series, to our knowledge, of the differential effect on donor-specific and third-party antibodies in heart transplant recipients treated with bortezomib. Institutional review board approval was obtained. A retrospective review was conducted of all adult patients receiving bortezomib for biopsy-proven or clinically suspected AMR from August 2010 to January 2012. Diagnosis of AMR consisted of positive endomyocardial biopsy by histology and immunohistochemistry, hemodynamic changes, or significant changes in donor-specific antibody (DSA) mean fluorescence intensity (MFI). An antibody MFI cutoff of Z5,000 was used, and a Z50% change in MFI was considered significant. Treatment consisted of plasmapheresis followed by bortezomib (0.7–1 mg/m intravenous push) on Day 1, 4, 7, and 10 followed by a 3-day course of plasmapheresis beginning 72 hours after the last bortezomib dose. Treatment efficacy was determined by clinical or histologic improvement or a significant change in DSA. Antibody MFI was determined via single-antigen Luminex assay (Luminex (R), One Lambda Inc., Canoga, Park, CA). Antibody samples were obtained on Day 5, 11, 14, and 30 and at each clinical follow-up. To study the antibody response to treatment, we constructed a matrix of data of all tested antibodies and used hierarchical clustering based on the Pearson correlation to generate a clustered heat map. The Pearson correlation is a standardized measure of linear dependency between 2 variables and was used because of its independence of scale, allowing for the identification of variables undergoing similar relative changes without dependence on absolute magnitudes or units. Wilcoxon signed rank test was used to calculate statistical significance of MFI changes. Statistical analysis was done using R (R Foundation for Statistical Computing, Vienna, Austria).

Pre-existing Psychiatric Illness is Associated With Increased Risk of Recurrent Takotsubo Cardiomyopathy

BACKGROUND The increased prevalence of psychiatric illness among patients with takotsubo cardiomyopathy (TC) has been previously described. OBJECTIVES We sought to assess the effect of pre-existing psychiatric illness on clinical outcomes following the diagnosis of TC. METHODS Adults diagnosed with TC at Vanderbilt University Medical Center between 1999 and 2015 were included in the study. Medical records were retrospectively reviewed to identify any pre-existing mood, anxiety, or schizophrenia-spectrum illness before TC presentation. Multivariable logistic regression was used to test for independent association of pre-existing psychiatric illness with 30-day mortality and recurrent TC; Cox proportional hazard analysis was used to evaluate for association with long-term mortality. RESULTS Among 306 patients diagnosed with TC during the study period, 114 (37%) had a pre-existing psychiatric illness. In all, 43 (14%) and 88 (29%) patients died within 30 days of index diagnosis and as of last medical record review, respectively. Of the 269 who survived their index hospitalization, 19 (7%) had a confirmed recurrent episode of TC. In multivariable analyses, pre-existing psychiatric illness was not associated with increased 30-day (P = 0.320) or long-term (P = 0.621) mortality. Pre-existing psychiatric illness was associated with higher risk of recurrent TC (odds ratio = 7.44, 95% CI: 2.30-24.01, P < 0.001). CONCLUSIONS Pre-existing psychiatric illness was associated with an increased risk of recurrent TC. No significant association was noted between pre-existing psychiatric illness and survival.

Molecular- and Organelle-Based Predictive Paradigm Underlying Recovery by Left Ventricular Assist Device Support

Advanced heart failure (HF) is a major cause of morbidity and mortality in the United States, and heart transplantation remains as the gold standard therapy. Because of scarcity of donor organs, the application of mechanical circulatory support devices has become a crucial approach in HF therapy as a bridge to transplantation. Therefore, mechanical circulatory support devices have existed both conceptually and experimentally for >40 years, along which an exponential evolution of mechanical circulatory support device technology has occurred. To mimic human physiology, the first generation of left ventricular assist devices (LVADs) were pulsatile volume displacement pumps (HeartMate XVE LVAS, Thoratec PVAD, and Novacor, etc). Insights demonstrating the inessentiality of the pulsatile nature of LVADs for survival from a physiological standpoint propelled the design of second-generation continuous flow devices (HeartMate II [Thoratec Inc], the Micromed DeBakey VAD, Berlin Heart Incor [Berlin Heart AG], and the Jarvik 2000 [Jarvik Heart Inc]), which emerged with superior safety and durability. Consequently, the HeartMate II was approved for bridge to transplantation and destination therapy in the United States.1 Recent reports2,3 have demonstrated a 2-year survival rate of 87% in destination therapy patients under intense surveillance, comparable with open heart transplantation survival statistics. In parallel, these promising outcomes of LVADs in HF therapy have spawned the translational research field of LV reverse remodeling, which has already shown great promise for elucidating underlying molecular and cellular mechanisms. HF is a highly complex clinical syndrome marked by a multitude of derangements, both in adult and pediatric populations.4 The clinical phenotype of HF begins with an injury or supranormal stressor on the heart and, through prolonged dyshomeostasis, eventuates in cellular and organ failure. The compensatory adaptive mechanisms including neurohormonal activation (eg, the …