Sujith Dassanayaka

O-GlcNAc and the cardiovascular system

The cardiovascular system is capable of robust changes in response to physiologic and pathologic stimuli through intricate signaling mechanisms. The area of metabolism has witnessed a veritable renaissance in the cardiovascular system. In particular, the post-translational β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents one such signaling pathway that has been implicated in the pathophysiology of cardiovascular disease. This highly dynamic protein modification may induce functional changes in proteins and regulate key cellular processes including translation, transcription, and cell death. In addition, its potential interplay with phosphorylation provides an additional layer of complexity to post-translational regulation. The hexosamine biosynthetic pathway generally requires glucose to form the nucleotide sugar, UDP-GlcNAc. Accordingly, O-GlcNAcylation may be altered in response to nutrient availability and cellular stress. Recent literature supports O-GlcNAcylation as an autoprotective response in models of acute stress (hypoxia, ischemia, oxidative stress). Models of sustained stress, such as pressure overload hypertrophy, and infarct-induced heart failure, may also require protein O-GlcNAcylation as a partial compensatory mechanism. Yet, in models of Type II diabetes, O-GlcNAcylation has been implicated in the subsequent development of vascular, and even cardiac, dysfunction. This review will address this apparent paradox and discuss the potential mechanisms of O-GlcNAc-mediated cardioprotection and cardiovascular dysfunction. This discussion will also address potential targets for pharmacologic interventions and the unique considerations related to such targets.

Mechanistic pathway(s) of acquired von willebrand syndrome with a continuous-flow ventricular assist device: in vitro findings.

In patients with a ventricular assist device (VAD), diminished high-molecular-weight von Willebrand factor (vWF) multimers may contribute to a bleeding diathesis. The mechanistic pathway(s) of vWF degradation and the role of ADAMTS-13, the vWF-cleaving metalloproteinase, are unknown. The objective of this study was to investigate the molecular mechanisms of VAD-induced vWF impairment in an in vitro system.Simple, mock circulatory loops (n = 4) were developed with a clinically approved, paracorporeal continuous-flow VAD. The loops were primed with anticoagulated, whole bovine blood (750 ml). The VAD was operated at constant blood flow and pressure. Blood samples were drawn at baseline and hourly for 6 hours. vWF multimers and ADAMTS-13 protein were quantified by agarose and polyacrylamide gel electrophoresis with immunoblotting. Plasma platelet factor 4 (PF4), a marker of platelet activation, was quantified via ELISA.Within 120 minutes, high-molecular-weight vWF multimers decreased, and low-molecular-weight multimers increased. Multiple low-molecular-weight vWF fragments emerged (~140, 176, 225, and 310 kDa). Total plasma ADAMTS-13 increased by 13 ± 3% (p < 0.05). Plasma PF4 increased by 21 ± 7% (p = 0.05).During VAD support, vWF degradation occurred quickly. Multiple mechanisms were responsible and included vWF cleavage by ADAMTS-13 (140 and 176 kDa fragments), and what may have been mechanical demolition of endogenous plasma vWF (225 kDa fragments) and nascent vWF (225 and 310 kDa fragments) from platelets. A modest increase in plasma ADAMTS-13 from activated platelets may have contributed to this process but was not the major mechanism. Mechanical demolition was likely the dominant process and warrants further evaluation.

MicroRNA-539 is up-regulated in failing heart, and suppresses O-GlcNAcase expression.

Background: Protein O-GlcNAcylation is nearly ubiquitous; however, regulation of the expression of key enzymes remains unknown. Results: miR-539 is up-regulated in the failing heart, binds to the 3′UTR, and negatively regulates O-GlcNAcase expression. Conclusion: Protein O-GlcNAcylation can be regulated by post-transcriptional mechanisms. Significance: miR-539 regulates one of the two enzymes responsible for O-GlcNAcylation in multicellular eukaryotes. Derangements in metabolism and related signaling pathways characterize the failing heart. One such signal, O-linked β-N-acetylglucosamine (O-GlcNAc), is an essential post-translational modification regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase (OGA), which modulate the function of many nuclear and cytoplasmic proteins. We recently reported reduced OGA expression in the failing heart, which is consistent with the pro-adaptive role of increased O-GlcNAcylation during heart failure; however, molecular mechanisms regulating these enzymes during heart failure remain unknown. Using miRNA microarray analysis, we observed acute and chronic changes in expression of several miRNAs. Here, we focused on miR-539 because it was predicted to target OGA mRNA. Indeed, co-transfection of the OGA-3′UTR containing reporter plasmid and miR-539 overexpression plasmid significantly reduced reporter activity. Overexpression of miR-539 in neonatal rat cardiomyocytes significantly suppressed OGA expression and consequently increased O-GlcNAcylation; conversely, the miR-539 inhibitor rescued OGA protein expression and restored O-GlcNAcylation. In conclusion, this work identifies the first target of miR-539 in the heart and the first miRNA that regulates OGA. Manipulation of miR-539 may represent a novel therapeutic target in the treatment of heart failure and other metabolic diseases.

Hemodynamic Support With a Microaxial Percutaneous Left Ventricular Assist Device (Impella) Protects Against Acute Kidney Injury in Patients Undergoing High-Risk Percutaneous Coronary InterventionNovelty and Significance

Rationale: Acute kidney injury (AKI) is common during high-risk percutaneous coronary intervention (PCI), particularly in those with severely reduced left ventricular ejection fraction. The impact of partial hemodynamic support with a microaxial percutaneous left ventricular assist device (pLVAD) on renal function after high-risk PCI remains unknown. Objective: We tested the hypothesis that partial hemodynamic support with the Impella 2.5 microaxial pLVAD during high-risk PCI protected against AKI. Methods and Results: In this retrospective, single-center study, we analyzed data from 230 patients (115 consecutive pLVAD-supported and 115 unsupported matched-controls) undergoing high-risk PCI with ejection fraction ⩽35%. The primary outcome was incidence of in-hospital AKI according to AKI network criteria. Logistic regression analysis determined the predictors of AKI. Overall, 5.2% (6) of pLVAD-supported patients versus 27.8% (32) of unsupported control patients developed AKI (P<0.001). Similarly, 0.9% (1) versus 6.1% (7) required postprocedural hemodialysis (P<0.05). Microaxial pLVAD support during high-risk PCI was independently associated with a significant reduction in AKI (adjusted odds ratio, 0.13; 95% confidence intervals, 0.09–0.31; P<0.001). Despite preexisting CKD or a lower ejection fraction, pLVAD support protection against AKI persisted (adjusted odds ratio, 0.63; 95% confidence intervals, 0.25–0.83; P=0.04 and adjusted odds ratio, 0.16; 95% confidence intervals, 0.12–0.28; P<0.001, respectively). Conclusions: Impella 2.5 (pLVAD) support protected against AKI during high-risk PCI. This renal protective effect persisted despite the presence of underlying CKD and decreasing ejection fraction.

A New Method to Stabilize C-Kit Expression in Reparative Cardiac Mesenchymal Cells

Cell therapy improves cardiac function. Few cells have been investigated more extensively or consistently shown to be more effective than c-kit sorted cells; however, c-kit expression is easily lost during passage. Here, our primary goal was to develop an improved method to isolate c-kitpos cells and maintain c-kit expression after passaging. Cardiac mesenchymal cells (CMCs) from wild-type mice were selected by polystyrene adherence properties. CMCs adhering within the first hours are referred to as rapidly adherent (RA); CMCs adhering subsequently are dubbed slowly adherent (SA). Both RA and SA CMCs were c-kit sorted. SA CMCs maintained significantly higher c-kit expression than RA cells; SA CMCs also had higher expression endothelial markers. We subsequently tested the relative efficacy of SA vs. RA CMCs in the setting of post-infarct adoptive transfer. Two days after coronary occlusion, vehicle, RA CMCs, or SA CMCs were delivered percutaneously with echocardiographic guidance. SA CMCs, but not RA CMCs, significantly improved cardiac function compared to vehicle treatment. Although the mechanism remains to be elucidated, the more pronounced endothelial phenotype of the SA CMCs coupled with the finding of increased vascular density suggest a potential pro-vasculogenic action. This new method of isolating CMCs better preserves c-kit expression during passage. SA CMCs, but not RA CMCs, were effective in reducing cardiac dysfunction. Although c-kit expression was maintained, it is unclear whether maintenance of c-kit expression per se was responsible for improved function, or whether the differential adherence property itself confers a reparative phenotype independently of c-kit.

E2F1 Transcription Factor Regulates O-linked N-acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Expression.

Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGA protein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts, yet the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored. Here, we used a reporter-based assay to analyze minimal promoters and leveraged in silico modeling to nominate several candidate transcription factor binding sites in both Ogt (i.e. the gene for OGT protein) and Mgea5 (i.e. the gene for OGA protein). We noted multiple E2F binding site consensus sequences in both promoters. We performed chromatin immunoprecipitation in both human and mouse cells and found that E2F1 bound to candidate E2F binding sites in both promoters. In HEK293 cells, we overexpressed E2F1, which significantly reduced OGT and MGEA5 expression. Conversely, E2F1-deficient mouse fibroblasts had increased Ogt and Mgea5 expression. Of the known binding partners for E2F1, we queried whether retinoblastoma 1 (Rb1) might be involved. Rb1-deficient mouse embryonic fibroblasts showed increased levels of Ogt and Mgea5 expression, yet overexpression of E2F1 in the Rb1-deficient cells did not alter Ogt and Mgea5 expression, suggesting that Rb1 is required for E2F1-mediated suppression. In conclusion, this work identifies and validates some of the promoter elements for mouse Ogt and Mgea5 genes. Specifically, E2F1 negatively regulates both Ogt and Mgea5 expression in an Rb1 protein-dependent manner.

Recent Developments in Heart Failure

Recent Developments in Cardiovascular Research: The goal of “Recent Developments” is to provide a concise but comprehensive overview of new advances in cardiovascular research, which we hope will keep our readers abreast of recent scientific discoveries and facilitate discussion, interpretation, and integration of the findings. This will enable readers who are not experts in a particular field to grasp the significance and effect of work performed in other fields. It is our hope and expectation that these “Recent Development” articles will help readers to gain a broader awareness and a deeper understanding of the status of research across the vast landscape of cardiovascular research— The Editors . Despite significant advances in cardiovascular medicine during the late 20th century, heart failure (HF) remains a leading cause of death in the United States and much of the rest of the world. Although improvements in acute management of cardiovascular disease have reduced death rates, efforts to halt the inexorable deterioration are largely futile. The current clinical approach focuses on disease management rather than curing HF because there is presently no cure.1 Primary treatment consists of angiotensin-converting enzyme inhibitors, β-blockers, and mineralocorticoids antagonists. And, although a new class of agents (ie, neprilysin inhibitor) has shown promise in a phase 3 clinical trial,2 collectively, these drugs only delay disease progression and death caused by HF. Given the general stagnation in the progress of clinical treatment of HF, we must undertake more daring and high-risk preclinical studies to achieve the collective dream of curing HF. This developments will highlight some recent progress in understanding the pathobiology of HF and advances in conceptual approaches for future treatments. The goal is to focus the readers’ attention on some of the more exciting and daring areas of cardiovascular research, which will likely dictate advances in the 21st …

E2F1 transcription factor regulates O-GlcNAc transferase and O-GlcNAcase expression

Protein O-GlcNAcylation, which is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), has emerged as an important posttranslational modification that may factor in multiple diseases. Until recently, it was assumed that OGT/OGA protein expression was relatively constant. Several groups, including ours, have shown that OGT and/or OGA expression changes in several pathologic contexts, yet the cis and trans elements that regulate the expression of these enzymes remain essentially unexplored. Here, we used a reporter-based assay to analyze minimal promoters and leveraged in silico modeling to nominate several candidate transcription factor binding sites in both Ogt (i.e. the gene for OGT protein) and Mgea5 (i.e. the gene for OGA protein). We noted multiple E2F binding site consensus sequences in both promoters. We performed chromatin immunoprecipitation in both human and mouse cells and found that E2F1 bound to candidate E2F binding sites in both promoters. In HEK293 cells, we overexpressed E2F1, which significantly reduced OGT and MGEA5 expression. Conversely, E2F1-deficient mouse fibroblasts had increased Ogt and Mgea5 expression. Of the known binding partners for E2F1, we queried whether retinoblastoma 1 (Rb1) might be involved. Rb1-deficient mouse embryonic fibroblasts showed increased levels of Ogt and Mgea5 expression, yet overexpression of E2F1 in the Rb1-deficient cells did not alter Ogt and Mgea5 expression, suggesting that Rb1 is required for E2F1-mediated suppression. In conclusion, this work identifies and validates some of the promoter elements for mouse Ogt and Mgea5 genes. Specifically, E2F1 negatively regulates both Ogt and Mgea5 expression in an Rb1 protein-dependent manner.

Cardiomyocyte Ogt limits ventricular dysfunction in mice following pressure overload without affecting hypertrophy

The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. The previous studies show that one such metabolic cue, O-GlcNAc, is elevated in the pressure-overloaded heart, and the increase in O-GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O-GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O-GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of OGT in the heart, we subjected cardiomyocyte-specific, inducibly deficient Ogt (i-cmOgt−/−) mice and Ogt competent littermate wild-type (WT) mice to transverse aortic constriction. Deletion of cardiomyocyte Ogt significantly decreased O-GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic and fibrotic signaling were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i-cmOgt−/− cardiomyocytes from surgically naïve mice. Interestingly, markers of cardiomyocyte dedifferentiation were elevated in Ogt-deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt-deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload-induced ventricular dysfunction.

Airn Regulates Igf2bp2 Translation in Cardiomyocytes

Rationale: Increasing evidence indicates the presence of lncRNAs in various cell types. Airn is an imprinting gene transcribed from the paternal chromosome. It is in antisense orientation to the imprinted, but maternally derived, Igf2r gene, on which Airn exerts its regulation in cis. Although Airn is highly expressed in the heart, functions aside from imprinting remain unknown. Objective: Here, we studied the functions of Airn in the heart, especially cardiomyocytes. Methods and Results: Silencing of Airn via siRNAs augmented cell death, vulnerability to cellular stress, and reduced cell migration. To find the cause of such phenotypes, the potential binding partners of Airn were identified via RNA pull-down followed by mass spectrometry, which indicated Igf2bp2 (insulin-like growth factor 2 mRNA-binding protein 2) and Rpa1 (replication protein A1) as potential binding partners. Further experiments showed that Airn binds to Igf2bp2 to control the translation of several genes. Moreover, silencing of Airn caused less binding of Igf2bp2 to other mRNAs and reduced translation of Igf2bp2 protein. Conclusions: Our study uncovers a new function of Airn and demonstrates that Airn is important for the physiology of cardiomyocytes.