Sarah Evans

SLC2A9 Is a High-Capacity Urate Transporter in Humans

Background Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man. Methods and Findings We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82). Conclusions This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.

Necrotic Myocardial Cells Release Damage‐Associated Molecular Patterns That Provoke Fibroblast Activation In Vitro and Trigger Myocardial Inflammation and Fibrosis In Vivo

Background Tissue injury triggers inflammatory responses that promote tissue fibrosis; however, the mechanisms that couple tissue injury, inflammation, and fibroblast activation are not known. Given that dying cells release proinflammatory “damage-associated molecular patterns” (DAMPs), we asked whether proteins released by necrotic myocardial cells (NMCs) were sufficient to activate fibroblasts in vitro by examining fibroblast activation after stimulation with proteins released by necrotic myocardial tissue, as well as in vivo by injecting proteins released by necrotic myocardial tissue into the hearts of mice and determining the extent of myocardial inflammation and fibrosis at 72 hours. Methods and Results The freeze–thaw technique was used to induce myocardial necrosis in freshly excised mouse hearts. Supernatants from NMCs contained multiple DAMPs, including high mobility group box-1 (HMGB1), galectin-3, S100β, S100A8, S100A9, and interleukin-1α. NMCs provoked a significant increase in fibroblast proliferation, α–smooth muscle actin activation, and collagen 1A1 and 3A1 mRNA expression and significantly increased fibroblast motility in a cell-wounding assay in a Toll-like receptor 4 (TLR4)- and receptor for advanced glycation end products–dependent manner. NMC stimulation resulted in a significant 3- to 4-fold activation of Akt and Erk, whereas pretreatment with Akt (A6730) and Erk (U0126) inhibitors decreased NMC-induced fibroblast proliferation dose-dependently. The effects of NMCs on cell proliferation and collagen gene expression were mimicked by several recombinant DAMPs, including HMGB1 and galectin-3. Moreover, immunodepletion of HMGB1 in NMC supernatants abrogated NMC-induced cell proliferation. Finally, injection of NMC supernatants or recombinant HMGB1 into the heart provoked increased myocardial inflammation and fibrosis in wild-type mice but not in TLR4-deficient mice. Conclusions These studies constitute the initial demonstration that DAMPs released by NMCs induce fibroblast activation in vitro, as well as myocardial inflammation and fibrosis in vivo, at least in part, through TLR4-dependent signaling.

Facilitative Glucose Transporter 9 Expression Affects Glucose Sensing in Pancreatic β-Cells

Facilitative glucose transporters (GLUTs) including GLUT9, accelerate the facilitative diffusion of glucose across the plasma membrane. Studies in GLUT2-deficient mice suggested the existence of another GLUT in the mammalian beta-cell responsible for glucose sensing. The objective of this study was to determine the expression and function of GLUT9 in murine and human beta-cells. mRNA and protein expression levels were determined for both isoforms of GLUT9 in murine and human isolated islets as well as insulinoma cell lines (MIN6). Immunohistochemistry and subcellular localization were performed to localize the protein within the cell. Small interfering RNA knockdown of GLUT9 was used to determine the effect of this transporter, in the presence of GLUT2, on cell metabolism and insulin secretion in MIN6 and INS cells. In this report we demonstrate that GLUT9a and GLUT9b are expressed in pancreatic islets and that this expression localizes to insulin-containing beta-cells. Subcellular localization studies indicate that mGLUT9b is found associated with the plasma membrane as well as in the high-density microsome fraction and low-density microsome fraction, whereas mGLUT9a appears to be located only in the high-density microsome and low-density microsome under basal conditions. Functionally GLUT9 appears to participate in the regulation of glucose-stimulated insulin secretion in addition to GLUT2. small interfering RNA knockdown of GLUT9 results in reduced cellular ATP levels that correlate with reductions in glucose-stimulated insulin secretion in MIN6 and INS cells. These studies confirm the expression of GLUT9a and GLUT9b in murine and human beta-cells and suggest that GLUT9 may participate in glucose-sensing in beta-cells.

Tumor Necrosis Factor Receptor Associated Factor 2 Signaling Provokes Adverse Cardiac Remodeling in the Adult Mammalian Heart

Background—Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor–associated factor 2 (TRAF2); however, virtually nothing is known about TRAF2 signaling in the adult mammalian heart. Methods and Results—We generated multiple founder lines of mice with cardiac-restricted overexpression of TRAF2 and characterized the phenotype of mice with higher expression levels of TRAF2 (myosin heavy chain [MHC]-TRAF2HC). MHC-TRAF2HC transgenic mice developed a time-dependent increase in cardiac hypertrophy, left ventricular dilation, and adverse left ventricular remodeling, and a significant decrease in LV+dP/dt and LV−dP/dt when compared with littermate controls (P<0.05 compared with littermate). During the early phases of left ventricular remodeling, there was a significant increase in total matrix metalloproteinase activity that corresponded with a decrease in total myocardial fibrillar collagen content. As the MHC-TRAF2HC mice aged, there was a significant decrease in total matrix metalloproteinase activity accompanied by an increase in total fibrillar collagen content and an increase in myocardial tissue inhibitor of metalloproteinase-1 levels. There was a significant increase in nuclear factor–&kgr;B activation at 4 to 12 weeks and jun N-terminal kinases activation at 4 weeks in the MHC-TRAF2HC mice. Transciptional profiling revealed that >95% of the hypertrophic/dilated cardiomyopathy–related genes that were significantly upregulated genes in the MHC-TRAF2HC hearts contained &kgr;B elements in their promoters. Conclusions—These results show for the first time that targeted overexpression of TRAF2 is sufficient to mediate adverse cardiac remodeling in the heart.

Dysferlin Mediates the Cytoprotective Effects of TRAF2 Following Myocardial Ischemia Reperfusion Injury

Background We have demonstrated that tumor necrosis factor (TNF) receptor‐associated factor 2 (TRAF2), a scaffolding protein common to TNF receptors 1 and 2, confers cytoprotection in the heart. However, the mechanisms for the cytoprotective effects of TRAF2 are not known. Methods/Results Mice with cardiac‐restricted overexpression of low levels of TRAF2 (MHC‐TRAF2LC) and a dominant negative TRAF2 (MHC‐TRAF2DN) were subjected to ischemia (30‐minute) reperfusion (60‐minute) injury (I/R), using a Langendorff apparatus. MHC‐TRAF2LC mice were protected against I/R injury as shown by a significant ≈27% greater left ventricular (LV) developed pressure after I/R, whereas mice with impaired TRAF2 signaling had a significantly ≈38% lower LV developed pressure, a ≈41% greater creatine kinase (CK) release, and ≈52% greater Evans blue dye uptake after I/R, compared to LM. Transcriptional profiling of MHC‐TRAF2LC and MHC‐TRAF2DN mice identified a calcium‐triggered exocytotic membrane repair protein, dysferlin, as a potential cytoprotective gene responsible for the cytoprotective effects of TRAF2. Mice lacking dysferlin had a significant ≈39% lower LV developed pressure, a ≈20% greater CK release, and ≈29% greater Evans blue dye uptake after I/R, compared to wild‐type mice, thus phenocopying the response to tissue injury in the MHC‐TRAF2DN mice. Moreover, breeding MHC‐TRAF2LC onto a dysferlin‐null background significantly attenuated the cytoprotective effects of TRAF2 after I/R injury. Conclusion The study shows that dysferlin, a calcium‐triggered exocytotic membrane repair protein, is required for the cytoprotective effects of TRAF2‐mediated signaling after I/R injury.

Widespread Down-regulation of Cardiac Mitochondrial and Sarcomeric Genes in Patients With Sepsis*

Objectives: The mechanism(s) for septic cardiomyopathy in humans is not known. To address this, we measured messenger RNA alterations in hearts from patients who died from systemic sepsis, in comparison to changed messenger RNA expression in nonfailing and failing human hearts. Design: Identification of genes with altered abundance in septic cardiomyopathy, ischemic heart disease, or dilated cardiomyopathy, in comparison to nonfailing hearts. Setting: ICUs at Barnes-Jewish Hospital, St. Louis, MO. Patients: Twenty sepsis patients, 11 ischemic heart disease, nine dilated cardiomyopathy, and 11 nonfailing donors. Interventions: None other than those performed as part of patient care. Measurements and Main Results: Messenger RNA expression levels for 198 mitochondrially localized energy production components, including Krebs cycle and electron transport genes, decreased by 43% ± 5% (mean ± SD). Messenger RNAs for nine genes responsible for sarcomere contraction and excitation-contraction coupling decreased by 43% ± 4% in septic hearts. Surprisingly, the alterations in messenger RNA levels in septic cardiomyopathy were both distinct from and more profound than changes in messenger RNA levels in the hearts of patients with end-stage heart failure. Conclusions: The expression profile of messenger RNAs in the heart of septic patients reveals striking decreases in expression levels of messenger RNAs that encode proteins involved in cardiac energy production and cardiac contractility and is distinct from that observed in patients with heart failure. Although speculative, the global nature of the decreases in messenger RNA expression for genes involved in cardiac energy production and contractility suggests that these changes may represent a short-term adaptive response of the heart in response to acute change in cardiovascular homeostasis.

Circulating p53-Responsive MicroRNAs as Predictive Biomarkers in Heart Failure After Acute Myocardial Infarction: The Long and Arduous Road From Scientific Discovery to Clinical Utility

The past decade has witnessed an exponential proliferation of new biomarkers that are useful in evaluating patients with heart failure (reviewed in 1). Indeed, 2 biomarkers, brain natriuretic peptide and N-terminalprobrain natriuretic peptide (BNP) are Food and Drug Administration–approved for the diagnosis of patients with heart failure, whereas BNP, N-terminalpro-BNP, galectin-3, and soluble ST-2 are Food and Drug Administration–approved for determining the prognosis of patients with heart failure. In this issue of Circulation Research , Matsumoto et al2 report that several p53-responsive microRNAs (miRs), including miR-192, miR-194, and miR-34a, were elevated in the sera of selected patients enrolled in the Osaka Acute Coronary Insufficiency study (n=8603 patients), who developed heart failure 1 year after acute myocardial infarction. Given that p53 has been implicated in the development of heart failure in mice,3 these findings are of potential interest both from a mechanistic standpoint, as well as clinically from a diagnostic standpoint. In this study, the authors examined a panel of 377 microRNAs in registry patients who developed heart failure (n=7 patients), and then confirmed their findings in a validation cohort of 21 patients. The authors reported previously that miR-192 was significantly ( P =0.04) elevated in selected registry patients.4 Noting that miR-192 was responsive to p53, in this study, the authors rescreened the sera of heart failure patients and identified significant increases in the levels of 2 additional p53-responsive microRNAs, namely miR-194 (increased ≈6-fold) and miR-34a (increased ≈3-fold). The presence or absence of heart failure in the registry was determined from a retrospective chart review; however, the criteria that were used to diagnose heart failure were not specified. Remarkably, the levels of miR-192, miR-194, and miR-34a were highly enriched in the exosome fraction of the heart failure patients when compared with control subjects, whereas the levels of miR-192, miR-194, …

TNF receptor–activated factor 2 mediates cardiac protection through noncanonical NF- κ B signaling

To elucidate the mechanisms responsible for cytoprotective effects of TNF receptor-activated factor 2 (TRAF2) in the heart, we employed genetic gain- and loss-of-function studies ex vivo and in vivo in mice with cardiac-restricted overexpression of TRAF2 (Myh6-TRAF2LC). Crossing Myh6-TRAF2LC mice with mice lacking canonical signaling (Myh6-TRAF2LC/Myh6-IκBαΔN) abrogated the cytoprotective effects of TRAF2 ex vivo. In contrast, inhibiting the JAK/STAT pathway did not abrogate the cytoprotective effects of TRAF2. Transcriptional profiling of WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts suggested that the noncanonical NF-κB signaling pathway was upregulated in the Myh6-TRAF2LC mouse hearts. Western blotting and ELISA for the NF-κB family proteins p50, p65, p52, and RelB on nuclear and cytoplasmic extracts from naive 12-week-old WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts showed increased expression levels and increased DNA binding of p52 and RelB, whereas there was no increase in expression or DNA binding of the p50 and p65 subunits. Crossing Myh6-TRAF2LC mice with RelB-/+ mice (Myh6-TRAF2LC/RelB-/+) attenuated the cytoprotective effects of TRAF2 ex vivo and in vivo. Viewed together, these results suggest that crosstalk between the canonical and noncanonical NF-κB signaling pathways is required for mediating the cytoprotective effects of TRAF2.

Load-Dependent Changes in Left Ventricular Structure and Function in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure

Background: To better understand reverse left ventricular (LV) remodeling, we developed a murine model wherein mice develop LV remodeling after transverse aortic constriction (TAC) and a small apical myocardial infarct (MI) and undergo reverse LV remodeling after removal of the aortic band. Methods and Results: Mice studied were subjected to sham (n=6) surgery or TAC+MI (n=12). Two weeks post-TAC+MI, 1 group underwent debanding (referred to as heart failure debanding [HF-DB] mice; n=6), whereas the aortic band remained in a second group (heart failure [HF] group; n=6). LV remodeling was evaluated by 2D echocardiography at 1 day, 2 weeks and 6 weeks post-TAC+MI. The hearts were analyzed by transcriptional profiling at 4 and 6 weeks and histologically at 6 weeks. Debanding normalized LV volumes, LV mass, and cardiac myocyte hypertrophy at 6 weeks in HF-DB mice, with no difference in myofibrillar collagen in the HF and HF-DB mice. LV ejection fraction and radial strain improved after debanding; however, both remained decreased in the HF-DB mice relative to sham and were not different from HF mice at 6 weeks. Hemodynamic unloading in the HF-DB mice was accompanied by a 35% normalization of the HF genes at 2 weeks and 80% of the HF genes at 4 weeks. Conclusions: Hemodynamic unloading of a pathophysiologically relevant mouse model of HF results in normalization of LV structure, incomplete recovery of LV function, and incomplete reversal of the HF transcriptional program. The HF-DB mouse model may provide novel insights into mechanisms of reverse LV remodeling.