Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel.

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Kansas City Cardiomyopathy Questionnaire Utility in Prediction of 30-Day Readmission Rate in Patients with Chronic Heart Failure.

Background. Heart failure (HF) is one of the most common diagnoses associated with hospital readmission. We designed this prospective study to evaluate whether Kansas City Cardiomyopathy Questionnaire (KCCQ) score is associated with 30-day readmission in patients hospitalized with decompensated HF. Methods and Results. We enrolled 240 patients who met the study criteria. Forty-eight (20%) patients were readmitted for decompensated HF within thirty days of hospital discharge, and 192 (80%) patients were not readmitted. Compared to readmitted patients, nonreadmitted patients had a higher average KCCQ score (40.8 versus 32.6, P = 0.019) before discharge. Multivariate analyses showed that a high KCCQ score was associated with low HF readmission rate (adjusted OR = 0.566, P = 0.022). The c-statistic for the base model (age + gender) was 0.617. The combination of home medication and lab tests on the base model resulted in an integrated discrimination improvement (IDI) increase of 3.9%. On that basis, the KCQQ further increased IDI of 2.7%. Conclusions. The KCCQ score determined before hospital discharge was significantly associated with 30-day readmission rate in patients with HF, which may provide a clinically useful measure and could significantly improve readmission prediction reliability when combined with other clinical components.

Blatchford Score Is Superior to AIMS65 Score in Predicting the Need for Clinical Interventions in Elderly Patients with Nonvariceal Upper Gastrointestinal Bleed.

Background. Blatchford and AIMS65 scores were developed to risk stratify patients with upper gastrointestinal bleed (UGIB). We sought to assess the performance of Blatchford and AIMS65 scores in predicting outcomes in elderly patients with nonvariceal UGIB. Methods. A retrospective cohort study of elderly patients (over 65 years of age) with nonvariceal UGIB admitted to a tertiary care center. Primary outcome was a combined outcome of in-hospital mortality, need for any therapeutic endoscopic, radiologic, or surgical intervention, rebleeding within 30 days, or blood transfusion. Secondary outcome was a combined outcome of in-hospital mortality or need for an intervention to control the bleed. Results. 164 patients were included. The primary outcome occurred in 119 (72.5%) patients. The secondary outcome occurred in 12 patients (7.2%). Blatchford score was superior to AIMS65 score in predicting the primary outcome (area under the receiver-operator curve (AUROC) 0.84 versus 0.68, resp., p < 0.001). Both scores performed poorly in predicting the secondary outcome (AUROC 0.56 versus 0.52, resp., p = 0.18). Conclusions. Blatchford score could be useful in predicting the need for hospital based interventions in elderly patients with nonvariceal UGIB. Blatchford and AIMS65 scores are poor predictors of the need for a therapeutic intervention to control bleeding.

AIP1 suppresses atherosclerosis by limiting hyperlipidemia-induced inflammation and vascular endothelial dysfunction.

OBJECTIVE: Apoptosis signal-regulating kinase 1-interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1(-/-)) mice to examine its effect on atherosclerotic lesions in an apolipoprotein E-null (ApoE(-/-)) mouse model of atherosclerosis. APPROACH AND RESULTS: ApoE(-/-) control mice developed atherosclerosis in the aortic roots and descending aortas on Western-type diet for 10 weeks, whereas the atherosclerotic lesions are significantly augmented in ApoE(-/-)AIP1(-/-) double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels, or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with nuclear factor-κB activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC, but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized low-density lipoprotein-induced nuclear factor-κB signaling, gene expression of inflammatory molecules, and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice. CONCLUSIONS: Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE(-/-)AIP1(-/-) mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction, and atherosclerosis.

Expression of microRNA-122 contributes to apoptosis in H9C2 myocytes.

The microRNAs (miRNAs) can post-transcriptionally regulate gene expression and heart development. The Pax-8 gene knockout mice have apparent heart abnormalities. This study investigated the role of miRNAs in regulation of cardiac apoptosis and development in the knockout mice. MicroRNA microarrays demonstrated differential expression of microRNAs between Pax-8(-/-) and Pax-8(+/-) mice, confirmed by real-time PCR. The miR-122 was up-regulated by 1.92 folds in Pax-8(-/-) mice. There were ventricular septum defects in Pax-8(-/-) mice, and increased numbers of apoptotic cells in the left ventricular wall and interventricular septum in Pax-8(-/-) mice. In H9C2 myocytes, treatment with miR-122 mimics or miR-122 inhibitor affects the expression of CCK-8 and activity of Caspase-3. The miR-122 is up-regulated in the myocytes of Pax-8(-/-) mice and may participate in the apoptotic gene expression and pathogenesis of heart development defect.

Glutathione S-transferases T1 null genotype is associated with susceptibility to aristolochic acid nephropathy.

OBJECTIVES: This study aims to determine whether six polymorphisms of the genes involved in drug metabolism are associated with susceptibility to the development and progression of aristolochic acid nephropathy (AAN). METHODS: In the study, 91 aristolochic acid nephropathy (AAN) cases and 152 healthy controls of Chinese Han population were examined. Six common polymorphisms of genes, including multidrug resistance gene 1 (MDR1), cytochrome P450 (CYP1A1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST) T1 and M1, were determined. Associations between their genotypes with AAN risk were calculated using an unconditional logistic regression model. RESULTS: Among the six candidate polymorphisms, only the distribution frequency of GSTT1 null genotype was significantly higher among AAN cases compared with controls (P = 0.041, 62.6% vs. 48.7%) and was associated with a 1.7-fold increased risk (OR = 1.728, 95%CI: 1.013-2.948, P = 0.045) of developing AAN, after adjustment for age and gender. The stratified analysis further showed that the GSTT1 null genotype was dominant in slow progressive AAN patients (OR = 2.497, 95%CI: 1.028-6.064, P = 0.043). The GSTM1 genotypes were not shown to influence the development of AAN. CONCLUSION: This study supports the hypothesis that polymorphisms related to drug metabolism such as GSTT1 may be an important factor influencing the development of AAN in the Chinese Han population exposed to AA.

Functional analysis of promoter mutations in the ACTN4 and SYNPO genes in focal segmental glomerulosclerosis.

BACKGROUND: To investigate the promoter mutations of ACTN4 and SYNPO genes in patients with idiopathic focal segmental glomerulosclerosis (FSGS), and to provide functional analysis of these mutations in the role of FSGS occurrence. METHODS: The study consisted of 82 Chinese idiopathic FSGS patients (55 patients had nephrotic syndrome: NS) and 90 healthy individuals. Genomic DNA extracted from peripheral leukocytes of patients of healthy individuals were used to analyse the ACTN4 and SYNPO gene promoter mutations by polymerase chain reaction (PCR) and direct sequencing. Mutations were matched with GenBank and TRANSFAC software database (www.genometix.de; www.gene-regulation.com). A dual luciferase assay system was used to analyse the effects of mutations based on PGL3-Basic vector, pRL-SV40 vector, a PC12 cell line and podocytes in vitro. Kidney alpha-actinin-4 and synaptopodin expression of mutated patients and genomic DNA of their parents were investigated. RESULTS: The study detected the ACTN4 gene promoter 1-34C>T, 1-590delA and (1-1044delT)+(1-797T>C)+(1-769A>G) heterozygous mutations in three patients, respectively, and the SYNPO gene promoter 1-24G>A and 1-851C>T heterozygous mutations in two patients, respectively (with adenine of translation start site ATG naming +1). The same mutations were not found in the control group of 90 healthy people. Excepting one patient with an ACTN4 gene promoter mutation who inherited her parents' 1-1044delT and 1-797T>C mutated chromosome, respectively, the same mutations were not found in patients' parents. Alpha-actinin-4 and synaptopodin protein expression are reduced in mutated patients' kidneys. Dual luciferase assays show that compared to the normal group (with the exception of the 1-1044delT group), luciferase activity in mutated groups decreased for the most part. (1-1044delT)+(1-797T>C)+(1-769A>G) mutations are associated with poor clinical outcomes, and patients with these mutations progress to end-stage renal failure. CONCLUSION: The study detected heterozygous mutations in the promoters of the ACTN4 and SYNPO genes in patients with idiopathic FSGS. These mutations affected gene transcription in vitro and may affect protein translation in vivo. So we presumed that the ACTN4 and SYNPO promoter mutations might also contribute to pathophysiology of idiopathic FSGS.

ACTN4 gene mutations and single nucleotide polymorphisms in idiopathic focal segmental glomerulosclerosis.

AIM: To investigate the association between mutations or single nucleotide polymorphisms (SNPs) of the gene ACTN4 in Chinese patients with idiopathic focal segmental glomerulosclerosis (FSGS). MATERIALS AND METHODS: Genomic DNA of 82 Chinese idiopathic FSGS patients and 70 healthy people were used to analyze ACTN4 gene mutations by polymerase chain reaction, direct sequencing and GenBank matching. Hair follicle DNA of novel mutated patients' parents were sequenced and alpha-actinin-4 expression in patients' kidney was examined by immunofluorescence. For SNPs, after the Hardy-Weinberg equilibrium test, allele association and the frequencies of genotypes were analyzed, followed by association analysis between genotypes and clinical diagnosis. RESULTS: We found a heterozygous candidate mutation 184T>A (S62T) in 1 patient and a 5' UTR candidate mutation 1-34C>T in another patient. Both patients had non-nephrotic syndrome FSGS with reduced kidney alpha-actinin-4 expression. Promoter activity analysis suggests that the 1-34C>T candidate mutation may affect the transcriptional regulation of ACTN4 gene. Additionally, 6 novel silent variants and 2 novel SNPs were also found in this study. Novel SNP 484 + 87C>G had a significant association with the level of urine protein excretion in these idiopathic FSGS patients. CONCLUSIONS: Our data suggest that mutations and SNP of ACTN4 gene may contribute to be associated with Chinese idiopathic FSGS.

Endothelial-specific expression of mitochondrial thioredoxin promotes ischemia-mediated arteriogenesis and angiogenesis.

OBJECTIVE: Thioredoxin-2 (Trx2), a major antioxidant protein in mitochondria, enhances nitric oxide bioavailability and inhibits ASK1-dependent apoptosis in endothelial cells (ECs). However, the in vivo role of Trx2 in angiogenesis has not been defined. Here we used EC-specific transgenesis of Trx2 (Trx2-TG) in mice to determine the in vivo function of Trx2 in arteriogenesis and angiogenesis. METHODS AND RESULTS: In a femoral artery ligation model, Trx2-TG mice had enhanced capacity in limb perfusion recovery and ischemic reserve capacity compared to the nontransgenic littermates. Ischemia-initiated arteriogenesis in the upper limb was augmented in Trx2-TG mice. Trx2-TG mice also showed significantly enhanced capillary formation and maturation in the lower limb. In nontransgenic limb, ischemia specifically induced a downregulation of Trx2 protein, leading to increased oxidative stress, ASK1 activation, and EC apoptosis. In contrast, Trx2-TG maintained a constitutive level of Trx2, reducing the ischemia-induced deleterious responses. We then defined the mechanism by which Trx2 increases angiogenesis using ECs isolated from Trx2-TG mice. Trx2-TG ECs showed increased NO and NO-dependent migration. In addition, these cells were more resistant to oxidative stress-induced activation of ASK1 signaling and apoptosis. Moreover, Trx2-augmented EC survival is NO-independent. To define the relative contributions of Trx2-increased NO and Trx2-reduced ASK1 apoptotic activity to angiogenesis in vivo, we examined Trx2 effects on ischemia-induced angiogenesis in eNOS-deficient mice. The eNOS deletion caused severe impairment in the functional flow recovery in response to ischemia. Trx2 expression in eNOS-KO mice still dramatically inhibited ischemia-induced ASK1 and EC apoptosis, leading to an enhanced functional flow recovery. CONCLUSIONS: These in vivo and in vitro data support that Trx2 maintains EC function by two parallel pathways-scavenging ROS to increase NO bioavailability and inhibiting ASK1 activity to enhance EC survival, facilitating ischemia-mediated arteriogenesis and angiogenesis.

AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice.

ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.

Endothelial-specific expression of mitochondrial thioredoxin improves endothelial cell function and reduces atherosclerotic lesions.

The function of the mitochondrial antioxidant system thioredoxin (Trx2) in vasculature is not understood. By using endothelial cell (EC)-specific transgenesis of the mitochondrial form of the thioredoxin gene in mice (Trx2 TG), we show the critical roles of Trx2 in regulating endothelium functions. Trx2 TG mice have increased total antioxidants, reduced oxidative stress, and increased nitric oxide (NO) levels in serum compared with their control littermates. Consistently, aortas from Trx2 TG mice show reduced vasoconstriction and enhanced vasodilation. By using ECs isolated from Trx2 TG mice, we further show that Trx2 increases the capacities of ECs in scavenging reactive oxygen species generated from mitochondria, resulting in increases in NO bioavailability in ECs. More importantly, Trx2 improves EC function and reduces atherosclerotic lesions in the apolipoprotein E-deficient mouse model. Our data provide the first evidence that Trx2 plays a critical role in preserving vascular EC function and prevention of atherosclerosis development, in part by reducing oxidative stress and increasing NO bioavailability.