Cardiomyocyte-specific Txnip C247S mutation improves left ventricular functional reserve in streptozotocin-induced diabetic mice.

Underlying molecular mechanisms for the development of diabetic cardiomyopathy remain to be determined. Long-term exposure to hyperglycemia causes oxidative stress, which leads to cardiomyocyte dysfunction. Previous studies established the importance of thioredoxin-interacting protein (Txnip) in cellular redox homeostasis and glucose metabolism. Txnip is a highly glucose-responsive molecule that interacts with the catalytic center of reduced thioredoxin and inhibits the antioxidant function of thioredoxin. Here, we show that the molecular interaction between Txnip and thioredoxin plays a pivotal role in the regulation of redox balance in the diabetic myocardium. High glucose increased Txnip expression, decreased thioredoxin activities, and caused oxidative stress in cells. The Txnip-thioredoxin complex was detected in cells with overexpressing wild-type Txnip but not Txnip cysteine 247 to serine (C247S) mutant that disrupts the intermolecular disulfide bridge. Then, diabetes was induced in cardiomyocyte-specific Txnip C247S knock-in mice and their littermate control animals by injections of streptozotocin (STZ). Prolonged hyperglycemia upregulated myocardial Txnip expression in both genotypes. The absence of Txnip's inhibition of thioredoxin in Txnip C247S mutant hearts promoted mitochondrial antioxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage by diabetes. Stress hemodynamic analysis uncovered that Txnip C247S knock-in hearts have a greater left ventricular contractile reserve than wild-type hearts under STZ-induced diabetic conditions. These results provide novel evidence that Txnip serves as a regulator of hyperglycemia-induced cardiomyocyte toxicities through direct inhibition of thioredoxin and identify the single cysteine residue in Txnip as a therapeutic target for diabetic injuries.NEW & NORTEWORTHY Thioredoxin-interacting protein (Txnip) has been of great interest as a molecular mechanism to mediate diabetic organ damage. Here, we provide novel evidence that a single mutation of Txnip confers a defense mechanism against myocardial oxidative stress in streptozotocin-induced diabetic mice. The results demonstrate the importance of Txnip as a cysteine-containing redox protein that regulates antioxidant thioredoxin via disulfide bond-switching mechanism and identify the cysteine in Txnip as a therapeutic target for diabetic cardiomyopathy.

Txnip C247S mutation protects the heart against acute myocardial infarction.

RATIONALE: Thioredoxin-interacting protein (Txnip) is a novel molecular target with translational potential in diverse human diseases. Txnip has several established cellular actions including binding to thioredoxin, a scavenger of reactive oxygen species (ROS). It has been long recognized from in vitro evidence that Txnip forms a disulfide bridge through cysteine 247 (C247) with reduced thioredoxin to inhibit the anti-oxidative properties of thioredoxin. However, the physiological significance of the Txnip-thioredoxin interaction remains largely undefined in vivo. OBJECTIVE: A single mutation of Txnip, C247S, abolishes the binding of Txnip with thioredoxin. Using a conditional and inducible approach with a mouse model of a mutant Txnip that does not bind thioredoxin, we tested whether the interaction of thioredoxin with Txnip is required for Txnip's pro-oxidative or cytotoxic effects in the heart. METHODS AND RESULTS: Overexpression of Txnip C247S in cells resulted in a reduction in ROS, due to an inability to inhibit thioredoxin. Hypoxia (1% O2, 24 h)-induced killing effects of Txnip were decreased by lower levels of cellular ROS in Txnip C247S-expressing cells compared with wild-type Txnip-expressing cells. Then, myocardial ischemic injuries were assessed in the animal model. Cardiomyocyte-specific Txnip C247S knock-in mice had better survival with smaller infarct size following myocardial infarction (MI) compared to control animals. The absence of Txnip's inhibition of thioredoxin promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage induced by MI. Furthermore, an unbiased RNA sequencing screen identified that hypoxia-inducible factor 1 signaling pathway was involved in Txnip C247S-mediated cardioprotective mechanisms. CONCLUSION: Txnip is a cysteine-containing redox protein that robustly regulates the thioredoxin system via a disulfide bond-switching mechanism in adult cardiomyocytes. Our results provide the direct in vivo evidence that regulation of redox state by Txnip is a crucial component for myocardial homeostasis under ischemic stress.

Prohemostatic Activity of Factor X in Combination With Activated Factor VII in Dilutional Coagulopathy.

BACKGROUND: Recombinant activated factor VII (rFVIIa) concentrate reduces allogeneic blood transfusions, but it may increase thromboembolic complications in complex cardiac surgery. The mixture of activated factor VII (FVIIa) and factor X (FX) (FVIIa/FX) (FVIIa:FX = 1:10) is a novel bypassing agent for hemophilia patients. We hypothesized that the combination of FX and FVIIa could improve thrombin generation (TG) in acquired multifactorial coagulation defects such as seen in cardiac surgery and conducted in vitro evaluation of FVIIa/FX in parallel with other coagulation factor concentrates using in vitro and in vivo diluted plasma samples. METHODS: Plasma samples were collected from 9 healthy volunteers and 12 cardiac surgical patients. We measured TG (Thrombinoscope) using in vitro 50% dilution plasma and in vivo dilution plasma after cardiopulmonary bypass, in parallel with thromboelastometry (ROTEM) and standard coagulation assays. In vitro additions of FVIIa/FX (0.35, 0.7, and 1.4 µg/mL, based on the FVIIa level), rFVIIa (1.4, 2.8, and 6.4 µg/mL), prothrombin complex concentrate (0.3 international unit), and 20% plasma replacement were evaluated. RESULTS: In diluted plasma, the addition of either FVIIa/FX or rFVIIa shortened the lag time and increased the peak TG, but the effect in lag time of FVIIa/FX at 0.35 µg/mL was more extensive than rFVIIa at 6.4 µg/mL. Prothrombin complex concentrate increased peak TG by increasing the prothrombin level but failed to shorten the lag time. No improvement in any of the TG variables was observed after 20% volume replacement with plasma. The addition of factor concentrates normalized prothrombin time/international normalized ratio but not with plasma replacement. In cardiac patients, similar patterns were observed on TG in post-cardiopulmonary bypass samples. FVIIa/FX shortened clotting time (CT) in a concentration-dependent manner on CT on thromboelastometry. Plasma replacement did not improve CT, but a combination of plasma and FVIIa/FX (0.35 µg/mL) more effectively shortened CT than FVIIa/FX alone. CONCLUSIONS: The combination of FVIIa and FX improved TG more efficiently than rFVIIa alone or plasma in dilutional coagulopathy models. The required FVIIa dose in FVIIa/FX was considerably lower than those reported during bypassing therapy in hemophilia patients (1.4-2.8 µg/mL). The combination of plasma could restore coagulation more efficiently compared to FVIIa/FX alone. Lesser FVIIa requirement to exert procoagulant activity may be favorable in terms of reducing systemic thromboembolic complications.

Potential contribution of erythrocyte microRNA to secondary erythrocytosis and thrombocytopenia in congenital heart disease.

BackgroundChildren with cyanotic heart disease develop secondary erythrocytosis and thrombocytopenia via unknown mechanisms. Mature erythrocyte microRNAs may reflect clinical pathologies and cell differentiation processes pre-enucleation. This study evaluated erythrocyte microRNAs in children with cyanotic heart disease.MethodsErythrocyte microRNAs from children with cyanotic and acyanotic heart disease and without cardiac disease were quantified with Ion PGM System (n=10 per group). Differential expression was confirmed by quantitative PCR (qPCR; n=20 per group).ResultsMir-486-3p, mir-486-5p, and mir-155-5p increased in patients with cyanotic heart disease compared with those without heart disease: fold differences (95% confidence interval): mir-486-3p: 1.92 (1.14-3.23), P=0.011; mir-486-5p: 2.27 (1.41-3.65), P<0.001; and mir-155-5p: 1.44 (1.03-2.03), P=0.028. Mir-486-5p was increased, and let-7e-5p and mir-1260a were decreased in patients with acyanotic heart disease compared with those without heart disease: mir-486-5p: 1.66 (1.03-2.66), P=0.035; let-7e-5p: 0.66 (0.44-0.99), P=0.049; and mir-1260a: 0.53 (0.29-0.99), P=0.045.ConclusionSeveral microRNA levels changed in children with cyanotic and acyanotic heart disease. Mir-486-3p and -5p are associated with hematopoietic differentiation. Mir-486-3p regulates the erythroid vs. megakaryocyte lineage fate decision. Mir-155 is a hypoxia-inducible microRNA, whose overexpression inhibits megakaryocyte differentiation. Erythrocyte microRNA expression changes may contribute to erythrocytosis and thrombocytopenia in children with cyanotic heart disease.

[Use and Safety of Preoperative Oral Rehydration Therapy Using a Jelly Type Oral Rehydration Solution].

BACKGROUND: Traditionally, perioperative nutritional management centered on fluid therapy, but in recent years, with the spread of enhanced recovery after surgery (ERAS) protocols, the utility of oral rehydration therapy (ORT) has been reported. There are few reports, however, on the safety of using jelly type oral rehydration solutions for ORT. We examined the effects of OS-1 jelly on gastric fluid and investigated its safety. METHODS: A total of 147 patients (age range, 4-91 years), scheduled for elective surgery at our institution for whom ORT was indicated, were enrolled in this study. If the surgery was scheduled for the morning, patients were given two bottles of 200 g OS-1 jelly during the previous evening meal. If surgery was scheduled for the afternoon, two additional 200 g bottles were given to the patient with the morning meal on the day of surgery. Patients were allowed to drink water until two hours before the surgery. Gastric fluid was aspirated with a gastric tube after anesthesia induction, after which, volume and pH were measured. RESULTS: In all cases, gastric content was aspirated as a liquid, not a jelly. The volume and pH were 11.4 ± 14.6 ml and 2.8 ± 2.2, respectively. No major difference was seen in comparison with the data for OS-1 liquid. No postoperative aspiration pneumonia or reflux of gastric contents at the time of anesthesia induction was seen in any of the patients. CONCLUSIONS: From the present findings, if the time of water intake is strictly controlled, preoperative rehydration therapy using jelly-type oral rehydration solution is thought to be safe and comparable to liquid solution regarding its effects on gastric fluid.

Combination of isoliquiritigenin and tumor necrosis factor-related apoptosis-inducing ligand induces apoptosis in colon cancer HT29 cells.

OBJECTIVES: Isoliquiritigenin is a chalcone derivative with potential in cancer chemoprevention. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent, some cancer cells are resistant to TRAIL treatment. Current studies have tried to overcome TRAIL-resistant cancer cells. Here, we show for the first time that isoliquiritigenin overcomes TRAIL resistance in colon cancer HT29 cells. METHODS: HT29 cells were treated with isoliquiritigenin and/or TRAIL, and apoptosis induction was detected by flow cytometry and fluorescence microscopy. Protein expression relating to the TRAIL pathway was analyzed by Western blotting. RESULTS: A single treatment with isoliquiritigenin scarcely induced apoptosis in HT29 cells. Combined treatment with suboptimal concentrations of isoliquiritigenin and TRAIL markedly induced apoptosis, however. The effect was blocked by a pan-caspase inhibitor and a caspase-3, 8, 9, or 10 inhibitor, suggesting that the combination facilitates caspase-dependent apoptosis. Furthermore, the apoptosis induced by isoliquiritigenin and TRAIL was blocked by a dominant negative form of the TRAIL receptor. This result indicates that the combined effect is caused by specific interaction between TRAIL and its receptors. Isoliquiritigenin increased the amount of DR5 protein among TRAIL receptors. Isoliquiritigenin did not significantly increase levels of the Bcl-2 family proteins Bcl-2, Bcl-xL, and BAX. CONCLUSIONS: Our results suggest that isoliquiritigenin has the potential to overcome resistance to TRAIL in cancer cells and its chemopreventive effects may depend on TRAIL function.