Azelnidipine protects HL-1 cardiomyocytes from hypoxia/reoxygenation injury by enhancement of NO production independently of effects on gene expression.

It remains to be elucidated whether Ca2+ antagonists induce pharmacological preconditioning to protect the heart against ischemia/reperfusion injury. The aim of this study was to determine whether and how pretreatment with a Ca2+ antagonist, azelnidipine, could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury in vitro. Using HL-1 cardiomyocytes, we studied effects of azelnidipine on NO synthase (NOS) expression, NO production, cell death and apoptosis during H/R. Action potential durations (APDs) were determined by the whole-cell patch-clamp technique. Azelnidipine enhanced endothelial NOS phosphorylation and NO production in HL-1 cells under normoxia, which was abolished by a heat shock protein 90 inhibitor, geldanamycin, and an antioxidant, N-acetylcysteine. Pretreatment with azelnidipine reduced cell death and shortened APDs during H/R. These effects of azelnidipine were diminished by a NOS inhibitor, L-NAME, but were influenced by neither a T-type Ca2+ channel inhibitor, NiCl2, nor a N-type Ca2+ channel inhibitor, ω-conotoxin. The azelnidipine-induced reduction in cell death was not significantly enhanced by either additional azelnidipine treatment during H/R or increasing extracellular Ca2+ concentrations. RNA sequence (RNA-seq) data indicated that azelnidipine-induced attenuation of cell death, which depended on enhanced NO production, did not involve any significant modifications of gene expression responsible for the NO/cGMP/PKG pathway. We conclude that pretreatment with azelnidipine protects HL-1 cardiomyocytes against H/R injury via NO-dependent APD shortening and L-type Ca2+ channel blockade independently of effects on gene expression.

C-reactive Protein Levels and Cardiovascular Outcomes After Febuxostat Treatment in Patients with Asymptomatic Hyperuricemia: Post-hoc Analysis of a Randomized Controlled Study.

PURPOSE: Inflammation plays an important role in the initiation and progression of atherosclerosis, leading to poor clinical outcomes. Hyperuricemia is associated with the activation of the Nod-like receptor protein 3 inflammasome. Here, we investigated whether inhibition of inflammation using febuxostat lowered the risk of cardiovascular events. METHODS: This is a post-hoc analysis of the randomized trial, Febuxostat for Cerebral and CaRdiorenovascular Events PrEvEntion StuDy (FREED). In total, 1067 patients (736 men and 331 women) were included in the analysis. We compared the serial changes in high-sensitivity C-reactive protein (hs-CRP) levels between febuxostat and non-febuxostat groups and assessed the correlation between the changes in uric acid (UA) and hs-CRP levels after febuxostat treatment. We also determined whether febuxostat could reduce a hard endpoint, defined as a composite of cardiovascular events and all-cause mortality. RESULTS: Serum UA levels in the febuxostat group were significantly lower than those in the non-febuxostat group after randomization (p < 0.05). However, hs-CRP levels were comparable between the two groups during the study. No significant correlation was observed between the changes in UA and hs-CRP levels after febuxostat treatment. The hard endpoints did not differ significantly between the two groups. In patients with baseline hs-CRP levels > 0.2 mg/dL or those administered 40 mg of febuxostat, the drug did not reduce hs-CRP levels or decrease the hard endpoint. CONCLUSION: Febuxostat reduced the UA levels but did not affect the CRP levels, and therefore may fail to improve cardiovascular outcomes after treatment. TRIAL REGISTRATION: ClinicalTrial.gov (NCT01984749). https://clinicaltrials.gov/ct2/show/NCT01984749.

Effects of Conditioned Medium of Adipose-Derived Stem Cells Exposed to Platelet-Rich Plasma on the Expression of Endothelial Nitric Oxide Synthase and Angiogenesis by Endothelial Cells.

ABSTRACT: Platelet-rich plasma (PRP) and adipose-derived stem cells (ADSCs) are known to secrete angiogenic factors that contribute to the treatment of intractable ulcers. The combination of PRP and ADSCs may enhance their angiogenic effects. However, it remains unclear whether treatment of ADSCs with PRP influences angiogenesis. We studied whether the conditioned medium from PRP-treated ADSCs under hypoxic conditions exerts angiogenic effects. Although PRP stimulated the proliferation of ADSCs obtained from rats, it decreased the mRNA levels of vascular endothelial growth factor, hepatocyte growth factor, and TGF-ß1, but not of basic fibroblast growth factor, under hypoxia. The conditioned medium of PRP-treated ADSCs inhibited endothelial nitric oxide synthase phosphorylation, decreased NO production, and suppressed tube formation in human umbilical vein endothelial cells. Transplantation of ADSCs alone increased both blood flow and capillary density of the ischemic limb; however, its combination with PRP did not further improve blood flow or capillary density. This suggests that both conditioned medium of ADSCs treated with PRP and combination of PRP with ADSCs transplantation may attenuate the phosphorylation of endothelial nitric oxide synthase and angiogenesis.

Clinical characteristics for distinguishing between acute cardiogenic pulmonary edema and community-acquired pneumonia in elderly patients: a prospective observational study.

Heart failure and pneumonia are highly prevalent in elderly patients. We conducted a study to evaluate the differences in the patterns of symptoms, laboratory findings, and computed tomography (CT) results in elderly patients with acute cardiogenic pulmonary edema (ACPE) and community-acquired pneumonia (CAP). From January 1, 2015 to December 31, 2017, we studied 140 patients aged >75 years who were diagnosed with ACPE and CAP. Symptoms, laboratory findings, mean ostial pulmonary vein (PV) diameter and patterns on CT images were assessed. The primary measures of diagnostic accuracy were assessed using the positive likelihood ratio (LR+). The cutoff value of ostial PVs for differentiating patients with ACPE from CAP was evaluated using the receiver operating characteristic (ROC) analysis. Ninety-three patients with ACPE, 36 with CAP, and 11 with complicated ACPE/CAP were included. In patients with ACPE, edema (LR+ 5.4) was a moderate factor for rule-in, and a high brain natriuretic peptide level (LR+ 4.2) was weak. In patients with CAP, cough (LR+ 5.7) and leukocytosis (LR+ 5.2) were moderate factors for rule-in, while fever (LR+ 3.8) and a high C-reactive protein level (LR+ 4.8) were weak factors. The mean diameter of ostial PVs in patients with ACPE was significantly larger than that of patients with CAP (15.8±â€Š1.8 mm vs 9.6±1.5 mm, p< 0.01). ROC analysis revealed that an ostial PV diameter cutoff of 12.5 mm was strong evidence for distinguishing ACPE from CAP with an area under the ROC curve of 0.99 and LR+ 36.0. In conclusion, as ACPE and CAP have similar symptoms and laboratory findings, dilated ostial PVs were useful in characterizing CT images to distinguish ACPE from CAP.

Optimal uric acid levels by febuxostat treatment and cerebral, cardiorenovascular risks: post hoc analysis of a randomized controlled trial.

OBJECTIVES: Hyperuricaemia is recognized as an independent risk marker for cardiovascular and renal diseases. However, uric acid is a powerful free-radical scavenger, and the optimal level of serum uric acid (SUA) determining outcomes is unknown. This study explored whether interventional treatments for excessive SUA reduction were harmful and what constituted the optimal lowering of SUA levels for the prevention of events in patients with asymptomatic hyperuricaemia. METHODS: This was a post hoc analysis of a randomized trial (Febuxostat for Cerebral and CaRdiorenovascular Events PrEvEntion StuDy [FREED]) in which 1070 older patients with asymptomatic hyperuricaemia were enrolled and allocated to febuxostat (n = 537) or non-febuxostat treatment group (n = 533). We assessed the relationship between the endpoint (withdrawal or study completion) SUA levels and clinical outcomes. Primary endpoint was defined as a composite of all-cause mortality, cerebral and cardiorenovascular events. RESULTS: In the febuxostat group, patients achieving SUA levels ≤4 mg/dl (hazard ratio: 2.01 [95% CI: 1.05, 3.87]), >4 to ≤5 mg/dl (2.12 [1.07, 4.20], >6 to ≤7 mg/dl (2.42 [1.05, 5.60]), and >7 mg/dl (4.73 [2.13, 10.5]) had significantly higher risks for a primary composite event than those achieving SUA levels >5 to ≤6 mg/dl (P = 0.003 [log-rank test]). This J-shaped relationship applied to patients with renal impairment (P = 0.007 [Gray's test]) and was not significant in the non-febuxostat treatment group (P = 0.212 [log-rank test]). CONCLUSION: Optimal SUA level by febuxostat treatment is 5-6 mg/dl for reducing all-cause mortality, cerebral, cardiovascular and renal events. Excessive SUA reduction may be harmful in older hyperuricaemic populations. TRIAL REGISTRATION: ClinicalTrial.gov, https://clinicaltrials.gov, NCT01984749.

Thrombin induces a temporal biphasic vascular response through the differential phosphorylation of endothelial nitric oxide synthase via protease-activated receptor-1 and protein kinase C.

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes.

BACKGROUND: Gout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1. METHODS AND RESULTS: Macrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1ß secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1ß. MSU reduced intracellular K+ concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1ß, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1ß and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5. CONCLUSIONS: Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF.

Hsp70 promotes maturation of uromodulin mutants that cause familial juvenile hyperuricemic nephropathy and suppresses cellular damage.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD. Here we studied effects of genetic expression and pharmacological induction of Hsp70 on the UMOD mutants C112Y and C217G. METHODS: We expressed wild type (WT), C112Y and C217G in HEK293 cells and studied their maturation and cellular damage using western blot and flow cytometry. RESULTS: Expression of C112Y or C217G increased pro-apoptotic proteins, decreased anti-apoptotic proteins, and induced cellular apoptosis as examined by annexin V staining and flow cytometry. Overexpression of Hsp70 or administration of an Hsp70 inducer geranylgeranylacetone (GGA) promoted maturation of the mutant proteins, increased their secreted forms, normalized the levels of pro- and anti-apoptotic proteins and suppressed apoptosis. CONCLUSION: These findings indicated that Hsp70 enhanced maturation of C112Y and C217G and reduced cellular apoptosis, suggesting that Hsp70 induction might be of a therapeutic value for treatment of FJHN.

High uric acid induces liver fat accumulation via ROS/JNK/AP-1 signaling.

Uric acid is the end metabolite derived from the oxidation of purine compounds. Overwhelming evidence shows the vital interrelationship between hyperuricemia (HUA) and nonalcoholic fatty liver disease (NAFLD). However, the mechanisms for this association remain unclear. In this study, we established a urate oxidase-knockout (Uox-KO) mouse model by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. To study the correlation between HUA and NAFLD, human HepG2 hepatoma cells were treated in culture medium with high level of uric acid. In vivo, the Uox-KO mice spontaneously developed hyperuricemia and aberrant lipid-metabolism, concomitant with abnormal hepatic fat accumulation. HUA activated c-Jun N-terminal kinase (JNK) in vivo and in vitro. Furthermore, inhibiting JNK activation by a JNK-specific inhibitor, SP600125, decreased fat accumulation and lipogenic gene expression induced by HUA. Overexpression of the lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase 1 was via activation of JNK, which was blocked by the JNK inhibitor SP600125. HUA activated AP-1 to upregulate lipogenic gene expression via JNK activation. In addition, HUA caused mitochondrial dysfunction and reactive oxygen species production. Pretreatment with the antioxidant N-acetyl-l-cysteine could ameliorate HUA-activated JNK and hepatic steatosis. These data suggest that ROS/JNK/AP-1 signaling plays an important role in HUA-mediated fat accumulation in liver.NEW & NOTEWORTHY Hyperuricemia and nonalcoholic fatty liver disease are global public health problems, which are strongly associated with metabolic syndrome. In this study, we demonstrate that uric acid induces hepatic fat accumulation via the ROS/JNK/AP-1 pathway. This study identifies a new mechanism of NAFLD pathogenesis and new potential therapeutic strategies for HUA-induced NAFLD.

Uric acid inhibits HMGB1-TLR4-NF-κB signaling to alleviate oxygen-glucose deprivation/reoxygenation injury of microglia.

Mounting evidence has implicated inflammation in ischemia-reperfusion injury following acute ischemic stroke (AIS). Microglia remain the primary initiator and participant of brain inflammation. Emerging evidence has indicated that uric acid has promise for the treatment of AIS, but its explicit mechanisms remain elusive. Here, we observed that uric acid reduced the severity of cerebral infarction and attenuated the activation of microglia in the cerebral cortex in a mouse middle cerebral-artery occlusion/reperfusion model. Thus, we speculated that uric acid may play a role by directly interfering with the inflammatory response of microglia. First, we investigated whether the HMGB1-TLR4-NF-κB signaling plays a role in oxygen glucose deprivation and reperfusion (OGD/R) injury of BV2 cells. Inhibition of the signaling significantly reduced the release of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1ß (IL1ß), and IL6 caused by OGD/R in BV2 cells. Second, uric acid weakened the decreased cell viability and lactate dehydrogenase release induced by OGD/R in BV2 cells. Finally, uric acid reduced the release of the proinflammatory cytokines TNF-α, IL1ß, and IL6 caused by OGD/R in BV2 cells by dampening HMGB1-TLR4-NF-κB signaling, which was reversed by probenecid treatment, an inhibitor of the uric acid channel. Hence, uric acid halted the release of inflammatory factors and the decreased cell viability induced by ODG/R via inhibiting the microglia HMGB1-TLR4-NF-κB signaling, thereby alleviating the damage to microglia. This may be part of the molecular mechanisms by which uric acid protects mice against the brain damage of middle cerebral-artery occlusion/reperfusion.

Uric Acid as a Risk Factor for Chronic Kidney Disease and Cardiovascular Disease　- Japanese Guideline on the Management of Asymptomatic Hyperuricemia.

Serum uric acid (UA) is taken up by endothelial cells and reduces the level of nitric oxide (NO) by inhibiting its production and accelerating its degradation. Cytosolic and plasma xanthine oxidase (XO) generates superoxide and also decreases the NO level. Thus, hyperuricemia is associated with impaired endothelial function. Hyperuricemia is often associated with vascular diseases such as chronic kidney disease (CKD) and cardiovascular disease (CVD). It has long been debated whether hyperuricemia is causally related to the development of these diseases. The 2020 American College of Rheumatology Guideline for the Management of Gout (ACR2020) does not recommend pharmacological treatment of hyperuricemia in patients with CKD/CVD. In contrast, the Japanese Guideline on Management of Hyperuricemia and Gout (JGMHG), 3rdedition, recommends pharmacological treatment of hyperuricemia in patients with CKD. In a FREED study on Japanese hyperuricemic patients with CVD, an XO inhibitor, febuxostat, improved the primary composite endpoint of cerebro-cardio-renovascular events, providing a rationale for the use of urate-lowering agents (ULAs). Since a CARES study on American gout patients with CVD treated with febuxostat revealed increased mortality, ACR2020 recommends switching to different ULAs. However, there was no difference in the mortality of Japanese patients between the febuxostat-treated group and the placebo or allopurinol-treated groups in either the FEATHER or FREED studies.

Esm1 and Stc1 as Angiogenic Factors Responsible for Protective Actions of Adipose-Derived Stem Cell Sheets on Chronic Heart Failure After Rat Myocardial Infarction.

BACKGROUND: Although adipose-derived stem cell (ADSC) sheets improve the cardiac function after myocardial infarction (MI), underlying mechanisms remain to be elucidated. The aim of this study was to determine the fate of transplanted ADSC sheets and candidate angiogenic factors released from ADSCs for their cardiac protective actions.Methods and Results:MI was induced by ligation of the left anterior descending coronary artery. Sheets of transgenic (Tg)-ADSCs expressing green fluorescence protein (GFP) and luciferase or wild-type (WT)-ADSCs were transplanted 1 week after MI. Both WT- and Tg-ADSC sheets improved cardiac functions evaluated by echocardiography at 3 and 5 weeks after MI. Histological examination at 5 weeks after MI demonstrated that either sheet suppressed fibrosis and increased vasculogenesis. Luciferase signals from Tg-ADSC sheets were detected at 1 and 2 weeks, but not at 4 weeks, after transplantation. RNA sequencing of PKH (yellow-orange fluorescent dye with long aliphatic tails)-labeled Tg-ADSCs identified mRNAs of 4 molecules related to angiogenesis, including those of Esm1 and Stc1 that increased under hypoxia. Administration of Esm1 or Stc1 promoted tube formation by human umbilical vein endothelial cells. CONCLUSIONS: ADSC sheets improved cardiac contractile functions after MI by suppressing cardiac fibrosis and enhancing neovascularization. Transplanted ADSCs existed for >2 weeks on MI hearts and produced the angiogenic factors Esm1 and Stc1, which may improve cardiac functions after MI.

Febuxostat for Cerebral and CaRdiorenovascular Events PrEvEntion StuDy.

AIMS: To compare the occurrence of cerebral, cardiovascular, and renal events in patients with hyperuricaemia treated with febuxostat and those treated with conventional therapy with lifestyle modification. METHODS AND RESULTS: This multicentre, prospective, randomized open-label, blinded endpoint study was done in 141 hospitals in Japan. A total of 1070 patients were included in the intention-to-treat population. Elderly patients with hyperuricaemia (serum uric acid >7.0 to ≤9.0 mg/dL) at risk for cerebral, cardiovascular, or renal disease, defined by the presence of hypertension, Type 2 diabetes, renal disease, or history of cerebral or cardiovascular disease, were randomized to febuxostat and non-febuxostat groups and were observed for 36 months. Cerebral, cardiovascular, and renal events and all deaths were defined as the primary composite event. The serum uric acid level at endpoint (withdrawal or completion of the study) in the febuxostat (n = 537) and non-febuxostat groups (n = 533) was 4.50 ± 1.52 and 6.76 ± 1.45 mg/dL, respectively (P < 0.001). The primary composite event rate was significantly lower in the febuxostat group than in non-febuxostat treatment [hazard ratio (HR) 0.750, 95% confidence interval (CI) 0.592-0.950; P = 0.017] and the most frequent event was renal impairment (febuxostat group: 16.2%, non-febuxostat group: 20.5%; HR 0.745, 95% CI 0.562-0.987; P = 0.041). CONCLUSION: Febuxostat lowers uric acid and delays the progression of renal dysfunction. REGISTRATION: ClinicalTrials.gov (NCT01984749).

ß-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction.

BACKGROUND: Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.Methods and Results:ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a ß-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts. CONCLUSIONS: ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

Uric Acid-Induced Enhancements of Kv1.5 Protein Expression and Channel Activity via the Akt-HSF1-Hsp70 Pathway in HL-1 Atrial Myocytes.

BACKGROUND: Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents. CONCLUSIONS: Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.

Restoration of mutant hERG stability by inhibition of HDAC6.

The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (IKr) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased IKr, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.

Isolation and characterization of ventricular-like cells derived from NKX2-5eGFP/w and MLC2vmCherry/w double knock-in human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5eGFP/w and MLC2vmCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5eGFP/w and MLC2vmCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources.

Protective Effects of Topiroxostat on an Ischemia-Reperfusion Model of Rat Hearts.

BACKGROUND: Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion. CONCLUSIONS: Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.

Dynamical mechanisms of phase-2 early afterdepolarizations in human ventricular myocytes: insights from bifurcation analyses of two mathematical models.

Early afterdepolarization (EAD) is known as a cause of ventricular arrhythmias in long QT syndromes. We theoretically investigated how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICaL), Na+/Ca2+ exchanger current (INCX), Na+-K+ pump current (INaK), intracellular Ca2+ (Cai) handling via sarcoplasmic reticulum (SR), and intracellular Na+ concentration (Nai) contribute to initiation, termination, and modulation of phase-2 EADs, using two human ventricular myocyte models. Bifurcation structures of dynamical behaviors in model cells were explored by calculating equilibrium points, limit cycles (LCs), and bifurcation points as functions of parameters. EADs were reproduced by numerical simulations. The results are summarized as follows: 1) decreasing IKs and/or IKr or increasing ICaL led to EAD generation, to which mid-myocardial cell models were especially susceptible; the parameter regions of EADs overlapped the regions of stable LCs. 2) Two types of EADs (termination mechanisms), IKs activation-dependent and ICaL inactivation-dependent EADs, were detected; IKs was not necessarily required for EAD formation. 3) Inhibiting INCX suppressed EADs via facilitating Ca2+-dependent ICaL inactivation. 4) Cai dynamics (SR Ca2+ handling) and Nai strongly affected bifurcations and EAD generation in model cells via modulating ICaL, INCX, and INaK Parameter regions of EADs, often overlapping those of stable LCs, shifted depending on Cai and Nai in stationary and dynamic states. 5) Bradycardia-related induction of EADs was mainly due to decreases in Nai at lower pacing rates. This study demonstrates that bifurcation analysis allows us to understand the dynamical mechanisms of EAD formation more profoundly. NEW & NOTEWORTHY: We investigated mechanisms of phase-2 early afterdepolarization (EAD) by bifurcation analyses of human ventricular myocyte (HVM) models. EAD formation in paced HVMs basically depended on bifurcation phenomena in non-paced HVMs, but was strongly affected by intracellular ion concentrations in stationary and dynamic states. EAD generation did not necessarily require IKs.