Although elevated soluble uric acid (SUA) levels enhance the production of IL-1β in macrophages stimulated with monosodium urate (MSU) crystals, the underlying mechanism remains unelucidated. The aim of this study was to examine the effects of SUA on inflammatory response-related gene expression in lipopolysaccharide-primed and MSU crystal-stimulated macrophages using mouse macrophage-like J774.1 cells. Differential gene expression in SUA-pretreated and untreated group cells was analyzed by RNA sequencing and quantitative reverse transcription-polymerase chain reaction. SUA upregulated the genes related to pro-inflammatory reactions and downregulated those related to anti-inflammatory reactions. SUA also upregulated M1 pro-inflammatory macrophage-related genes and enhanced mRNA expression of CD44 responsible for phagocytosis of MSU crystals. These results suggest that SUA enhances gouty inflammation via promoting the expression of genes related to pro-inflammatory reactions, polarization toward the M1 phenotype, and MSU crystal phagocytosis in macrophages. Enhanced expression of the transcription factor genes Nfkb1 and Stat1 may underlie the SUA-induced M1 polarization and resulting enhancement of inflammasome activation and IL-1β transcription.

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.

Background:Doxorubicin (Dox) is effective against different types of cancers, but it poses cardiotoxic side effects, frequently resulting in irreversible heart failure. However, the complexities surrounding this cardiotoxicity, especially at sublethal dosages, remain to be fully elucidated. We investigated early cellular disruptions in response to sublethal Dox, with a specific emphasis on the role of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in initiating mitochondrial dysfunction.Methods:This study utilized the H9c2 cardiomyocyte model to identify a sublethal concentration of Dox and investigate its impact on mitochondrial health using markers such as mitochondrial membrane potential (MMP), mitophagy initiation, and mitochondrial calcium dynamics. We examined the roles of and interactions between CaMKII, dynamin-related protein 1 (Drp1), and the mitochondrial calcium uniporter (MCU) in Dox-induced mitochondrial disruption using specific inhibitors, such as KN-93, Mdivi-1, and Ru360, respectively.Results:Exposure to a sublethal dose of Dox reduced the MMP red-to-green fluorescence ratio in H9c2 cells by 40.6% compared with vehicle, and increased the proportion of cells undergoing mitophagy from negligible levels compared with vehicle to 62.2%. Mitochondrial calcium levels also increased by 8.7-fold compared with the vehicle group. Notably, the activation of CaMKII, particularly its phosphorylated form, was pivotal in driving these mitochondrial changes, as inhibition using KN-93 restored MMP and decreased mitophagy. However, inhibition of Drp1 and MCU functions had a limited impact on the observed mitochondrial disruptions.Conclusion:Sublethal administration of Dox is closely linked to CaMKII activation through phosphorylation, emphasizing its pivotal role in early mitochondrial disruption. These findings present a promising direction for developing therapeutic strategies that may alleviate the cardiotoxic effects of Dox, potentially increasing its clinical efficacy.

AbstractPlatelet-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-β1, 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.

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.

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.

Background：The biological actions of fucoidan depend on its molecular weight. 20-30 kDa fucoidan has been reported to stimulate angiogenesis in ischemic limbs. We purified very high molecular weight fucoidan (HMWF) from mozuku (brown algae of the Okinawan coontail family) to assess its effect on angiogenesis. Methods and Results：We examined the angiogenic effects of mozuku HMWF (300 kDa) and akamoku (Sargassum seaweed) HMWF (80 kDa) in a mouse ischemic limb model by measuring laser Doppler blood flow (LDBF) and capillary density. We also studied the angiogenic actions of mozuku HMWF administered pre- and post-ischemia as compared to post-ischemia treatment. Mozuku HMWF increased both LDBF and capillary density in the ischemic leg, whereas akamoku HMWF did not. Treatment with mozuku HMWF pre- and post-ischemia increased both LDBF and capillary density, which was not seen in post-ischemia treatment alone. Conclusions：This study demonstrated the therapeutic effect of pre- and post-ischemia treatment with mozuku HMWF in ischemic limbs, and the timing of administration is important for its angiogenic activity.

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.

Cell-based therapy using adipose-derived stem cells (ADSCs) has emerged as a novel therapeutic approach to treat heart failure after myocardial infarction (MI). The purpose of this study was to determine whether inhibition of α1-adrenergic receptors (α1-ARs) in ADSCs attenuates ADSC sheet-induced improvements in cardiac functions and inhibition of remodeling after MI. ADSCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ADSCs, we determined the mRNA levels of vascular endothelial growth factor (VEGF)-A and α1-AR under normoxia or hypoxia and the effects of norepinephrine and an α1-blocker, doxazosin, on the mRNA levels of angiogenic factors. Hypoxia increased α1-AR and VEGF mRNA levels in ADSCs. Norepinephrine further increased VEGF mRNA expression under hypoxia; this effect was abolished by doxazosin. Tube formation of human umbilical vein endothelial cells was promoted by conditioned media of ADSCs treated with the α1 stimulant phenylephrine under hypoxia but not by those of ADSCs pretreated with phenylephrine plus doxazosin. In in vivo studies using rats with MI, transplanted ADSC sheets improved cardiac functions, facilitated neovascularization, and suppressed fibrosis after MI. These effects were abolished by doxazosin treatment. Pathway analysis from RNA sequencing data predicted significant upregulation of α1-AR mRNA expression in transplanted ADSC sheets and the involvement of α1-ARs in angiogenesis through VEGF. In conclusion, doxazosin abolished the beneficial effects of ADSC sheets on rat MI hearts as well as the enhancing effect of norepinephrine on VEGF expression in ADSCs, indicating that ADSC sheets promote angiogenesis and prevent cardiac dysfunction and remodeling after MI via their α1-ARs.

Background: Monocarboxylate transporter 9 (MCT9), an orphan transporter member of the solute carrier family 16 (SLC16), possibly reabsorbs uric acid in the renal tubule and has been suggested by genome-wide association studies to be involved in the development of hyperuricemia and gout. In this study we investigated the mechanisms regulating the expression of human (h) MCT9, its degradation, and physiological functions. Methods and Results: hMCT9-FLAG was stably expressed in HEK293 cells and its degradation, intracellular localization, and urate uptake activities were assessed by pulse-chase analysis, immunofluorescence, and [14C]-urate uptake experiments, respectively. hMCT9-FLAG was localized on the plasma membrane as well as in the endoplasmic reticulum and Golgi apparatus. The proteasome inhibitors MG132 and lactacystine increased levels of hMCT9-FLAG protein expression with enhanced ubiquitination, prolonged their half-life, and decreased [14C]-urate uptake. [14C]-urate uptake was increased by both heat shock (HS) and the HS protein inducer geranylgeranylacetone (GGA). Both HS and GGA restored the [14C]-urate uptake impaired by MG132. Conclusions: hMCT9 does transport urate and is degraded by a proteasome, inhibition of which reduces hMCT9 expression on the cell membrane and urate uptake. HS enhanced urate uptake through hMCT9.

Myocardial ischemia/reperfusion injury worsens in the absence of nitric oxide synthase (NOS). Cilnidipine, a Ca2+ channel blocker, has been reported to activate endothelial NOS (eNOS) and increases nitric oxide (NO) in vascular endothelial cells. We examined whether pretreatment with cilnidipine could attenuate cardiac cell deaths including apoptosis caused by hypoxia/reoxygenation (H/R) injury. HL-1 mouse atrial myocytes as well as H9c2 rat ventricular cells were exposed to H/R, and cell viability was evaluated by an autoanalyzer and flow cytometry; eNOS expression, NO production, and electrophysiological properties were also evaluated by western blotting, colorimetry, and patch clamping, respectively, in the absence and presence of cilnidipine. Cilnidipine enhanced phosphorylation of eNOS and NO production in a concentration-dependent manner, which was abolished by siRNAs against eNOS or an Hsp90 inhibitor, geldanamycin. Pretreatment with cilnidipine attenuated cell deaths including apoptosis during H/R; this effect was reproduced by an NO donor and a xanthine oxidase inhibitor. The NOS inhibitor L-NAME abolished the protective action of cilnidipine. Pretreatment with cilnidipine also attenuated H9c2 cell death during H/R. Additional cilnidipine treatment during H/R did not significantly enhance its protective action. There was no significant difference in the protective effect of cilnidipine under normal and high Ca2+ conditions. Action potential duration (APD) of HL-1 cells was shortened by cilnidipine, with this shortening augmented after H/R. L-NAME attenuated the APD shortening caused by cilnidipine. These findings indicate that cilnidipine enhances NO production, shortens APD in part by L-type Ca2+ channel block, and thereby prevents HL-1 cell deaths during H/R.

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 beta-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.

Introduction: Human induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called "iCell cardiomyocytes" under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents. Methods: iCell cardiomyocytes were cultured for 8-14 days (38-44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (I-Na) and L-type Ca2+ channel current (I-CaL) were evaluated by the whole-cell patch clamp. Results: iCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of I-Na, I-CaL and rapidly-activating delayed-rectifier K+ channel current were -62.7 +/- 13.7, -8.1 +/- 0.7, and 3.0 +/- 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K + channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked I-Na in a concentration-dependent manner with IC50 of 0.87 +/- 0.37 and 0.88 +/- 0.16 mu M, respectively; the class lb agent mexiletine revealed weak I-Na block with a higher IC50 of 30.0 +/- 3.0 mu M. Pirmenol, pilsicainide and mexiletine blocked I-CaL with IC50 of 2.00 +/- 0.39, 7.7 +/- 2.5 and 5.0 +/- 0.1 mu M, respectively. Conclusions: In iCell cardiomyocytes, I-Na was blocked by the class Ia and Ic antiarrhythmic agents and I-CaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity. (C) 2019, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration.

Background: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs).Purpose and method: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays.Results: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7mg/dl for 24h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect.Conclusion: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

BACKGROUND:Pilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr ) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. METHODS:HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments. RESULTS:Acute exposure to pilsicainide at 0.03-10 μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03-10 μM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W. CONCLUSIONS:Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

BACKGROUND:Besides its antiarrhythmic action, carvedilol has an activity to suppress cardiac tissue damage. However, it is unknown whether it has any effect on cellular apoptosis and ion channel remodelling.PURPOSE:To know whether carvedilol has any effect on apoptosis and ion channel remodeling of HL-1 cells expressing E334K MyBPC, and comparing it with bisoprolol.METHOD:We examined effects of carvedilol and bisoprolol on the levels of pro- and anti-apoptotic proteins and ion channels as well as apoptosis of HL-1 cells transfected with E334K MyBPC using Western blot and flow cytometry.RESULTS:Carvedilol decreased the protein levels of p53, Bax and cytochrome c and increased that of Bcl-2 in HL-1 cells expressing E334K MyBPC. Bisoprolol failed to affect the protein levels. Both carvedilol and bisoprolol increased the protein levels of Cav1.2 but not that of Nav1.5. Carvedilol was stronger than bisoprolol at decreasing the number of annexin-V positive cells in HL-1 cells expressing E334K MyBPC.CONCLUSION:Carvedilol suppressed apoptosis of HL-1 cells expressing E334K MyBPC through modification of pro- and anti-apoptotic proteins, whose was associated with an increase of Cav 1.2 protein expression.

Olprinone is an inotropic agent that inhibits phosphodiesterase (PDE) III and causes vasodilation. Olprinone has been shown to be less proarrhythmic and possibly affect expression of functional Kv1.5 channels that confer the ultra-rapid delayed-rectifier K+ channel current (IKur) responsible for action potential repolarization. To reveal involvement of Kv1.5 channels in the less arrhythmic effect of olprinone, we examined effects of the agent on the stability of Kv1.5 channel proteins expressed in COS7 cells. Olprinone at 30–1000nM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that olprinone delayed degradation of Kv1.5 channels. Olprinone increased the immunofluorescent signal of Kv1.5 channels in the endoplasmic reticulum (ER) and Golgi apparatus as well as on the cell surface. Kv1.5-mediated membrane currents, measured as 4-aminopyridine-sensitive currents, were increased by olprinone without changes in their activation kinetics. A protein transporter inhibitor, colchicine, abolished the olprinone-induced increase of Kv.1.5-mediated currents. The action of olprinone was inhibited by 4-aminopyridine, and was not mimicked by the application of 8-Bromo-cAMP. Taken together, we conclude that olprinone stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, and thereby increases the density of Kv1.5 channels on the cell membrane. The enhancement of Kv1.5 currents could underlie less arrhythmogenicity of olprinone.

Background A KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed‐rectifier K + channel current (I Ks ). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them. Methods KCNE1‐D85N or the wild‐type protein was coexpressed in COS7 cells with KCNQ1 to form K + channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch‐clamp techniques. Results The protein level of KCNE1‐D85N was lower than that of the wild‐type, in spite of the comparable levels of their mRNA. KCNE1‐D85N was highly ubiquitinated and rapidly degraded as compared to the wild‐type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady‐state level. Both KCNE1‐D85N and the wild‐type proteins were co‐immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1‐D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch‐clamp experiments demonstrated that the membrane current corresponding to I Ks was much smaller in cells expressing KCNE1‐D85N than in those expressing the wild‐type. Verapamil (0.5–10 μM) increased the protein level of KCNE1‐D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1‐D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil. Conclusion KCNE1‐D85N is less stable than the wild‐type protein, and is rapidly degraded through the ubiquitin‐proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1‐D85N.