Mitochondrial Responses to Sublethal Doxorubicin in H9c2 Cardiomyocytes: The Role of Phosphorylated CaMKII.

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.

Inhibition of the uric acid efflux transporter ABCG2 enhances stimulating effect of soluble uric acid on IL-1ß production in murine macrophage-like J774.1 cells.

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1ß secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1ß production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1ß production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1ß, mature IL-1ß, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1ß, mature IL-1ß and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1ß and mature IL-1ß in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1ß production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1ß production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1ß transcription in macrophage-like J774.1 cells.

Epstein-Barr virus reactivation in peripheral B lymphocytes induces IgM-type thyrotropin receptor autoantibody production in patients with Graves' disease.

Epstein-Barr virus (EBV) is a human herpes virus that latently infects B lymphocytes. When EBV is reactivated, host B cells differentiate into plasma cells and produce IgM-dominant antibodies as well as many progeny virions. The aims of the present study were to confirm the IgM dominance of thyrotropin-receptor antibodies (TRAbs) produced by EBV reactivation and investigate the roles of TRAb-IgM in Graves' disease. Peripheral blood mononuclear cells (PBMCs) containing TRAb-producing cells were stimulated for EBV reactivation, and TRAb-IgM and TRAb-IgG were measured by ELISA. TRAb-IgM were purified and TSH-binding inhibitory activities were assessed using a radio-receptor assay. Porcine thyroid follicular epithelial cells were cultured with TRAb-IgM and/or complements to measure the intracellular levels of cAMP and the amount of LDH released. TRAb-IgM/TRAb-IgG (the MG ratio) was examined in sequential serum samples of Graves' disease and compared among groups of thyroid function. The results obtained showed that IgM-dominant TRAb production was induced by EBV reactivation. TRAb-IgM did not inhibit TSH binding to TSH receptors and did not transduce hormone-producing signals. However, it destroyed thyroid follicular epithelial cells with complements. The MG ratio was significantly higher in samples of hyperthyroidism or hypothyroidism than in those with normal function or in healthy controls. A close relationship was observed between TRAb-IgM produced by EBV reactivation and the development and exacerbation of Graves' disease. The present results provide novel insights for the development of prophylaxis and therapeutics for Graves' disease.

α1-Adrenergic receptor mediates adipose-derived stem cell sheet-induced protection against chronic heart failure after myocardial infarction in rats.

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.

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.

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.

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.

Hyperuricemia as a Risk Factor for Atrial Fibrillation Due to Soluble and Crystalized Uric Acid.

Among the several independent risk factors for atrial fibrillation (AF), hyperuricemia has been widely accepted as associated with the incidence of paroxysmal or persistent AF, as well as with the risk of AF in patients undergoing cardiovascular surgery. The electrophysiological mechanism of AF involves electrical remodeling of the arrhythmogenic substrate and abnormal automaticity as trigger. Both electrical and structural remodeling mediated by oxidative stress derived from either xanthine oxidoreductase (XOR), soluble uric acid (UA) or monosodium urate (MSU) crystals might be plausible explanations for the association of AF with hyperuricemia. XOR generates reactive oxygen species (ROS) that lead to atrial structural remodeling via inflammation. Soluble UA accumulates intracellularly through UA transporters (UAT), shortening the atrial action potential via enhanced expression and activity of Kv1.5 channel proteins. Intracellular accumulation of soluble UA generates ROS in atrial myocytes via nicotinamide adenine dinucleotide phosphate oxidase, which phosphorylates ERK/Akt and heat shock factor 1 (HSF1), thereby increasing transcription and translation of Hsp70, which stabilizes Kv1.5. In macrophages, MSU activates the NLRP3 inflammasome and proteolytic processing mediated by caspase-1 with enhanced interleukin (IL)-1ß and IL-18 secretion. Use of an XOR inhibitor, antioxidants, a UAT inhibitor such as a uricosuric agent, and an NLRP3 inflammasome inhibitor, might become a potential strategy to reduce the risk of hyperuricemia-induced AF, and control serum UA level.

Pretreatment with an angiotensin II receptor blocker abolished ameliorating actions of adipose-derived stem cell sheets on cardiac dysfunction and remodeling after myocardial infarction.

INTRODUCTION: Cell sheets using myoblasts have been developed for the treatment of heart failure after myocardial infarction (MI) bridging to heart transplantation. Stem cells are supposed to be better than myoblasts as a source of cells, since they possess a potential to proliferate and differentiate into cardiomyocytes, and also have capacity to secrete angiogenic factors. Adipose-derived stem cells (ASCs) obtained from fat tissues are expected to be a new cell source for ASC sheet therapies. Administration of angiotensin II receptor blockers (ARBs) is a standard therapy for heart failure after MI. However, it is not known whether ARBs affect the cell sheet therapy. This study aimed to examine ameliorating effects of ASC sheets on heart failure and remodeling after MI, and how pretreatment with ARBs prior to the creation of MI and ASC sheet transplantation modifies the effects of ASC sheets. METHODS: ASCs were isolated from fat tissues of wild-type rats, and ASC sheets were engineered on temperature-responsive dishes. In in vitro studies using cultured cells, mRNA levels of vascular endothelial growth factor (VEGF) in ASCs were determined by RT-PCR in the presence of angiotensin II and/or an ARB, irbesartan, under normoxia and hypoxia; mRNA and protein levels of angiotensin II receptor type 1a (AT1aR), type 1b (AT1bR) and type 2 (AT2R) were also determined by RT-PCR and western blotting. In in vivo studies using a rat MI model, effects of transplanted ASC sheets and/or irbesartan on cardiac functions and remodeling after MI were evaluated by echocardiography, histological analysis and molecular biological techniques. RESULTS: In the in vitro studies, ASCs expressed higher levels of VEGF mRNA under hypoxia. They also expressed mRNA and protein of AT1aR but not AT1bR or AT2R. Under normoxia, angiotensin II increased the level of VEGF mRNA in ASCs, which was abolished by irbesartan. Under hypoxia, irbesartan reduced the level of VEGF mRNA in ASCs regardless of whether angiotensin II was present or not. In the in vivo studies, ASC sheets improved cardiac functions after MI, leading to decreased interstitial fibrosis and increased capillary density in border zones. These effects of ASC sheets were abolished by oral administration of irbesartan before MI and their transplantation. CONCLUSIONS: ASC sheets ameliorated cardiac dysfunctions and remodeling after MI via increasing VEGF expression, which was abolished by pretreatment with irbesartan before the creation of MI and transplantation.

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.

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

AIMS: We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. METHODS AND RESULTS: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. CONCLUSION: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

Novel effects of extracts from poisonous mushrooms on expression and function of the human ether-a-go-go-related gene channel.

UNLABELLED: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current I(Kr), which plays a pivotal role in cardiac action potential repolarization. Inherited mutations of this gene cause Long QT syndrome type 2. hERG expression is altered by several types of drugs as well as by temperature. Heat shock protein 70 (Hsp70) and Heat shock cognate protein 70 (Hsc70) have reciprocal effects on hERG proteins. We examined the effects of poisonous mushrooms on hERG protein expression and its channel function. METHODS: We evaluated the effects of several types of poisonous mushrooms on the expression and function of wild-type hERG by Western blotting, reverse transcription polymerase chain reaction (PCR), and patch clamping in transfected HEK293 cells and mouse HL-1 cardiomyocytes. RESULTS: Extracts of Gymnopilus junonius (junonius) increased expression of both hERG and Hsp70 in HEK293 cells with concomitant decrease in Hsc70, whereas extracts of Amanita ibotengutake (ibotengutake) decreased hERG proteins with increase in Hsc70. Knockdown of Hsp70 and Hsc70 by small interfering RNA abolished the effects of the two mushrooms on hERG, respectively. Certain fractions of junonius increased expression of hERG proteins. hERG currents were increased by extracts of junonius, resulting in shortening of action potential duration (APD). In contrast, hERG currents were decreased and APD was prolonged by extracts of ibotengutake. CONCLUSION: junonius enhanced the expression and function of hERG by increasing Hsp70 and decreasing Hsc70. Ibotengutake decreased hERG expression via increase in Hsc70. Constituents of junonius may have the potential for use in treatment of arrhythmia.

Reciprocal control of hERG stability by Hsp70 and Hsc70 with implication for restoration of LQT2 mutant stability.

RATIONALE: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current I(Kr). It is highly expressed in cardiomyocytes and its mutations cause long QT syndrome type 2. Heat shock protein (Hsp)70 is known to promote maturation of hERG. Hsp70 and heat shock cognate (Hsc70) 70 has been suggested to play a similar function. However, Hsc70 has recently been reported to counteract Hsp70. OBJECTIVE: We investigated whether Hsc70 counteracts Hsp70 in the control of wild-type and mutant hERG stability. METHODS AND RESULTS: Coexpression of Hsp70 with hERG in HEK293 cells suppressed hERG ubiquitination and increased the levels of both immature and mature forms of hERG. Immunocytochemistry revealed increased levels of hERG in the endoplasmic reticulum and on the cell surface. Electrophysiological studies showed increased I(Kr). All these effects of Hsp70 were abolished by Hsc70 coexpression. Heat shock treatment of HL-1 mouse cardiomyocytes induced endogenous Hsp70, switched mouse ERG associated with Hsc70 to Hsp70, increased I(Kr), and shortened action potential duration. Channels with disease-causing missense mutations in intracellular domains had a higher binding capacity to Hsc70 than wild-type channels and channels with mutations in the pore region. Knockdown of Hsc70 by small interfering RNA or heat shock prevented degradation of mutant hERG proteins with mutations in intracellular domains. CONCLUSIONS: These results indicate reciprocal control of hERG stability by Hsp70 and Hsc70. Hsc70 is a potential target in the treatment of LQT2 resulting from missense hERG mutations.

Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies.

The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.

Stabilizing effects of eicosapentaenoic acid on Kv1.5 channel protein expressed in mammalian cells.

We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3-10 muM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30-100 muM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.

Pendrin is a novel autoantigen recognized by patients with autoimmune thyroid diseases.

CONTEXT: Pendrin is an apical protein of thyroid follicular cells, responsible for the efflux of iodide into the follicular lumen via an iodide-chloride transport mechanism. It is unknown whether pendrin is recognized by autoantibodies. OBJECTIVE: Our objective was to examine the prevalence of pendrin antibodies in autoimmune thyroid diseases and compare with that of thyroglobulin, thyroperoxidase, TSH receptor, and sodium iodide symporter antibodies. DESIGN: In a prevalent case-control study, we analyzed the sera of 140 autoimmune thyroid disease cases (100 with Graves' disease and 40 with Hashimoto's thyroiditis) and 80 controls (50 healthy subjects, 10 patients with papillary thyroid cancer, 10 with systemic lupus erythematosus, and 10 with rheumatoid arthritis). Pendrin antibodies were measured by immunoblotting using extract of COS-7 cells transfected with pendrin and a rabbit polyclonal pendrin antibody. RESULTS: Pendrin antibodies were found in 81% of the cases and 9% of controls (odds ratio = 44; P < 0.0001). Among cases, pendrin antibodies were more frequent and of higher titers in Hashimoto's thyroiditis than in Graves' disease. Pendrin antibodies correlated significantly with thyroglobulin, thyroperoxidase, and sodium iodide symporter antibodies but not with TSH receptor antibodies. Pendrin antibodies were equally effective as thyroglobulin and thyroperoxidase antibodies in diagnosis of autoimmune thyroid diseases, especially Hashimoto's thyroiditis. CONCLUSIONS: The study identifies pendrin as a novel autoantigen recognized by patients with autoimmune thyroid diseases and proposes the use of pendrin antibodies as an accurate diagnostic tool.

Lentiviral vectors bearing the cardiac promoter of the Na+-Ca2+ exchanger report cardiogenic differentiation in stem cells.

Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have acquired a cardiac phenotype. Lentiviral vectors achieved significantly higher transduction efficiencies in CDCs than any of the nine adeno-associated viral (AAV) serotypes tested. To define the most suitable vector system for reporting cardiogenic differentiation, we compared the cell specificity of five commonly-used cardiac-specific promoters in the context of lentiviral vectors. The promoter of the cardiac sodium-calcium exchanger (NCX1) conveyed the highest degree of cardiac specificity, as assessed by transducing seven cell types with each vector and measuring fluorescence intensity by flow cytometry. NCX1-GFP-positive CDC subpopulations, demonstrating prolonged expression of a variety of cardiac markers, could be isolated and expanded in vitro. Finally, we used chemical biology to validate that lentiviral vectors bearing the cardiac NCX1-promoter can serve as a highly accurate biosensor of cardiogenic small molecules in stem cells. The ability to accurately report cardiac fate and selectively enrich for cardiomyocytes and their precursors has important implications for drug discovery and the development of cell-based therapies.

Macroreentrant atrial tachycardia with an isolated pathway mimicking focal activation on three-dimensional electroanatomical mapping.

A 76-year-old man with two different sustained atrial arrhythmias that occurred after coronary artery bypass grafting underwent electrophysiological studies. Macroreentrant atrial tachycardias were detected with an isolated slow pathway mimicking focal activation on three-dimensional electroanatomical mapping. The slow conduction pathway in the right atrial free wall was assumed to represent tissue damaged by right atrial cannulation during previous coronary artery bypass grafting.