Pretreatment with cilnidipine attenuates hypoxia/reoxygenation injury in HL-1 cardiomyocytes through enhanced NO production and action potential shortening.

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.

Evidence for Urate Uptake Through Monocarboxylate Transporter 9 Expressed in Mammalian Cells and Its Enhancement by Heat Shock.

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.

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.

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).

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.

BACKGROUND: The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). METHODS: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. RESULTS: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. CONCLUSIONS: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.

A case of variant biochemical phenotype of Niemann-Pick disease type C accompanying savant syndrome.

A 40-year-old man was referred to our hospital because of vertical supranuclear gaze palsy, frequent sudden loss of muscle tonus and ataxia for several years. He had a history of prolonged neonatal jaundice. He was given a diagnosis of autism in his childhood, followed by a diagnosis of schizophrenia in his teenage. He also developed a savant skill of calendar calculating. (123)I-IMP-SPECT showed decreased cerebral blood flow in the left frontotemporal lobe as often seen in savant syndrome. Although genetic analysis of NPC1 and NPC2 revealed no pathogenic mutation, filipin staining of cultured fibroblasts from his biopsied skin revealed a certain amount of intracellular cholesterol storage pattern, indicating a variant biochemical phenotype of Niemann-Pick disease type C (NPC). The diagnosis of adulthood onset NPC is difficult and challenging, especially for neurologists, because the symptoms and signs are not as clear as those in the classical childhood onset NPC and this subtype is not yet widely known. However, the diagnosis can be made by a combination of filipin staining of fibroblast and/or gene analysis. As a disease-specific therapy for NPC has been approved in Japan, the diagnosis of NPC is of significance.

Endoplasmic reticulum-associated degradation of Niemann-Pick C1: evidence for the role of heat shock proteins and identification of lysine residues that accept ubiquitin.

Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.

Molecular basis of 1-deoxygalactonojirimycin arylthiourea binding to human α-galactosidase a: pharmacological chaperoning efficacy on Fabry disease mutants.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene often leading to missense α-galactosidase A (α-Gal A) variants that undergo premature endoplasmic reticulum-associated degradation due to folding defects. We have synthesized and characterized a new family of neutral amphiphilic pharmacological chaperones, namely 1-deoxygalactonojirimycin-arylthioureas (DGJ-ArTs), capable of stabilizing α-Gal A and restoring trafficking. Binding to the enzyme is reinforced by a strong hydrogen bond involving the aryl-N'H thiourea proton and the catalytic aspartic acid acid D231 of α-Gal A, as confirmed by a 2.55 Å resolution cocrystal structure. Selected candidates enhanced α-Gal A activity and ameliorate globotriaosylceramide (Gb3) accumulation and autophagy impairments in FD cell cultures. Moreover, they acted synergistically with the proteostasis regulator 4-phenylbutyric acid, appearing to be promising leads as pharmacological chaperones for FD.

Analysis of Lujo virus cell entry using pseudotype vesicular stomatitis virus.

UNLABELLED: Several arenaviruses are known to cause viral hemorrhagic fever (VHF) in sub-Saharan Africa and South America, where VHF is a major public health and medical concern. The biosafety level 4 categorization of these arenaviruses restricts their use and has impeded biological studies, including therapeutic drug and/or vaccine development. Due to difficulties associated with handling live viruses, pseudotype viruses, which transiently bear arenavirus envelope proteins based on vesicular stomatitis virus (VSV) or retrovirus, have been developed as surrogate virus systems. Here, we report the development of a pseudotype VSV bearing each envelope protein of various species of arenaviruses (AREpv), including the newly identified Lujo virus (LUJV) and Chapare virus. Pseudotype arenaviruses generated in 293T cells exhibited high infectivity in various mammalian cell lines. The infections by New World and Old World AREpv were dependent on their receptors (human transferrin receptor 1 [hTfR1] and α-dystroglycan [αDG], respectively). However, infection by pseudotype VSV bearing the LUJV envelope protein (LUJpv) occurred independently of hTfR1 and αDG, indicating that LUJpv utilizes an unidentified receptor. The pH-dependent endocytosis of AREpv was confirmed by the use of lysosomotropic agents. The fusion of cells expressing these envelope proteins, except for those expressing the LUJV envelope protein, was induced by transient treatment at low pH values. LUJpv infectivity was inhibited by U18666A, a cholesterol transport inhibitor. Furthermore, the infectivity of LUJpv was significantly decreased in the Niemann-Pick C1 (NPC1)-deficient cell line, suggesting the necessity for NPC1 activity for efficient LUJpv infection. IMPORTANCE: LUJV is a newly identified arenavirus associated with a VHF outbreak in southern Africa. Although cell entry for many arenaviruses has been studied, cell entry for LUJV has not been characterized. In this study, we found that LUJpv utilizes neither αDG nor hTfR1 as a receptor and found unique characteristics of LUJV glycoprotein in membrane fusion and cell entry. Proper exclusion of cholesterol or some kinds of lipids may play important roles in LUJpv cell entry.