Systemic Deletion of ARRDC4 Improves Cardiac Reserve and Exercise Capacity in Diabetes.

BACKGROUND: Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism. OBJECTIVE: To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac and skeletal myopathy. METHODS AND RESULTS: High glucose promoted translocation of MondoA into the nucleus, which upregulated Arrdc4 transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of ARRDC4 was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial Arrdc4 expression in multiple types of mouse models of diabetes. We then analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. However, deletion of Arrdc4 augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that Arrdc4-knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type (WT) animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression (beyond levels observed during diabetes) using adenoassociated virus suggests that high levels of myocardial ARRDC4 have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the ARRDC4 mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1. CONCLUSIONS: ARRDC4 serves as a regulator of hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.

Interaction of ARRDC4 With GLUT1 Mediates Metabolic Stress in the Ischemic Heart.

BACKGROUND: An ancient family of arrestin-fold proteins, termed alpha-arrestins, may have conserved roles in regulating nutrient transporter trafficking and cellular metabolism as adaptor proteins. One alpha-arrestin, TXNIP (thioredoxin-interacting protein), is known to regulate myocardial glucose uptake. However, the in vivo role of the related alpha-arrestin, ARRDC4 (arrestin domain-containing protein 4), is unknown. METHODS: We first tested whether interaction with GLUTs (glucose transporters) is a conserved function of the mammalian alpha-arrestins. To define the in vivo function of ARRDC4, we generated and characterized a novel Arrdc4 knockout (KO) mouse model. We then analyzed the molecular interaction between arrestin domains and the basal GLUT1. RESULTS: ARRDC4 binds to GLUT1, induces its endocytosis, and blocks cellular glucose uptake in cardiomyocytes. Despite the closely shared protein structure, ARRDC4 and its homologue TXNIP operate by distinct molecular pathways. Unlike TXNIP, ARRDC4 does not increase oxidative stress. Instead, ARRDC4 uniquely mediates cardiomyocyte death through its effects on glucose deprivation and endoplasmic reticulum stress. At baseline, Arrdc4-KO mice have mild fasting hypoglycemia. Arrdc4-KO hearts exhibit a robust increase in myocardial glucose uptake and glycogen storage. Accordingly, deletion of Arrdc4 improves energy homeostasis during ischemia and protects cardiomyocytes against myocardial infarction. Furthermore, structure-function analyses of the interaction of ARRDC4 with GLUT1 using both scanning mutagenesis and deep-learning Artificial Intelligence identify specific residues in the C-terminal arrestin-fold domain as the interaction interface that regulates GLUT1 function, revealing a new molecular target for potential therapeutic intervention against myocardial ischemia. CONCLUSIONS: These results uncover a new mechanism of ischemic injury in which ARRDC4 drives glucose deprivation-induced endoplasmic reticulum stress leading to cardiomyocyte death. Our findings establish ARRDC4 as a new scaffold protein for GLUT1 that regulates cardiac metabolism in response to ischemia and provide insight into the therapeutic strategy for ischemic heart disease.

Phase transitions and dynamics in ionic liquid crystals confined in nanopores.

We investigate the phase-transition behavior of ionic liquid crystals, namely 1-methyl-3-alkylimidazolium tetrafluoroborate, [Cnmim]BF4, confined in cylindrical nanopores using differential scanning calorimetry, x-ray scattering, and dielectric relaxation spectroscopy. Here, n is the number of carbon atoms in the alkyl part of this ionic liquid crystal. For n = 10 and 12, the isotropic liquid phase changes to the smectic phase and then to a metastable phase for the cooling process. During the subsequent heating process, the metastable phase changes to the isotropic phase via crystalline phases. The transition temperatures for this ionic liquid crystal confined in nanopores decrease linearly with the increase in the inverse pore diameter, except for the transitions between the smectic and isotropic phases. In the metastable phase, the relaxation rate of the α-process shows the Vogel-Fulcher-Tammann type of temperature dependence for some temperature ranges. The glass transition temperature evaluated from the dynamics of the α-process decreases with the decrease in the pore diameter and increases with the increase in the carbon number n. The effect of confinement on the chain dynamics can clearly be observed for this ionic liquid crystal. For n = 10, the melting temperature of the crystalline phase is slightly higher than that of the smectic phase for the bulk, while, in the nanopores, the melting temperature of the smectic phase is higher than that of the crystalline phase. This suggests that the smectic phase can be thermodynamically stable, thanks to the confinement effect.

Txnip C247S mutation protects the heart against acute myocardial infarction.

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

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

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

Clinical and microbiological effect of pulsed xenon ultraviolet disinfection to reduce multidrug-resistant organisms in the intensive care unit in a Japanese hospital: a before-after study.

BACKGROUND: No-touch environmental disinfection using ultraviolet devices has been highlighted in the past several years to control the transmission of multidrug-resistant organisms (MDROs). However, its effectiveness in non-US healthcare settings is yet to be examined. This study aimed to evaluate the effectiveness of disinfection by portable pulsed xenon ultraviolet (PX-UV) devices in controlling transmission of MDROs in a non-US healthcare setting. METHODS: All patients admitted in the intensive care unit in a 629-bed tertiary referral hospital in Japan from August 2016 to February 2019 were enrolled. During the study period, PX-UV disinfection was added to manual terminal cleaning after every patient transfer/discharge. For microbiological evaluation, surfaces were selected for sampling by contact plates before/after manual cleaning and after PX-UV. After overnight incubation, colonies on the plates were counted. RESULTS: The incidence of newly acquired methicillin-resistant Staphylococcus aureus (MRSA) declined significantly (13.8 to 9.9 per 10,000 patient days, incidence rate ratio 0.71, p = 0.002), as well as that of newly acquired drug-resistant Acinetobacter (48.5 to 18.1, 0.37, p < 0.001). The percent reduction of the microbiological burden by manual cleaning was 81%, but a further 59% reduction was achieved by PX-UV. CONCLUSIONS: PX-UV is effective in further reducing the microbial burden and controlling MDROs in a non-US healthcare setting.

Spherical-cap droplets of a photo-responsive bent liquid crystal dimer.

Using a photo-responsive dimer exhibiting the transition between nematic (N) and twist-bend nematic (NTB) phases, we prepared spherical cap-shaped droplets on solid substrates exposed to air. The internal director structures of these droplets vary depending on the phase and on the imposed boundary conditions. The structural switching between the N and NTB phases was successfully performed either by temperature control or by UV light-irradiation. The N phase is characterized by an extremely small bend elastic constant K3, and surprisingly, we found that the droplet-air interface induces a planar alignment, in contrast to that seen for typical calamitic liquid crystals. As a consequence, the director configuration was stabilized in a structure substantially different from that normally found in conventional nematic liquid crystalline droplets. In the twist-bend nematic droplets characteristic structures with macroscopic length scales were formed, and they were well controlled by the droplet size. These results indicated that a continuum theory is effective in describing the stabilization mechanism of the macroscopic structure even in the twist-bend nematic liquid crystal droplets exhibiting director modulations on a scale of several molecular lengths.

Direct observation of mobility of thin polymer layers via asymmetric interdiffusion using neutron reflectivity measurements.

In this study, we investigated the diffusion dynamics at the interface between deuterated poly(methyl methacrylate) (d-PMMA) and protonated poly(methyl methacrylate) (h-PMMA) in two-layered thin films of d- and h-PMMA layers via neutron reflectivity (NR) measurements during isothermal annealing above the glass transition temperature Tg. When Tg of d-PMMA was higher than that of h-PMMA, the d-PMMA layer thickness increased with increasing annealing time ta and, simultaneously, the h-PMMA layer thickness decreased. However, the opposite ta dependence of the layer thicknesses was observed, if the Tg of d-PMMA was decreased by the increase in the fraction of the low-molecular weight d-PMMA: With increasing ta, the d-PMMA layer thickness decreased and the h-PMMA layer thickness increased when Tg of d-PMMA was lower than that of h-PMMA. This change in the ta dependence of the layer thickness was related to the change in the mobility of the d-PMMA layer accompanied by the change in the Tg value of d-PMMA. With the decrease in the d-PMMA layer thickness from 49 nm to 13 nm, when the h-PMMA layer thickness was maintained, the ta dependence of the layer thickness changed and the mobility of the d-PMMA layer dramatically increased. These results suggest that the mobility of thin polymer films can be determined by the observation of interfacial dynamics via NR measurements.

Deletion of thioredoxin-interacting protein improves cardiac inotropic reserve in the streptozotocin-induced diabetic heart.

Although the precise pathogenesis of diabetic cardiac damage remains unclear, potential mechanisms include increased oxidative stress, autonomic nervous dysfunction, and altered cardiac metabolism. Thioredoxin-interacting protein (Txnip) was initially identified as an inhibitor of the antioxidant thioredoxin but is now recognized as a member of the arrestin superfamily of adaptor proteins that classically regulate G protein-coupled receptor signaling. Here we show that Txnip plays a key role in diabetic cardiomyopathy. High glucose levels induced Txnip expression in rat cardiomyocytes in vitro and in the myocardium of streptozotocin-induced diabetic mice in vivo. While hyperglycemia did not induce cardiac dysfunction at baseline, ß-adrenergic challenge revealed a blunted myocardial inotropic response in diabetic animals (24-wk-old male and female C57BL/6;129Sv mice). Interestingly, diabetic mice with cardiomyocyte-specific deletion of Txnip retained a greater cardiac response to ß-adrenergic stimulation than wild-type mice. This benefit in Txnip-knockout hearts was not related to the level of thioredoxin activity or oxidative stress. Unlike the ß-arrestins, Txnip did not interact with ß-adrenergic receptors to desensitize downstream signaling. However, our proteomic and functional analyses demonstrated that Txnip inhibits glucose transport through direct binding to glucose transporter 1 (GLUT1). An ex vivo analysis of perfused hearts further demonstrated that the enhanced functional reserve afforded by deletion of Txnip was associated with myocardial glucose utilization during ß-adrenergic stimulation. These data provide novel evidence that hyperglycemia-induced Txnip is responsible for impaired cardiac inotropic reserve by direct regulation of insulin-independent glucose uptake through GLUT1 and plays a role in the development of diabetic cardiomyopathy.

Stability of a double twisted structure in spherical cholesteric droplets.

We found for the first time the stabilization of a double twisted structure in cholesteric liquid crystals confined to small spherical droplets under weak anchoring conditions. The direct observation of the droplets using a polarized microscope revealed the physical properties of the structure. The experimental results showed that the stability of the double twisted structure is determined by the relationship between the helical pitch length and the droplet size. We theoretically analyzed the structural stability by the calculation of the Frank elastic free energy including the surface elastic term, and succeeded in explaining the experimental results. In this paper, we concluded that the stability of the double twisted structure is determined by the competition between the surface and the bulk elasticity.

A Twist-Bend Nematic (NTB ) Phase of Chiral Materials.

New chiral dimers consisting of a rod-like and cholesterol mesogenic units are reported to form a chiral twist-bend nematic phase (NTB *) with heliconical structure. The compressibility of the NTB phase made of bent dimers was found to be as large as in smectic phases, which is consistent with the nanoperiodic structure of the NTB phase. The atomic force microscopy observations in chiral bent dimers revealed a periodicity of about 50 nm, which is significantly larger than the one reported previously for non-chiral compounds (ca. 10 nm).

Director/barycentric rotation in cholesteric droplets under temperature gradient.

When a chiral liquid crystal is given a transport current, a unidirectional molecular motion is known to take place, which is called the Lehmann effect. In this paper, we study the mysterious heat-current-driven Lehmann effect using two types of hemispherical cholesteric droplets using polarizing, reflecting, confocal and fluorescent microscopies. Both the droplets, coexisting with the isotropic phase and contacting on a glass substrate, are characterized by the concavo-convex modulated surface and the inside orientational helix. Further, the only difference between them is the helical axis direction; i.e., one is perpendicular and the other is parallel to the substrate. Under the temperature gradient perpendicular to the substrate, the droplet whose helical axis is parallel to the heat current exhibited pure director rotation, while that with the axis perpendicular to the current rotated independently as a rigid body. In the two droplets, the rotational conversion efficiency from the temperature gradient into the angular velocity showed very different dependences on the chirality strength and on the droplets' size, suggesting that the rotations of the two droplets may be driven by independent torques with different origins. This is the first observation that the cholesteric droplets under the temperature gradient exhibit the two rotational modes, the pure director rotation and the molecular barycentric motion, which can be switched to each other by changing the heat-current direction parallel and perpendicular to the helical axis.

Morphogenesis of a chiral liquid crystalline droplet with topological reconnection and Lehmann rotation.

Morphogenesis is a hierarchical phenomenon that produces various macroscopic structures in living organisms, with high reproducibility. This study demonstrates that such structural formation can also be observed in a chiral liquid crystalline droplet under a temperature gradient. Through specific control of the temperature change process, we were able to switch the final structure obtained as a result of the formation via the appearance and reconnection of loop defects in the transient state during structure formation. Simultaneously, the existence of the gradient resulted in a characteristic rotational phenomenon called Lehmann rotation, which was prominently induced in the transient state. By demonstrating three-dimensional measurements of the flow field, we revealed the existence of Marangoni convection in the state. Consequently, it is indicated that the convection results in high-speed Lehmann rotation and large structural deformation with topological changes, thereby playing a significant role in the structure formation.

Marangoni convection driven by temperature gradient near an isotropic-nematic phase transition point.

Marangoni flow driven by a temperature gradient was observed near the isotropic-nematic phase transition point. By applying the gradient to a liquid crystalline material in sandwich cells, it was possible to measure the flow field near the air interface using the photobleaching method. In the isotropic phase, the direction of the observed flow was opposite to that in the nematic phase. Moreover, when the measurement was performed in the coexistence state of these phases, the flow direction depended on the coating materials of the cell substrates. These singular flow properties are explained well by the singular changes in surface tension and the shape of the air interface near the transition point.

Attenuated hypertrophic response to pressure overload in a lamin A/C haploinsufficiency mouse.

Inherited mutations cause approximately 30% of all dilated cardiomyopathy cases, with autosomal dominant mutations in the LMNA gene accounting for more than one third of these. The LMNA gene encodes the nuclear envelope proteins lamins A and C, which provide structural support to the nucleus and also play critical roles in transcriptional regulation. Functional deletion of a single allele is sufficient to trigger dilated cardiomyopathy in humans and mice. However, whereas Lmna(-/-) mice develop severe muscular dystrophy and dilated cardiomyopathy and die by 8 weeks of age, heterozygous Lmna(+/-) mice have a much milder phenotype, with changes in ventricular function and morphology only becoming apparent at 1 year of age. Here, we studied 8- to 20-week-old Lmna(+/-) mice and wild-type littermates in a pressure overload model to examine whether increased mechanical load can accelerate or exacerbate myocardial dysfunction in the heterozygotes. While overall survival was similar between genotypes, Lmna(+/-) animals had a significantly attenuated hypertrophic response to pressure overload as evidenced by reduced ventricular mass and myocyte size. Analysis of pressure overload-induced transcriptional changes suggested that the reduced hypertrophy in the Lmna(+/-) mice was accompanied by impaired activation of the mechanosensitive gene Egr-1. In conclusion, our findings provide further support for a critical role of lamins A and C in regulating the cellular response to mechanical stress in cardiomyocytes and demonstrate that haploinsufficiency of lamins A and C alone is sufficient to alter hypertrophic responses and cardiac function in the face of pressure overload in the heart.

Differential rotation in cholesteric pillars under a temperature gradient.

Steady rotation is induced in cholesteric droplets dispersed in a specific liquid solvent under a temperature gradient. In this phenomenon, two rotational modes have been considered: (1) collective rotation of the local director field and (2) rigid-body rotation of the whole droplet structure. However, here we present another rotational mode induced in a pillar-shaped cholesteric droplet confined between substrates under a temperature gradient, that is, a differential rotation where the angular velocity varies as a function of the radial coordinate in the pillar. A detailed flow field analysis revealed that every pillar under a temperature gradient involves a double convection roll. These results suggested that the differential rotation in the cholesteric pillars was driven by the inhomogeneous material flow induced by a temperature gradient. The present experimental study indicates that the coupling between the flow and the director motion plays a key role in the rotation of the cholesteric droplets under the temperature gradient.

Self-excited oscillation of the director field in cholesteric liquid crystalline droplets under a temperature gradient.

In this study, we demonstrate a self-excited oscillation induced in cholesteric liquid crystalline droplets under a temperature gradient. At equilibrium, a winding Maltese cross pattern with a point defect was observed via polarised microscopy in the droplets dispersed in an isotropic solvent. When the temperature gradient was applied, the pattern was deformed owing to the Marangoni convection induced by the gradient. Here, when both the droplet size and temperature gradient were sufficiently large, the periodic movement of the defect together with the pattern deformation was observed, which demonstrated the self-excited oscillation of the director field. To describe this phenomenon, we theoretically analysed the flow and director fields by using Onsager's variational principle. This principle enabled the simplified description of the phenomenon; consequently, the time evolution of the director field could be expressed by the phenomenological equations for the two parameters characterising the field. These equations represented the van der Pol equation, which well expressed the mechanism of the self-excited oscillation.