Midori Edamatsu

Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway

BACKGROUND Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, resulting in the loss of cardiac function. In the current study we sought to identify if excessive autophagy plays a major role in inducing this progressive loss. METHODS AND RESULTS Immunofluorescence and western blotting analysis of the right atrial appendages collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery showed a marked increase in the level of autophagy in the diabetic heart, as evidenced by increased expression of autophagy marker LC3B-II and its mediator Beclin-1 and decreased expression of p62, which incorporates into autophagosomes to be efficiently degraded. Moreover, a marked activation of pro-apoptotic caspase-3 was observed. Electron microscopy showed increased autophagosomes in the diabetic heart. In vivo measurement of autophagic flux by choloroquine injection resulted in further enhancement of LC3B-II in the diabetic myocardium, confirming increased autophagic activity in the type-2 diabetic heart. Importantly, in-vitro genetic depletion of beclin-1 in high glucose treated adult rat cardiomyocytes markedly inhibited the level of autophagy and subsequent apoptotic cell death. CONCLUSIONS These findings demonstrate the pathological role of autophagy in the type-2 diabetic heart, opening up a potentially novel therapeutic avenue for the treatment of diabetic heart disease.

Cellular localization of facilitated glucose transporter 1 (GLUT-1) in the cochlear stria vascularis: its possible contribution to the transcellular glucose pathway.

Immunoreactivity for the facilitated glucose transporter 1 (GLUT-1) has been found in the cochlear stria vascularis, but whether the strial marginal cells are immunopositive for GLUT-1 remains uncertain. To determine the cellular localization of GLUT-1 and to clarify the glucose pathway in the stria vascularis of rats and guinea pigs, immunohistochemistry was performed on sections, dissociated cells, and whole-tissue preparations. Immunoreactivity for GLUT-1 in sections was observed in the basal side of the strial tissue and in capillaries in both rats and guinea pigs. However, the distribution of the positive signals within the guinea pig strial tissue was more diffuse than that in rats. Immunostaining of dissociated guinea pig strial cells revealed GLUT-1 in the basal cells and capillary endothelial cells, but not in the marginal cells. These results indicated that GLUT-1 was not expressed in the marginal cells, and that another isoform of GLUT was probably expressed in these cells. Three-dimensional observation of whole-tissue preparations demonstrated that cytoplasmic prolongations from basal cells extended upward to the apical surface of the stria vascularis from rats and guinea pigs, and that the marginal cells were surrounded by these protrusions. We speculate that these upward extensions of basal cells have been interpreted as basal infoldings of marginal cells in previous reports from other groups. The three-dimensional relationship between marginal cells and basal cells might contribute to the transcellular glucose pathway from perilymph to intrastrial space.

Multiple expression of glucose transporters in the lateral wall of the cochlear duct studied by quantitative real-time PCR assay.

We have investigated the gene expression of the facilitated glucose transporter (GLUT), H+-coupled myo-inositol cotransporter (HMIT), and Na+ glucose cotransporter (SGLT) in the lateral wall of the cochlear duct by conventional RT-PCR and quantitative real-time PCR. The isoforms GLUT1, -3, -4, -5, -8, -10, -12 and HMIT were detected in both the stria vascularis and the spiral ligament, whereas no SGLT isoforms could be detected in these tissues. Quantitative real-time PCR analysis revealed significant differences in the gene expression of GLUT1, -4, -5, -10, and HMIT isoforms between the stria vascularis and the spiral ligament. This result reflects the tissue-dependent distributions of GLUT isoforms. These findings strongly suggest that a number of GLUT isoforms participate in glucose transport in the stria vascularis and the spiral ligament.

Data supporting the activation of autophagy genes in the diabetic heart

This data article contains full list of autophagy related genes that are altered in diabetic heart. This article also shows data from in vitro cultured cardiomyocytes that are exposed the high glucose treatment to simulate hyperglycemic state in vitro. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in “Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway” (Munasinghe et al., in press) [1].

Progenitor cells from atria, ventricle and peripheral blood of the same patients exhibit functional differences associated with cardiac repair

AIM Deciding the best cell type for cardiac regeneration remains a big challenge. No studies have directly compared the functional efficacy of cardiac progenitor cells (CPCs) with extra-cardiac stem cells isolated from the same patient. METHODS AND RESULTS We compared the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CPCs and left ventricular (LV) CPCs isolated from the same patients (n=14). Within the same heart, RAA and LV CPCs exhibited marked differences in surface marker expression, with RAA CPCs exhibiting better expansion potential and migration properties. When subjected to hypoxia and serum starvation to simulate in vivo ischemic environment, RAA and LV CPCs exhibited similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under hypoxia. RT-profiler array showed comparable gene expression pattern in RAA and LV CPCs, while they were differentially expressed in EPCs. Further, treating human umbilical vein endothelial cells with conditioned medium (CM) from LV showed maximum angiogenic potential, while cardiomyocytes treated with CM from RAA showed greatest survival under hypoxic conditions. CONCLUSIONS Results from this study provide the first evidence that progenitor cells from different regions exhibit functional differences within the same patient.

Differential localizations of the myo-inositol transporters HMIT and SMIT1 in the cochlear stria vascularis

The cochlear stria vascularis produces endolymph and thereby plays an active role in inner ear homeostasis. We recently reported that the H+/myo-inositol cotransporter (HMIT) gene is expressed in the stria vascularis. Here, we examined the protein localization of HMIT and Na+/myo-inositol cotransporter 1 (SMIT1) in the stria vascularis by immunohistochemistry. HMIT and SMIT1 were detected in the lateral wall of the cochlear duct. HMIT was widely detected throughout the stria vascularis, while SMIT1 was enriched in the strial basal cells. To examine the localization of HMIT in the stria vascularis in more detail, dissociated strial cells were immunostained, which resulted in the detection of HMIT immunoreactivity in marginal cells. These results indicate that HMIT is expressed in marginal cells and basal cells of the stria vascularis, while SMIT1 expression is enriched in basal cells. We speculate that HMIT and SMIT1 may play important roles in the homeostasis of cochlear fluids, for example by participating in pH regulation and osmoregulation.

A deficiency of the link protein Bral2 affects the size of the extracellular space in the thalamus of aged mice

Bral2 is a link protein stabilizing the binding between lecticans and hyaluronan in perineuronal nets and axonal coats (ACs) in specific brain regions. Using the real‐time iontophoretic method and diffusion‐weighted magnetic resonance, we determined the extracellular space (ECS) volume fraction (α), tortuosity (λ), and apparent diffusion coefficient of water (ADCW) in the thalamic ventral posteromedial nucleus (VPM) and sensorimotor cortex of young adult (3–6 months) and aged (14–20 months) Bral2‐deficient (Bral2−/−) mice and age‐matched wild‐type (wt) controls. The results were correlated with an analysis of extracellular matrix composition. In the cortex, no changes between wt and Bral2−/− were detected, either in the young or aged mice. In the VPM of aged but not in young Bral2−/− mice, we observed a significant decrease in α and ADCW in comparison with age‐matched controls. Bral2 deficiency led to a reduction of both aggrecan‐ and brevican‐associated perineuronal nets and a complete disruption of brevican‐based ACs in young as well as aged VPM. Our data suggest that aging is a critical point that reveals the effect of Bral2 deficiency on VPM diffusion. This effect is probably mediated through the enhanced age‐related damage of neurons lacking protective ACs, or the exhausting of compensatory mechanisms maintaining unchanged diffusion parameters in young Bral2−/− animals. A decreased ECS volume in aged Bral2−/− mice may influence the diffusion of neuroactive substances, and thus extrasynaptic and also indirectly synaptic transmission in this important nucleus of the somatosensory pathway.

Hapln4/Bral2 is a selective regulator for formation and transmission of GABAergic synapses between Purkinje and deep cerebellar nuclei neurons

Purkinje cells (PCs) convey the sole output of the cerebellar cortex to the deep cerebellar nuclei (DCN). DCN neurons are enwrapped in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast‐spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the DCN and medial nucleus of the trapezoid body, which are the post‐synaptic neurons of large axo‐somatic synapses adapted for fast signaling. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast‐signaling synapses. To elucidate the role of the PNN at these synapses, we investigated the electrophysiological and morphological properties of DCN synapses in hyaluronan and proteoglycan binding link protein 4 (Hapln4/Bral2) knockout (KO) mice around postnatal day (P)14. Hapln4/Bral2 is important for PNN structure, as it stabilizes the interaction between hyaluronan and proteoglycan. Here, using immunohistochemistry we show that Hapln4/Bral2 localized closely with GABAergic terminals. In DCN neurons of Hapln4/Bral2 KO mice, inhibitory synaptic strengths were reduced as compared to those in wild‐type mice, whereas the properties of excitatory synapses were unaffected. The reduced IPSC amplitudes were mainly because of reduced numbers of releasable vesicles. Moreover, Hapln4/Bral2 deficiency reduced the number of PC GABAergic terminals in the DCN. These results demonstrate that Hapln4/Bral2 is a PNN component that selectively contributes to formation and transmission of PC‐DCN synapses in the cerebellum.