The results demonstrate the critical role of the N-terminal domain of Nav1.5 channels in Nav1.5-Kir2.x-reciprocal interactions and suggest that the molecular mechanisms controlling atrial and ventricular cellular excitability may be different.

Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and suggested that this transcription factor could contribute to the CAF-induced IKs increase and ICa,L reduction observed in humans.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role.

Background— Atrial fibrillation is characterized by progressive atrial structural and electrical changes (atrial remodeling) that favor arrhythmia recurrence and maintenance. Reduction of L-type Ca 2+ current ( I Ca,L ) density is a hallmark of the electrical remodeling. Alterations in atrial microRNAs could contribute to the protein changes underlying atrial fibrillation–induced atrial electrical remodeling. This study was undertaken to compare miR-21 levels in isolated myocytes from atrial appendages obtained from patients in sinus rhythm and with chronic atrial fibrillation (CAF) and to determine whether L-type Ca 2+ channel subunits are targets for miR-21. Methods and Results— Quantitative polymerase chain reaction analysis showed that miR-21 was expressed in human atrial myocytes from patients in sinus rhythm and that its expression was significantly greater in CAF myocytes. There was an inverse correlation between miR-21 and the mRNA of the α1c subunit of the calcium channel (CACNA1C) expression and I Ca,L density. Computational analyses predicted that CACNA1C and the mRNA of the β2 subunit of the calcium channel (CACNB2) could be potential targets for miR-21. Luciferase reporter assays demonstrated that miR-21 produced a concentration-dependent decrease in the luciferase activity in Chinese Hamster Ovary cells transfected with CACNA1C and CACNB2 3′ untranslated region regions. miR-21 transfection in HL-1 cells produced changes in I Ca,L properties qualitatively similar to those produced by CAF (ie, a marked reduction of I Ca,L density and shift of the inactivation curves to more depolarized potentials). Conclusions— Our results demonstrated that CAF increases miR-21 expression in enzymatically isolated human atrial myocytes. Moreover, it decreases I Ca,L density by downregulating Ca 2+ channel subunits expression. These results suggested that this microRNA could participate in the CAF-induced I Ca,L downregulation and in the action potential duration shortening that maintains the arrhythmia.

The present results demonstrate that CAF increases the effects of β1-Adrenoceptor stimulation on repolarizing currents by means of a chamber-specific up-regulation of the receptors. This, together with the ion channel derangements produced by CAF, could contribute to the long-term stabilization of the arrhythmia by shortening the AP duration.

BACKGROUND:Lung cancer is the leading cause of cancer death in the world. The objective of this study was to investigate the expression of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) in patients with nonsmall cell lung cancer (NSCLC) and its correlation with the prognosis for patients with lung cancer.METHODS:The expression status of VEGFs and VEGFRs was examined in 48 nonconsecutive specimens of primary lung cancer by immunohistochemistry. Correlations between the expression of VEGFs and VEGFRs and clinicopathologic parameters were analyzed.RESULTS:Nineteen of 48 samples (39.6%) were moderately/highly immunoreactive for VEGF‐A, 6 samples (12.5%) were reactive for VEGF‐B, 14 samples (29.2%) were reactive for VEGF‐C, 11 samples (22.9%) were reactive for VEGF‐D, 20 samples (41.7%) were reactive for VEGFR1, 26 samples (54.2%) were reactive for VEGFR2, 20 samples (41.7%) were reactive for VEGFR3, and 19 samples (39.6%) were reactive for nuclear expression of VEGFR3. Patients with moderate/high VEGF‐C, VEGFR1, and VEGFR2 expression had worse survival, whereas patients with moderate/high VEGF‐D and nuclear VEGFR3 expression had better survival. After adjusting according to tumor stage, VEGF‐B and VEGF‐D expression had a significant correlation with worse survival in patients with stage I and II disease. Patients with stage III and IV disease who had VEGFR1 and VEGFR2 expression had worse survival, whereas the expression of VEGF‐D was correlated significantly with better survival. Finally, stage, VEGF‐D expression, and VEGFR1 expression were significantly independent prognostic predictors.CONCLUSIONS:The results of the current study indicated that the over‐expression of VEGFs and VEGFRs plays an important role in the survival of patients with NSCLC. The inclusion of angiogenic factors in the standard pathologic study of lung cancer may improve the clinical evaluation of patients with NSCLC. Cancer 2009. © 2009 American Cancer Society.

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

(MVTL) S U M M A R Y Increased protein synthesis is regulated, in part, by two eukaryotic translation initiation factors (eIFs): eIF4E and eIF2a. One or both of these factors are often overexpressed in several types of cancer cells; however, no data are available at present regarding eIF4E and eIF2a levels in brain tumors. In this study, we analyzed the expression, subcellular localization and phosphorylation states of eIF4E and eIF2a in 64 brain tumors (26 meningiomas, 16 oligodendroglial tumors, and 22 astrocytomas) and investigated the correlation with the expression of MIB-1, p53, and cyclin D1 proteins as well. There are significant differences in the phosphorylated eIF4E levels between the tumors studied, being the highest in meningiomas and the lowest in the oligodendroglial tumors. Relative to subcellular localization, eIF4E is frequently found in the nucleus of the oligodendroglial tumors and rarely in the same compartment of the meningiomas, whereas eIF2a showed an inverse pattern. Finally, cyclin D1 levels directly correlate with the phosphorylation status of both factors. The different expression, phosphorylation, or/and subcellular distribution of eIF2a and eIF4E within the brain types of tumors studied could indicate that different pathways are activated for promoting cell cycle proliferation, for instance, leading to increased cyclin D1 expression. (J Histochem Cytochem 57:503-512, 2009)