Search

Voltage-gated ion channels are key molecules for the generation of cellular electrical excitability. Many pharmaceutical drugs target these channels by blocking their ion-conducting pore, but in many cases, channel-opening compounds would be more beneficial. Here, to search for new channel-opening compounds, we screen 18,000 compounds with high-throughput patch-clamp technology and find several potassium-channel openers that share a distinct biaryl-sulfonamide motif. Our data suggest that the negatively charged variants of these compounds bind to the top of the voltage-sensor domain, between transmembrane segments 3 and 4, to open the channel. Although we show here that biaryl-sulfonamide compounds open a potassium channel, they have also been reported to block sodium and calcium channels. However, because they inactivate voltage-gated sodium channels by promoting activation of one voltage sensor, we suggest that, despite different effects on the channel gates, the biaryl-sulfonamide motif is a general ion-channel activator motif. Because these compounds block action potential-generating sodium and calcium channels and open an action potential-dampening potassium channel, they should have a high propensity to reduce excitability. This opens up the possibility to build new excitability-reducing pharmaceutical drugs from the biaryl-sulfonamide scaffold., Funding Agencies|Swedish Research Council [2016-02615]; Swedish Heart-Lung Foundation [20150672]; Swedish Brain Foundation [2016-0326]; Swedish Society for Medical Research

AimAtrial fibrillation is the most common persistent cardiac arrhythmia, and it is not well controlled by present drugs. Because some resin acids open voltage-gated potassium channels and reduce neuronal excitability, we explored the effects of the resin acid isopimaric acid (IPA) on action potentials and ion currents in cardiomyocytes. MethodsSpontaneously beating mouse atrial HL-1 cells were investigated with the whole-cell patch-clamp technique. Results1-25 mol L-1 IPA reduced the action potential frequency by up to 50%. The effect of IPA on six different voltage-gated ion channels was investigated; most voltage-dependent parameters of ion channel gating were shifted in the negative direction along the voltage axis, consistent with a hypothesis that a lipophilic and negatively charged compound binds to the lipid membrane close to the positively charged voltage sensor of the ion channels. The major finding was that IPA inactivated sodium channels and L- and T-type calcium channels and activated the rapidly activating potassium channel and the transient outward potassium channel. Computer simulations of IPA effects on all of the ion currents were consistent with a reduced excitability, and they also showed that effects on the Na channel played the largest role to reduce the action potential frequency. Finally, induced arrhythmia in the HL-1 cells was reversed by IPA. ConclusionLow concentrations of IPA reduced the action potential frequency and restored regular firing by altering the voltage dependencies of several voltage-gated ion channels. These findings can form the basis for a new pharmacological strategy to treat atrial fibrillation., Funding Agencies|Swedish Research Council; Swedish Heart-Lung Foundation; Swedish Brain Foundation; ALF

Cardiovascular diseases, including myocardial infarction, are the cause of significant morbidity and mortality globally. Tissue engineering is a key emerging treatment method for supporting and repairing the cardiac scar tissue caused by myocardial infarction. Creating cell supportive scaffolds that can be directly implanted on a myocardial infarct is an attractive solution. Hydrogels made of collagen are highly biocompatible materials that can be molded into a range of shapes suitable for cardiac patch applications. The addition of mechanically reinforcing materials, carbon nanotubes, at subtoxic levels allows for the collagen hydrogels to be strengthened, up to a toughness of 30 J m-1 and a two to threefold improvement in Youngs' modulus, thus improving their viability as cardiac patch materials. The addition of carbon nanotubes is shown to be both nontoxic to stem cells, and when using single-walled carbon nanotubes, supportive of live, beating cardiac cells, providing a pathway for the further development of a cardiac patch., Funding agencies: Linkoping Initiative in Life Science Technologies (LIST); Central ALF Matching Grant from Landstinget i Ostergotland [LIO-344071]; European Research Agency [304209]; GeCONiI [POIG.02.03.01-24-099/13]

Cell therapy is a promising approach for the treatment of patients suffering from myocardial infarction. Most recent therapies involve direct injection of cells into the damaged heart tissue to induce regeneration and help restore its functions, however, anoikis and the harsh environment at the sight of injection limit the therapeutic efficacy of current techniques. Biopolymeric microspheres such as alginate have been widely used for cells encapsulation and delivery for cell therapy applications. However, majority of these techniques are not standardized that is a big challenge for translation into clinically-relevant treatment options. In addition, purely-alginate base microspheres are limited by poor biodegradability and lack of strong interaction between the encapsulated cells and their surrounding alginate matrix. In this work, we have shown that the addition of type I collagen into alginate microspheres, systematically optimized by a multivariate experimental design, improves the biocompatibility of the microspheres towards induced pluripotent stem cells (iPS), cardiomyocytes, and blood outgrowth endothelial cells (BOEC), whilst improving diffusion between outside environment and the inner sphere. The addition of collagen allows for multiple routes for sphere degradation leading to potentially greater control over cell release once delivered. Mathematical models were developed and utilized to effectively evaluate and predict the influence of various factors such as polymer ratios, micronization air flow rate, and air-gap distance on spheres size and shape, which play a key role in cell viability, degradation rate of microspheres, as well as controlled production of the cell cocoons toward clinically-relevant cell therapies for treatment of myocardial infarction. (C) 2016 Elsevier B.V. All rights reserved., Funding Agencies|European Research Agency for EU FP7-PEOPLE-IIF-Marie Curie Actions-International Incoming Fellowship (IIF) [302782]; Central ALF Matching Grant from Landstinget i Ostergotland [LIO-344071]; Vice-rector matching fund from Linkoping University

Transport of ions across the cell membrane is essential for the regulation of cell death and tissue homeostasis, and alterations in the function of voltage-gated ion channels and of the intracellular ionic compositions interfere with these processes. Opening of K, Na , or Cl channels have been linked to the apoptotic process and in many cases, opening of these channels precede caspase-3 activation and are thus early events in the apoptotic process. Consistent with the role of these channels in apoptosis, inhibition of these channels prevents or delays the apoptotic process. However, the role of ion channels during apoptosis has been difficult to explore, mainly due to unspecific/non-selective ion channe blockers. In the present investigation, the molecular identity of a voltage-gated Na channel in oocytes from Xenopus laevis, which is crucial for the apoptotic response to mechanical stress, was identified. Specific down regulation of SCN2A Na channel expression by miRNA prevented apoptosis, suggesting that Na+ influx is essential for apoptosis in Xenopus oocytes.

n/a

Valproic acid (VPA) is an anticonvulsant used to treat bipolar disorder, a psychiatric disease associated with disturbances in circadian rhythmicity. Little is known about how VPA affects circadian rhythms. The authors cultured tissues containing the master brain pacemaker for circadian rhythmicity, the suprachiasmatic nuclei (SCN), and skin fibroblasts from transgenic PERIOD2::LUCIFERASE (PER2::LUC) mice and studied the effect of VPA on the circadian PER2::LUC rhythm by measuring bioluminescence. VPA (1 mM) significantly phase advanced the PER2::LUC rhythm when applied at a time point corresponding to the lowest (trough, similar to ZT 0) PER2::LUC expression but phase delayed the PER2::LUC rhythm when the drug was administered at the time of highest (peak, similar to ZT 12) protein expression. In addition, it significantly increased the overall amplitude of PER2::LUC oscillations at time points at or close to ZT 12 but had no effect on period. Real-time PCR analyses on mouse and human fibroblasts revealed that expressions of other clock genes were increased after 2 h treatment with VPA. Because VPA is known to inhibit histone deacetylation, the authors treated cultures with an established histone deacetylation inhibitor, trichostatin A (TSA; 20 ng/mL), to compare the effect of VPA and TSA on molecular rhythmicity. They found that TSA had similar effects on the PER2::LUC rhythm as VPA. Furthermore, VPA and TSA significantly increased acetylation on histone H3 but in comparison little on histone H4. Lithium is another commonly used treatment for bipolar disorder. Therefore, the authors also studied the impact of lithium chloride (LiCl; 10 mM) on the PER2::LUC rhythm. LiCl delayed the phase, but in contrast to VPA and TSA, LiCl lengthened the PER2::LUC period and had no effect on histone acetylation. These results demonstrate that VPA can delay or advance the phase, as well as increase the amplitude, of the PERIOD2::LUCIFERASE rhythm depending on the circadian time of

n/a

n/a

microRNAs (miRNAs) are small non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis and proliferation. Several miRNAs display defective expression patterns in human tumors with the consequent alteration of target oncogene or tumor suppressor genes. Many of these miRNAs modulate the major proliferation pathways through direct interaction with critical regulators such as RAS, PI3K/PTEN or ABL, as well as members of the retinoblastoma pathway, Cyclin-CDK complexes or cell cycle inhibitors of the INK4 or Cip/Kip families. A complex interplay between miRNAs and MYC or E2F family members also exists to modulate cell cycle-dependent transcription during normal or tumoral proliferation. The ability of miRNAs to modulate these proliferation pathways may have relevant implications not only in physiological or developmental processes but also in tumor progression or cancer therapy.

Udgivelsesdato: 2005-Nov-7, Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ras, R-Ras, Rap) both inhibited autaptic responses. In a proportion of the neurons (25%, TcdA-10463; 54%, TcsL-1522), the inhibition was associated with a shift from activity-dependent depression to facilitation, indicating that the synaptic release probability was reduced. Overexpression of a dominant negative Ral mutant, Ral A28N, caused a strong inhibition of autaptic responses, which was associated with a shift to facilitation in a majority (80%) of the neurons. These results indicate that Ral, along with at least one other non-Rab GTPase, participates in presynaptic regulation in hippocampal neurons.

The central nervous system (CNS) can be the target for several infections that include those with RNA viruses. The parenchyma of the CNS is considered an immuneprivileged site since it is protected behind the blood-brain barrier, expresses no or only low levels of major histocompatibility complex (MHC) molecules and contains a paucity of antigenpresenting cells that prime an immune response. Nevertheless, viral infections in the brain can be controlled to be cleared or become persistent. This poses the questions concerning by which mechanisms an RNA virus, which must undergo a continuous replication to exist, can be controlled in the brain, and how the function in neurons and neuronal networks is affected by an infection that has either been cleared or become persistent. This thesis work deals mainly with the second question. Effects on synaptic formation and function were analyzed in primary hippocampal cultures after infections with the neuroadapted WSN/33 strain of influenza A virus, and after exposure to certain molecules involved in host defence against a virus. The latter involved interferon-gamma (IFN-gamma), which has been implied in the control of certain microbe infections in the nervous system in recent studies. In this respect, I have in particular been interested in determining whether viral infections during restricted periods of development, corresponding to those of peaks of synapse formation, can cause persistent changes in synaptic activities. The viral WSN/33 strain used in this study has previously been shown to selectively infect the hippocampus and the substantia nigra in mice following intracerebral and systemic injections. In my thesis the following observations were made: 1) Infected hippocampal neurons showed reduced voltage-dependent Ca2+ currents in whole-cell patch clamp recordings. 2) By over-expressing the viral nucleoprotein (NP) using the Semliki Forest virus expression system, this protein could be localized to the dendritic spines