A kalcium homeosztázisban szerepet játszó fehérjék vizsgálata siRNS technikával. = The role of intracellular calcium stores in the regulation of calcium homeostasis in muscle cells under physiological and pathological conditions

A protein kináz C (PKC) izoenzimek eltérő módon befolyásolják epidermális keratinocyták proliferációját és differenciálódását, melynek hátterében a megváltozott kalciumhomeosztázis és purinoreceptor expresszió áll. Epidermális keratinocyták (HaCaT és NHEK) osztódását rövid UVB besugárzás szignifikánsan csökkenti. A csökkent nyugalmi kalciumszint, alacsonyabb ATP-indukálta kalciumtranziens amplitúdók mellett a P2X3 és P2X7 receptorok degradációját, valamint a P2X1 downregulációját tapasztaltuk, melyek felelősek lehetnek a besugárzás onkogén hatásáért. Sejttenyészetben és szöveti preparátumban található melanoma malignum sejtek erős TASK-3 immunpozitivitást mutatnak, mely érinti a melanoma sejtek magját is, de legkifejezettebb a mitokondriumokban. A csatorna expressziójának gátlása RNS-interferenciával a melanoma sejtek számát, míg HEK293 sejteken a TASK-3 csatornákon átfolyó áramot szignifikánsan csökkentette. Triadin 95-öt overexpresszáló C2C12 sejtvonalon és egér primer sejtkultúrán az elemi kalciumfelszabadulási események amplitudója és frekvenciája lecsökkent a kontrollhoz képest. Patkány primer izomrostokon specifikus shRNS transzfekciót követően a spontán kialakuló kalcium hullámok és kalcium spark-ok szignifikánsan nagyobb amplitúdóval és frekvenciával jellemezhetők, mint kontroll izomrostokon. A nyugalmi kalciumkoncentrációt és a kálium indukálta depolarizációt szintén jelentősen befolyásolta a triadin expressziójának megváltoztatása. | Protein kinase C izoenzymes have different role in regulating the proliferation and differentiation of keratinocytes mainly due to the altered calcium homeostais and purinergic receptor expression. Changes of the membrane potential, ion current and intracellular calcium concentration caused by hyposmotic solutions are more pronounced in cells where the PKC system was activated. Proliferation of epidermal keratinocytes (HaCaT and NHEK) is significantly reduced following short time UVB irradiation. The oncogen properties of irradiation could be due to the decreased calcium concentration, ATP-induced calcium transient amplitude, degradation of the P2X3 and P2X7 receptors and the downregulation of P2X1 receptor. Cultured and tissue melanoma cells show strong TASK-3 immunopositivity in the cytoplasm, mainly in the mitochondria. Reducing the expression of the channel protein by RNA interference decreased the number of viable cells, and the total ion current on HEK293 cells overexpressing TASK-3. C2C12 and mouse primary cultured myotubes overexpressing triadin 95 generated elementary calcium release events with lower amplitude and frequency than control cells. However, the amplitude and frequency of calcium transients and sparks were significantly higher in rat primary muscle cells following triadin 95-specific shRNA transfection than in control myotubes. Changes of the triadin expression altered the intracellular calcium concentration and potassium induced depolarization

Intracelluláris kalcium koncentráció szabályozása vázizom sejtekben = Regulation of intracellular calcium concentration in skeletal muscle cells

Az elektromechanikai csatolásban kulcsszerepet játszik dihidropiridin (DHP) receptor 2-3 hurokja és a rianodin receptor közötti kölcsönhatás. A DHP receptor putatív kölcsönható részével analóg a Scorpio Maurus Palmatus nevű skorpió toxinja a "maurocalcin" (MCa). Megállapítottuk, hogy a MCa befolyásolja a szarkoplazmás retikulum calcium csatornájának kapuzását, hosszantartó szubkonduktancia állapotok létrehozásával. Ezen szubkonduktancia állapotok átlagos ideje 10-12 másodperc a vad tipusú toxin esetén, míg a különböző pontmutánsok hatása csökkent attól függően, hogy a mutáció helye milyen messze van a kritikus 24-es pozíciótól. A csatorna (RyR1) ion-vezető pórusának méretét - a lantanida-kontrakciót felhazsnálva -gadolinium és európium ionra vonatkozó kinetikai paraméterek meghatározásával kezdtük el. Megállapítottuk, hogy a gadoliniumra vonatkozó fél-gátló koncentráció a cis és a trans oldalra nézve megegyezik és mintegy 5 mikromól. Ugyanakkor az euróium a trans oldal felől hatástalan és a cis oldalra vonatkozó fél-gátló koncentráció mintegy három nagyságrenddel alacsonyabb mint gadoliniumra. A két lantanida hatásának ilyen mértékére nem találtunk magyarázatot. | The interaction between the 2-3 loop of the Dihidropyridin receptor (DHPR) and the ryanodine receptor (RyR1) plays essential role int he electro-mechanical coupling. The toxin of the Scorpio Maurus Palmatus (maurocalcine = MCa) has very similar structure to the putative interaction domain of the DHPR and induces long lasting subconductance states ()LLSS) on the RyR1. The average length of these LLSS are about 10-12 seconds in case of the wild type toxin, while in case of mutated ones the length is dependent ont he distance of the mutation site from the critical 24th amino acid residue. We planned to estimate the diameter of the conductive channel of the Ryanodine receptor (RyR1) using Gadolinium and europium ions - based ont he lanthanide contraction. It was shown, that the half inhibitory gadolinum concentration is about 5 microM on the cis and on the trans side too. Similar studies carried out using Europium ions reveled, that Europium does not influence the channel from the trans side, but inhibits from the cis side - surprisingly much stronger, since the half-inhibitory concentration is a few nanomole. We have no exlponation for these striking findings

Astaxanthin Exerts Anabolic Effects via Pleiotropic Modulation of the Excitable Tissue

Astaxanthin is a lipid-soluble carotenoid influencing lipid metabolism, body weight, and insulin sensitivity. We provide a systematic analysis of acute and chronic effects of astaxanthin on different organs. Changes by chronic astaxanthin feeding were analyzed on general metabolism, expression of regulatory proteins in the skeletal muscle, as well as changes of excitation and synaptic activity in the hypothalamic arcuate nucleus of mice. Acute responses were also tested on canine cardiac muscle and different neuronal populations of the hypothalamic arcuate nucleus in mice. Dietary astaxanthin significantly increased food intake. It also increased protein levels affecting glucose metabolism and fatty acid biosynthesis in skeletal muscle. Inhibitory inputs innervating neurons of the arcuate nucleus regulating metabolism and food intake were strengthened by both acute and chronic astaxanthin treatment. Astaxanthin moderately shortened cardiac action potentials, depressed their plateau potential, and reduced the maximal rate of depolarization. Based on its complex actions on metabolism and food intake, our data support the previous findings that astaxanthin is suitable for supplementing the diet of patients with disturbances in energy homeostasis

A triadin szerepe a szarkoplazmatikus retikulumból történő kalcium-felszabadulás folyamatában harántcsíkolt izmon = The role of surface membrane and sarcoplasmic reticular proteins in acquired and hereditary muscle disorders

A szarkoplazmatikus retikulum (SR) kalcium csatornája (RyR) több fehérjével is kölcsönhatásban áll. Ide tartozik a 95 kDa-os molekulasúlyú triadint (Trisk 95), melynek alternatív splicingjával többféle molekulasúlyú izoformája jöhet létre. A Trisk 95 funkciójának felderítésére létrehozott triadin knockout (KO) egerek izmai gyengébbek és a kalciumhomeosztázisban résztvevő fehérjék expressziós szintje változott. Eredményeink szerint a triadin hiánya izom myopathiához vezethet és az általunk létrehozott állatmodellel ez jól tanulmányozható. A Trisk 95 további szerepét C2C12 izomsejteken és primer vázizomtenyészeteken tanulmányoztuk. A fehérje stabil overexpressziója csökkentette az elemi kalcium felszabadulási események (ECRE) amplitúdóját és frekvenciáját, és a depolarizáció által kiváltott Ca2+-tranzienseket. Ugyanakkor az endogén triadinexpresszió shRNS-sel történő gátlása után az ECRE-k jelentősen gyakoribbak voltak. Így feltételezhető, hogy a Trisk 95 fehérje negatívan szabályozza a RyR működését. A 32 kDa-os molekulasúlyú triadint (Trisk 32) L6.G8 myoblastokban termeltettük túl. A transzfekció nem befolyásolta a sejtek életképességét, ugyanakkor a sejtek proliferációs képessége lecsökkent. Kimutattuk a Trisk 32 és az IP3R kolokalizációját és közvetlen fizikai kapcsolatát. Funkcionális kapcsolatukat is bizonyítottuk, mivel a Trisk 32 overexpressziója szignifikánsan nagyobb amplitúdójú és felszálló meredekségű Ca2+-tranzienseket eredményezett az IP3 útvonalon keresztül. | The calcium release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum (SR) is associated with different proteins. One of them is the 95 kDa molecular weight triadin (Trisk 95) which has various isoforms with different molecular weights, all of them are splice variants of the same gene. To explore the function of Trisk 95 we generated a triadin knockout (KO) mouse which is characterized by a weaker skeletal muscle force than the control animals. Also, in case of the KO animals, the expression levels of the proteins which play role in calcium homeostasis, was changed. These results indicate that the lack of triadin can cause muscle myopathy and our animal model is suitable to investigate it. To further study the role of Trisk 95, the protein was investigated in C2C12 skeletal muscle cells and in primary skeletal muscle cultures. The constant overexpression of the protein decreased the amplitude and frequency of the elementary calcium release events (ECRE). Furthermore, the silencing of endogenous triadin expression with shRNA led to more frequent ECREs. These results suggest that Trisk 95 is a negative regulator of RyR function. Finally the 32 kDa molecular weight triadin (Trisk 32) was overexpressed in L6.G8 myoblasts. We showed the colocalization and direct physical interaction of Trisk 32 and IP3 receptor. Their functional interaction was also proved as the overexpression of Trisk 32 caused greater Ca2+-transients

Astaxanthin Exerts Anabolic Effects via Pleiotropic Modulation of the Excitable Tissue

Astaxanthin is a lipid-soluble carotenoid influencing lipid metabolism, body weight, and insulin sensitivity. We provide a systematic analysis of acute and chronic effects of astaxanthin on different organs. Changes by chronic astaxanthin feeding were analyzed on general metabolism, expression of regulatory proteins in the skeletal muscle, as well as changes of excitation and synaptic activity in the hypothalamic arcuate nucleus of mice. Acute responses were also tested on canine cardiac muscle and different neuronal populations of the hypothalamic arcuate nucleus in mice. Dietary astaxanthin significantly increased food intake. It also increased protein levels affecting glucose metabolism and fatty acid biosynthesis in skeletal muscle. Inhibitory inputs innervating neurons of the arcuate nucleus regulating metabolism and food intake were strengthened by both acute and chronic astaxanthin treatment. Astaxanthin moderately shortened cardiac action potentials, depressed their plateau potential, and reduced the maximal rate of depolarization. Based on its complex actions on metabolism and food intake, our data support the previous findings that astaxanthin is suitable for supplementing the diet of patients with disturbances in energy homeostasis

Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis

: During muscle cell differentiation, the alternatively spliced, acidic β-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the β-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid β-domain variants, as well as by a disordered β-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the β-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that β-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process

Sarcolemmal Ca2+-entry through L-type Ca2+ channels controls the profile of Ca2+-activated Cl- current in canine ventricular myocytes

Ca2+-activated Cl- current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca2+]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca2+]i was monitored using Fura-2-AM. Setting [Ca2+]i to the systolic level measured in the bulk cytoplasm (1.1muM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca2+-entry through L-type Ca2+ channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca2+-entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA