Calcium Handling Defects and Cardiac Arrhythmia Syndromes

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Blockade of sodium‑calcium exchanger via ORM-10962 attenuates cardiac alternans

Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium‑calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug's mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium‑calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans

The development of L-type Ca2+ current mediated alternans does not depend on the restitution slope in canine ventricular myocardium

Cardiac alternans have crucial importance in the onset of ventricular fibrillation. The early explanation for alternans development was the voltage‑driven mechanism, where the action potential (AP) restitution steepness was considered as crucial determining factor. Recent results suggest that restitution slope is an inadequate predictor for alternans development, but several studies still claim the role of membrane potential as underlying mechanism of alternans. These controversial data indicate that the relationship of restitution and alternans development is not completely understood. APs were measured by conventional microelectrode technique from canine right ventricular papillary muscles. Ionic currents combined with fluorescent measurements were recorded by patch‑clamp technique. APs combined with fluorescent measurements were monitored by sharp microelectrodes. Rapid pacing evoked restitution‑independent AP duration (APD) alternans. When non‑alternating AP voltage command was used, Ca2+i‑transient (CaT) alternans were not observed. When alternating rectangular voltage pulses were applied, CaT alternans were proportional to ICaL amplitude alternans. Selective ICaL inhibition did not influence the fast phase of APD restitution. In this study we found that ICaL has minor contribution in shaping the fast phase of restitution curve suggesting that ICaL—if it plays important role in the alternans mechanism—could be an additional factor that attenuates the reliability of APD restitution slope to predict alternans

Effect of the intracellular calcium concentration chelator BAPTA acetoxy-methylester on action potential duration in canine ventricular myocytes

Intracellular calcium concentration ([Ca(2+)]i) is often buffered by using the cell-permeant acetoxy-methylester form of the Ca(2+) chelator BAPTA (BAPTA-AM) under experimental conditions. This study was designed to investigate the time-dependent actions of extracellularly applied BAPTA-AM on action potential duration (APD) in cardiac cells. Action potentials were recorded from enzymatically isolated canine ventricular myocytes with conventional sharp microelectrodes. The effect of BAPTA-AM on the rapid delayed rectifier K(+) current (IKr) was studied using conventional voltage clamp and action potential voltage clamp techniques. APD was lengthened by 5 muM BAPTA-AM - but not by BAPTA - and shortened by the Ca(2+) ionophore A23187 in a time-dependent manner. The APD-lengthening effect of BAPTA-AM was strongly suppressed in the presence of nisoldipine, and enhanced in the presence of BAY K8644, suggesting that a shift in the [Ca(2+)]i-dependent inactivation of L-type Ca(2+) current may be an important underlying mechanism. However, in the presence of the IKr-blocker dofetilide or E-4031 APD was shortened rather than lengthened by BAPTA-AM. Similarly, the APD-lengthening effect of 100 nM dofetilide was halved by the pretreatment with BAPTA-AM. In line with these results, IKr was significantly reduced by extracellularly applied BAPTA-AM under both conventional voltage clamp and action potential voltage clamp conditions. This inhibition of IKr was partially reversible and was not related to the Ca(2+) chelator effect BAPTA-AM. The possible mechanisms involved in the APD-modifying effects of BAPTA-AM are discussed. It is concluded that BAPTA-AM has to be applied carefully to control [Ca(2+)]i in whole cell systems because of its direct inhibitory action on IKr

Late sodium current in human, canine and guinea pig ventricular myocardium

Although late sodium current (INa-late) has long been known to contribute to plateau formation of mammalian cardiac action potentials, lately it was considered as possible target for antiarrhythmic drugs. However, many aspects of this current are still poorly understood. The present work was designed to study the true profile of INa-late in canine and guinea pig ventricular cells and compare them to INa-late recorded in undiseased human hearts. INa-late was defined as a tetrodotoxin-sensitive current, recorded under action potential voltage clamp conditions using either canonic- or self-action potentials as command signals. Under action potential voltage clamp conditions the amplitude of canine and human INa-late monotonically decreased during the plateau (decrescendo-profile), in contrast to guinea pig, where its amplitude increased during the plateau (crescendo profile). The decrescendo-profile of canine INa-late could not be converted to a crescendo-morphology by application of ramp-like command voltages or command action potentials recorded from guinea pig cells. Conventional voltage clamp experiments revealed that the crescendo INa-late profile in guinea pig was due to the slower decay of INa-late in this species. When action potentials were recorded from multicellular ventricular preparations with sharp microelectrode, action potentials were shortened by tetrodotoxin, which effect was the largest in human, while smaller in canine, and the smallest in guinea pig preparations. It is concluded that important interspecies differences exist in the behavior of INa-late. At present canine myocytes seem to represent the best model of human ventricular cells regarding the properties of INa-late. These results should be taken into account when pharmacological studies with INa-late are interpreted and extrapolated to human. Accordingly, canine ventricular tissues or myocytes are suggested for pharmacological studies with INa-late inhibitors or modifiers. Incorporation of present data to human action potential models may yield a better understanding of the role of INa-late in action potential morphology, arrhythmogenesis, and intracellular calcium dynamics

Sexual Dimorphism in Bidirectional Sr-Mitochondria Crosstalk in Ventricular Cardiomyocytes

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females

Az NHERF2 fehérje bakteriális overexpressziójának optimalizálása

Az endotél sejtek összefüggő monolayert alakítanak ki az erek belső falán. Az endotél sejtek rétege elválasztja a keringő vért a szövetektől, és csak bizonyos makromolekuláknak és sejteknek biztosítja az átjárást a véráram és a szövetek között, továbbá szabályozó szerepük van különböző patológiás és élettani folyamatokban is. Az oldott anyagok transzportja az endotéliumon keresztül többféle módon történhet, legjelentősebb a paracelluláris útvonal a sejt-sejt kapcsolatoknál. A vaszkuláris endotéliumban adherens és szoros kapcsolatok/junctionok is találhatóak, melyek fontos szerepet töltenek be a paracelluláris permeábilitás szabályozásában. A citoszkeleton elemeinek átszerveződése megváltoztatja az endotél sejtek alakját, így a sejtek közötti rések méretét, melyeken keresztül a paracelluláris anyagátvitel zajlik. Az endotélium barrier funkcióját többféle jelátviteli útvonal szabályozza, leggyakrabban kinázok (pl. miozin könnyű lánc kináz, protein kináz C, stb.) és foszfatázok (protein foszfatáz 1 és 2A) befolyásolják reverzibilis foszforilációval a sejt-sejt kapcsolatoknál lévő fehérjéket és a citoszkeleton-asszociált fehérjéket. A jelátvitelben részt vevő proteinek gyakran több fehérjéből álló komplexekben képesek működni, melyeket vázproteinek állványoznak fel. A scaffold fehérjék alapvető funkciója a jelátviteli útvonalak komponenseinek egymás közelébe helyezése, fehérje komplexek létrehozása a jelátvitel számára. Több adaptor fehérje tartalmaz PDZ fehérje-fehérje interakciós doméneket. A PDZ domén fehérjék nagyon változatos molekulákkal képesek kölcsönhatni, pl. növekedési faktor-, G-protein kapcsolt-, neurotranszmitter receptorokkal, transzport fehérjékkel, adhéziós molekulákkal, a citoszkeleton elemeivel és transzkripciós faktorokkal. A Na+/H+ Exchanger Regulatory Factor (NHERF) család tagjai nagy mennyiségben jelen vannak az epitéliumban. Az NHERF1 és NHERF2 adaptor fehérjék két tandem PDZ interakciós domént tartalmaznak és egy C-terminális végen lévő ERM-kötő domént, mely az ezrin, radixin és moesin fehérjékkel képes kölcsönhatni. Az ezrin, radixin és moesin az ERM fehérje család tagjai. Szerepük van az aktin és a plazmamembrán kapcsolat kialakításában és fenntartásában, valamint a jelátvitelben részt vevő fehérjék működésének szabályozásában. Az NHERF3 és NHERF4 fehérjékben négy darab PDZ domén található, ERM-kötő doménjük nincs. A család tagjai kölcsönhatnak membrán proteinekkel, ioncsatornákkal, receptorokkal és különböző G-protein kapcsolt receptorokkal is. Továbbá vannak nem membránhoz kapcsolt célpontjai is, még magban lévő fehérjékkel is kölcsönhatnak, és szabályozhatják egyes anyagoknak a sejtmag és a citoplazma közötti ingázását, és/vagy segítik a felépítését és az állványozását a multiprotein transzkripciós komplexeknek, melyek szükségesek a gének aktiválásához. Munkacsoportunk korábban az ERM fehérjéket és azoknak a kötőpartnereit vizsgálta. Az NHERF1, másnéven ERM Binding Phosphoprotein of 50 kD (EBP50) és NHERF2 egyaránt a citoplazmában lokalizálódik az epitélium sejtjeiben, feladatuk vázproteinként a plazmamembrán fehérjék és a citoszkeletális fehérjék közötti kapcsolat kialakítása és fenntartása. Az endotéliumban betöltött szerepük és elhelyezkedésük nem tisztázott, ezért kezdte el a munkacsoportunk komolyabb érdeklődéssel vizsgálni az NHERF1 és NHERF2 fehérjéket endotél sejtekben. Az eddigi eredmények azt bizonyítják, hogy az NHERF2 funkciója az endotél sejtekben jelentősen különbözik az EBP50 funkciójától.BSc/BABiológiag