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1.
Int J Mol Sci ; 24(11)2023 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-37298207

RESUMO

Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca2+ concentration and an assessment of the rat cardiomyocytes' shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed for Naja oxiana cobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.


Assuntos
Proteínas Cardiotóxicas de Elapídeos , Contratura , Toxinas Biológicas , Ratos , Animais , Proteínas Cardiotóxicas de Elapídeos/farmacologia , Proteínas Cardiotóxicas de Elapídeos/toxicidade , Cálcio , Miócitos Cardíacos , Venenos Elapídicos/química , Peptídeos , Cálcio da Dieta
2.
Membranes (Basel) ; 12(12)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36557146

RESUMO

Various models, including stem cells derived and isolated cardiomyocytes with overexpressed channels, are utilized to analyze the functional interplay of diverse ion currents involved in cardiac automaticity and excitation-contraction coupling control. Here, we used ß-NAD and ammonia, known hyperpolarizing and depolarizing agents, respectively, and applied inhibitory analysis to reveal the interplay of several ion channels implicated in rat papillary muscle contractility control. We demonstrated that: 4 mM ß-NAD, having no strong impact on resting membrane potential (RMP) and action potential duration (APD90) of ventricular cardiomyocytes, evoked significant suppression of isometric force (F) of paced papillary muscle. Reactive blue 2 restored F to control values, suggesting the involvement of P2Y-receptor-dependent signaling in ß-NAD effects. Meantime, 5 mM NH4Cl did not show any effect on F of papillary muscle but resulted in significant RMP depolarization, APD90 shortening, and a rightward shift of I-V relationship for total steady state currents in cardiomyocytes. Paradoxically, NH4Cl, being added after ß-NAD and having no effect on RMP, APD, and I-V curve, recovered F to the control values, indicating ß-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca2+-activated K+ channels (SK, IK, and BK), or NCX exchanger reverse mode prevented this effect, indicating consistent cooperation of all currents mediated by these channels and NCX. We suggest that the activation of Kir2.x and HCN channels by extracellular K+, that creates positive and negative feedback, and known ammonia and K+ resemblance, may provide conditions required for the activation of all the chain of channels involved in the interplay. Here, we present a mechanistic model describing an interplay of channels and second messengers, which may explain discovered antagonism of ß-NAD and ammonia on rat papillary muscle contractile activity.

3.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923625

RESUMO

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.


Assuntos
Potenciais de Ação , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Adrenérgicos alfa 2/genética
4.
PLoS One ; 11(3): e0152018, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27015602

RESUMO

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.


Assuntos
Compostos Azo/química , Brometos/química , Luz , Metilaminas/química , Miócitos Cardíacos/citologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potenciais de Ação , Animais , Animais Recém-Nascidos , Cálcio/química , Fibronectinas/química , Ventrículos do Coração/patologia , Íons , Potenciais da Membrana , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Potássio/química , Ratos , Sódio/química
5.
J Mol Cell Cardiol ; 68: 66-74, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24412533

RESUMO

Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α2 (α2-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine.


Assuntos
Sinalização do Cálcio , Receptores de Imidazolinas/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Agmatina/farmacologia , Animais , Benzofuranos/farmacologia , Células Cultivadas , Imidazóis/farmacologia , Receptores de Imidazolinas/agonistas , Receptores de Imidazolinas/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar
6.
Biophys Chem ; 117(2): 173-90, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15936868

RESUMO

A kinetic model of Ca2+-dependent inactivation (CDI) of L-type Ca2+ channels was developed. The model is based on the hypothesis that postulates the existence of four short-lived modes with lifetimes of a few hundreds of milliseconds. Our findings suggest that the transitions between the modes is primarily determined by the binding of Ca2+ to two intracellular allosteric sites located in different motifs of the CI region, which have greatly differing binding rates for Ca2+ (different k(on)). The slow-binding site is controlled by local Ca2+ near a single open channel that is consistent with the "domain" CDI model, and Ca2+ binding to the fast-binding site(s) depends on Ca2+ arising from distant sources that is consistent with the "shell" CDI model. The model helps to explain numerous experimental findings that are poorly understood so far.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/fisiologia , Cálcio/química , Canais de Cálcio Tipo L/química , Ativação do Canal Iônico/fisiologia , Cinética , Potenciais da Membrana/fisiologia , Fatores de Tempo
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