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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 Ca 2+ 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.

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 β-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 β-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 β-NAD effects. Meantime, 5 mM NH 4 Cl 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, NH 4 Cl, being added after β-NAD and having no effect on RMP, APD, and I-V curve, recovered F to the control values, indicating β-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca 2+ -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 β-NAD and ammonia on rat papillary muscle contractile activity.

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca 2+ 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 Ca 2+ current ( I CaL ). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of I CaL 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 I CaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.

Long-chain acylcarnitines (LCAC) are implicated in ischemia-reperfusion (I/R)-induced myocardial injury and mitochondrial dysfunction. Yet, molecular mechanisms underlying involvement of LCAC in cardiac injury are not sufficiently studied. It is known that in cardiomyocytes, palmitoylcarnitine (PC) can induce cytosolic Ca 2+ accumulation, implicating L-type calcium channels, Na + /Ca 2+ exchanger, and Ca 2+ -release from sarcoplasmic reticulum (SR). Alternatively, PC can evoke dissipation of mitochondrial potential (ΔΨ m ) and mitochondrial permeability transition pore (mPTP). Here, to dissect the complex nature of PC action on Ca 2+ homeostasis and oxidative phosphorylation (OXPHOS) in cardiomyocytes and mitochondria, the methods of fluorescent microscopy, perforated path-clamp, and mitochondrial assays were used. We found that LCAC in dose-dependent manner can evoke Ca 2+ -sparks and oscillations, long-living Ca 2+ enriched microdomains, and, finally, Ca 2+ overload leading to hypercontracture and cardiomyocyte death. Collectively, PC-driven cardiotoxicity involves: (I) redistribution of Ca 2+ from SR to mitochondria with minimal contribution of external calcium influx; (II) irreversible inhibition of Krebs cycle and OXPHOS underlying limited mitochondrial Ca 2+ buffering; (III) induction of mPTP reinforced by PC-calcium interplay; (IV) activation of Ca 2+ -dependent phospholipases cPLA2 and PLC. Based on the inhibitory analysis we may suggest that simultaneous inhibition of both phospholipases could be an effective strategy for protection against PC-mediated toxicity in cardiomyocytes.

α2-adrenoceptor (α2-AR) isoforms, abundant in sympathetic synapses and noradrenergic neurons of the central nervous system, are integral in the presynaptic feed-back loop mechanism that moderates norepinephrine surges. We recently identified that postsynaptic α2-ARs, found in the myocellular sarcolemma, also contribute to a muscle-delimited feedback control capable of attenuating mobilization of intracellular Ca 2+ and myocardial contractility. This previously unrecognized α2-AR-dependent rheostat is able to counteract competing adrenergic receptor actions in cardiac muscle. Specifically, in ventricular myocytes, nitric oxide (NO) and cGMP are the intracellular messengers of α2-AR signal transduction pathways that gauge the kinase-phosphatase balance and manage cellular Ca 2+ handling preventing catecholamine-induced Ca 2+ overload. Moreover, α2-AR signaling counterbalances phospholipase C - PKC-dependent mechanisms underscoring a broader cardioprotective potential under sympathoadrenergic and angiotensinergic challenge. Recruitment of such tissue-specific features of α2-AR under sustained sympathoadrenergic drive may, in principle, be harnessed to mitigate or prevent cardiac malfunction. However, cardiovascular disease may compromise peripheral α2-AR signaling limiting pharmacological targeting of these receptors. Prospective cardiac-specific gene or cell-based therapeutic approaches aimed at repairing or improving stress-protective α2-AR signaling may offer an alternative towards enhanced preservation of cardiac muscle structure and function., (Copyright © 2019. Published by Elsevier Inc.)

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C-7°C-30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states.

Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca 2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca 2+ current (I CaL ) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced β-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of β- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated I CaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca 2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Antioxidant properties of recombinant peroxiredoxin-6 and chimeric protein PSH combining peroxidase and superoxide dismutase activities were studied on the model of retrograde perfusion of isolated rat heart under conditions of H2O2-induced oxidative stress. The exogenous antioxidant proteins exhibited cardioprotective properties manifested in heart rate normalization, maintenance of contractile activity of the myocardium, and prevention of H2O2-induced LPO in oxidative stress. Localization of peroxiredoxin-6 and PSH in the cardiac tissue was determined and myocardial structures most effectively protected by the antioxidant enzymes from ischemia/reperfusion-induced damages were identified. The results suggest that modified peroxiredoxins are promising components of perfusion media for preservation of isolated organs.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.