Alexey S. Averin

Phospholipases A₂ (PLA₂s) are a large family of snake toxins manifesting diverse biological effects, which are not always related to phospholipolytic activity. Snake venom PLA₂s (svPLA₂s) are extracellular proteins with a molecular mass of 13–14 kDa. They are present in venoms in the form of monomers, dimers, and larger oligomers. The cardiovascular system is one of the multiple svPLA₂ targets in prey organisms. The results obtained previously on the cardiovascular effects of monomeric svPLA₂s were inconsistent, while the data on the dimeric svPLA₂ crotoxin from the rattlesnake Crotalus durissus terrificus showed that it significantly reduced the contractile force of guinea pig hearts. Here, we studied the effects of the heterodimeric svPLA₂ HDP-1 from the viper Vipera nikolskii on papillary muscle (PM) contractility and the tension of the aortic rings (ARs). HDP-1 is structurally different from crotoxin, and over a wide range of concentrations, it produced a long-term, stable, positive inotropic effect in PMs, which did not turn into contractures at the concentrations studied. This also distinguishes HDP-1 from the monomeric svPLA₂s, which at high concentrations inhibited cardiac function. HDP-1, when acting on ARs preconstricted with 10 μM phenylephrine, induced a vasorelaxant effect, similar to some other svPLA₂s. These are the first indications of the cardiac and vascular effects of true vipers’ heterodimeric svPLA₂s.

The present study examined the effects of the в-adrenergic receptor agonist, isoproterenol, on contractile activity of right ventricle papillary muscles in the rat heart at near-physiological temperature (30°C) and under conditions of deep hypothermia (10°C). Isoproterenol exerts a pronounced positive inotropic effect at 30°C. After agonist addition, the force of contraction increases from 1.2 ± 0.1 mN in control to 2.4 ± 0.4 mN that leads to a reliable acceleration of time parameters of contraction. So, time-to-maximum contraction decreased from 101 ± 6 ms to 85 ± 4 ms; time-to-50% relaxation declined from 55 ± 3 ms to 36 ± 1 ms. Under hypothermic conditions, isoproterenol produced a powerful negative inotropic effect, reducing the force of contraction from 2.2 ± 0.4 mN to 1.2 ± 0.4 mN. Similarly as at 30°C, there was a tendency for increase in contraction speed, so time-to-maximum contraction decreased from 717 ± 52 ms to 624 ± 50 ms, and time-to-50% relaxation was shortened from 667 ± 86 ms to 450 ± 40 ms. Thus, under conditions of deep hypothermia at 10°C, the isoproterenol-induced inotropy changes from positive to negative, while negative lusitropic effect remains clear.

The effects of the β-adrenergic receptor agonist isoproterenol on the contractile activity of the papillary muscle of the rat heart right ventricle were studied both at a temperature close to physiological (30°C) and under conditions of deep hypothermia (10°C). Isoproterenol had a pronounced positive inotropic effect at 30°C, the contraction force increased from 1.2 ± 0.1 mN in the control to 2.4 ± 0.4 mN after the addition of the agonist and there was a significant acceleration of the time parameters of contraction, that is, the time to reach the maximum contraction decreased from 101 ± 6 ms to 85 ± 4 ms; the relaxation time decreased by 50% from 55 ± 3 ms to 36 ± 1 ms. Under hypothermic conditions, isoproterenol caused a powerful negative inotropic effect, reducing the contraction force from 2.2 ± 0.4 mN to 1.2 ± 0.4 mN. The tendency to accelerate contraction persisted as at 30°C: the time to reach the maximum contraction decreased from 717 ± 52 ms to 624 ± 50 ms, and the relaxation time decreased by 50% from 667 ± 86 ms to 450 ± 40 ms. Thus, under conditions of deep hypothermia at 10°C, the direction of the inotropic response to isoproterenol changed from positive to negative, while the lusitropic effect remained negative.

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.

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 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 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, 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 β-NAD and ammonia on rat papillary muscle contractile activity.

The effects of 1.5 mM nickel chloride on the contraction force, the force–frequency relation, and post-rest potentiation in the myocardium were studied in active Urocitellus undulates ground squirrels with different responses to hypothermia. In a group of ground squirrels that retained a negative trend of the force–frequency relation upon cooling, the contraction forces were 52 ± 15 and 4 ± 3% of the contraction force in a control upon stimulation at frequencies of 0.1 and 1 Hz, respectively, at 20°C. A reduction of myocardial contractility was far greater in a group where the force–frequency relation changed from a biphasic pattern to a positive trend. Contractions were almost completely suppressed; the contraction forces were 6 ± 3 and 1 ± 0.5% at 0.1 and 1.0 Hz, respectively. When stimulation at a frequency of 0.3 Hz was performed in the presence of 1.5 mM Ni²⁺, post-rest potentiation decreased, on average, by 45–55% in the group with a negative force–frequency relation and by 90–98% in the group with a positive force–frequency relation. At a stimulation frequency of 1.0 Hz, there were 45–55 and 65–85% reductions, respectively. The results showed that the myocardium was significantly less dependent on extracellular Ca²⁺ input in ground squirrels that retained a negative force–frequency relation as compared with active ground squirrels that displayed a transition to a positive force–frequency relation upon cooling.

Cardiotoxins (CaTxs) are a group of snake toxins that affect the cardiovascular system (CVS). Two types (S and P) of CaTxs are known, but the exact differences in the effects of these types on CVS have not been thoroughly studied. We investigated cellular mechanisms of action on CVS for Naja oxiana cobra CaTxs CTX-1 (S-type) and CTX-2 (P-type) focusing on the papillary muscle (PM) contractility and contraction of aortic rings (AR) supplemented by pharmacological analysis. It was found that CTX-1 and CTX-2 exerted dose-dependent effects manifested in PM contracture and AR contraction. CTX-2 impaired functions of PM and AR more strongly than CTX-1. Effects of CaTxs on PM were significantly reduced by nifedipine, an L-type Ca²⁺ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na⁺/Ca²⁺ exchange. Furthermore, 2-aminoethoxydiphenyl borate, an inhibitor of store-operated calcium entry, partially restored PM contractility damaged by CaTxs. The CaTx influence on AR contracture was significantly reduced by nifedipine and KB-R7943. The involvement of reverse-mode Na⁺/Ca²⁺ exchange in the effect of CaTxs on the rat aorta was shown for the first time. The results obtained indicate that CaTx effects on CVS are mainly associated with disturbance of transporting systems responsible for the Ca²⁺ influx.

Snake venoms, as complex mixtures of peptides and proteins, affect various vital systems of the organism. One of the main targets of the toxic components from snake venoms is the cardiovascular system. Venom proteins and peptides can act in different ways, exhibiting either cardiotoxic or cardioprotective effects. The principal classes of these compounds are cobra cardiotoxins, phospholipases A2, and natriuretic, as well as bradykinin-potentiating peptides. There is another group of proteins capable of enhancing angiogenesis, which include, e.g., vascular endothelial growth factors possessing hypotensive and cardioprotective activities. Venom proteins and peptides exhibiting cardiotropic and vasoactive effects are promising candidates for the design of new drugs capable of preventing or constricting the development of pathological processes in cardiovascular diseases, which are currently the leading cause of death worldwide. For example, a bradykinin-potentiating peptide from Bothrops jararaca snake venom was the first snake venom compound used to create the widely used antihypertensive drugs captopril and enalapril. In this paper, we review the current state of research on snake venom components affecting the cardiovascular system and analyse the mechanisms of physiological action of these toxins and the prospects for their medical application.

—The effects of a four-fold decrease in the concentration of Ca²⁺ in a perfusing medium under pronounced hypothermia at 20°C on the contractile responses of the papillary muscles of the right ventricle of a guinea pig, that is, the frequency–force dependence in the frequency range of 0.1–1.0 Hz and the effect of potentiation by a pause at stimulation frequencies of 0.3 and 0.8 Hz, were studied. It was shown that with a decrease in the concentration of extracellular Ca²⁺ there was a significant decrease in the contraction force of papillary muscles from 40 to 70%, reaching a maximum of 72 ± 3% at a frequency of 0.4 Hz; at the same time, the positive frequency–forcer dependence remained. The absolute values of the effect of potentiation by a pause decreased and the decrease was most pronounced at a lower stimulation frequency (79 ± 6% and 40 ± 19% at 0.3 and 0.8 Hz, respectively). Thus, it was shown that the predominant dependence of contraction on extracellular sources of Ca²⁺ in the myocardium of guinea pigs persisted under pronounced hypothermia; at the same time, an increase in the frequency of stimulation led to an increase in the content of Ca²⁺ in the sarcoplasmic reticulum.