Cardiorespiratory arrest in rats caused by EqT was first described by Sket et al. [
47] but, at that time, the underlying mechanism was not fully understood. In vitro studies revealed that EqT (80–200 ng/mL) increases the permeability and resistance of the lung vasculature and produces, at concentrations higher than 150 ng/mL, interstitial and alveolar pulmonary oedema [
48]. At low concentrations (0.1–3 µg/mL), EqT induces a transient negative inotropic effect followed by a long-lasting positive inotropic effect in isolated guinea pig atrium. It was proposed that the formation of prostaglandin E2 is responsible for this effect, since it could be inhibited by indomethacin, a well-known inhibitor of prostaglandin synthesis [
49]. The positive inotropic effect described by these authors was also seen in experiments performed on isolated guinea pig hearts using EqT II [
50], but only when the toxin had been applied at low concentrations (picomolar range). Higher concentrations of EqT II caused a pronounced negative inotropic effect. Cardiorespiratory effects, similar to those produced by EqT [
47], were later confirmed using EqT II [
51] and EqT III [
52]. EqT II causes negative inotropic and chronotropic effects such as bradycardia, action potential conduction disturbances and extrasystoles. Similarly, the lethal dose of EqT III also produces arrhythmias, a drop of arterial blood pressure and cardiac arrest. The two isotoxins are haemolytic, and it is well known that the hyperkalaemia caused by the lysis of erythrocytes can produce serious arrhythmias leading to cardiac failure. A detailed study of the role of haemolysis in EqT lethality revealed an only marginal role of hyperkalaemia in the cardiotoxic effects of these toxins [
52]. This is in agreement with data showing that the more toxic EqT II is less haemolytic than EqT III. As EqT III is the least toxic but causes the most pronounced hyperkalaemia, it seems that the elevation of plasma K
+ concentration is not the primary cause of cardiorespiratory arrest. In vivo, EqT II and EqT III cause similar alterations of electrocardiogram (ECG), breathing and blood pressure, indicating that the same cardiotoxicity mechanism may be involved.
EqTs belong to the group of pore forming toxins that enable passage of cations, mainly Ca
2+, through phospholipid bilayers, including the plasma membrane. Therefore, another possible pathophysiological mechanism of the cardiotoxicity may be a decreased coronary perfusion due to vasoconstriction. Vasoconstrictor effects of EqT II may also include endothelin-dependent pathway [
53]. EqT II-triggered endothelin release is probably one of the mechanisms involved in the lowering of coronary flow induced by this toxin [
54], since endothelin is well known to activate L-type calcium channels in smooth muscle cells. As EqTs form cation selective pores in cellular membranes, a direct effect on smooth muscle cells in the vascular wall may also play a major role. To answer this question, porcine coronary arteries were exposed to EqT III (1–100 nM), and the resting tension of smooth muscle as well as the maximum force of contractions were measured. The results revealed that EqT III also directly triggers contraction of isolated porcine coronary arteries at nanomolar concentrations. This mechanism probably explains most of the EqT III cardiotoxic effects [
52]. On Langendorff rat heart preparations, EqT II (0.1–10 nM) was reported to cause arrhythmia as well as decreased coronary perfusion rate and left ventricular pressure in a dose-dependent manner. At higher concentrations, EqT II produces cardiac arrest within a few seconds. As in the rat heart, EqT II also decreases coronary flow in the porcine heart. This effect could be abolished by an antagonist of L-type voltage-dependent calcium channels (i.e., Ca
V1.2 channels) such as nicardipine. Additionally, it was shown that EqT II increases the tension of spontaneous contractions and induced long-lasting contracture of guinea pig
taenia caeci smooth muscle, accompanied by a marked increase in [Ca
2+]
i [
55]. After i.v. administration, EqT II first enters into the right atrium and then the right ventricle of the heart before reaching the pulmonary circulation. EqTs have high binding affinity for sphingomyelin-rich cell membranes [
5,
56,
57,
58] and thus rapidly bind to blood cells and endothelium. In order to assess the possibility that a sufficient concentration of unbound EqT II is still present in the arterial blood and in the systemic circulation to produce direct cardiotoxic effects, perfusion experiments were performed on isolated rat lungs. After in vitro perfusion of the lung with a solution containing 100 nM EqT II, the toxin concentration in perfusates ranged between 0.8 and 5 nM. Effluent from the lungs contained enough EqT II to produce cardiotoxic effects on isolated Langendorff heart, as described previously [
51]. This is in accordance with the findings that the lethal effects of EqT II are mainly attributed to its vasoconstrictor effects and direct cardiotoxicity. The mechanism of EqT II-induced respiratory arrest is not yet sufficiently explained. After i.v. administration of EqT I, EqT II or EqT III, the respiratory activity stops within a few seconds. It was shown, at least for EqT I, that electrical stimulation of the phrenic nerve triggers normal diaphragm muscle contraction indicating that neuromuscular transmission and function are unaffected by the toxin. Because respiratory arrest causes cardiac hypoxia, the alterations in blood pressure and electrical activity, similar to those observed after administration of one mouse LD
50 of EqTs, could cause cardiac hypoxia. However, experiments performed on artificially ventilated animals, showed that artificial ventilation did not prevent the changes in ECG and blood pressure. Therefore, hypoxia was not confirmed as a primary cause for cardiotoxicity. Respiratory arrest may also be caused by respiratory reflexes activated through J-receptors in lung parenchyma, which are strongly stimulated under pathophysiological conditions like pulmonary oedema. EqTs are relatively large molecules (with molecular masses of around 20 kDa) and, due to their size, it is unlikely that they could pass the brain-blood barrier unless endothelial damage gives access to the neurons of the respiratory centre in the
medulla oblongata. Recent preliminary results have shown that EqT II causes swelling and lysis of endothelial cells, an effect that may give toxin access to neuronal cells. Direct effects of EqTs on the respiratory centre cannot be excluded since EqT II has been reported to produce swelling of differentiated neuroblastoma NG108-15 cells [
59]. Moreover, axonal swelling at the node of Ranvier of myelinated nerve fibres has also been observed in vitro after application of EqT II [
60].