Reduction in arterial blood pressure can result from a reduced total peripheral resistance (TPR) and/or a reduced cardiac output. Already in 1890, Takahashi and Inoko concluded that the heart does not contribute to the TTX-mediated blood pressure reduction [26]. The authors explained the hypotensive action of the toxin exclusively with a reduced vasomotor tone, resulting in a significant TPR reduction. Compression of the abdominal aorta or volume expansion instantaneously normalized a markedly lowered carotid blood pressure and a slightly lowered pulse rate in TTX-poisoned cats and dogs [26]. A direct negative inotropic or chronotropic action of TTX in intoxicated animals was excluded also in most if not all subsequent studies. For example, Cheng and colleagues [43,46] found that the blood flow in the ascending aorta was not measurably altered when injecting a dose of 0.25 MLD into right external jugular vein (Table 2), demonstrating that the simultaneously observed arterial hypotension was caused by peripheral vasodilatation only.

The mechanisms behind the TTX-mediated vasodilatation were investigated by several groups. The significance of each mechanism, however, was discussed controversially [39,41]. Firstly, TTX could act on medullary structures known to be involved in cardiovascular control after the toxin crossed the blood-brain barrier [26,27,35,36,41,45]. Hypotension occurred nearly instantaneously when low doses of TTX were injected into the brain or carotid artery of cats and rats [35,48]. In another study, a fast vasodepressive response and a delayed but significant bradycardia was observed when 12.5–25 pmol TTX were injected into the cat nucleus reticularis lateralis (NRL), a ventromedullary vasopressive region [40]. Microinjection of the same TTX dose into the nucleus tractus solitarii (NTS), a well known vasodepressive and cardio-inhibitory area, caused an increase in blood pressure. Secondly, vasodilatation resulted mainly from a conduction block in sympathetic fibers [27,35,37,39,41,43,46]. Thirdly, low doses of TTX may directly affect vascular smooth muscles [39]. Several authors, however, did not observe any effect of TTX on smooth muscle tone [27,37,43].

Slowing of the heart rate may also contribute to hypotension. However, administration of small sub-lethal doses produced hypotension only, and apparent changes of heart rate were rarely seen. TTX in doses of 0.1–0.3 MLD, corresponding to about 16–47 nM in the whole body’s extracellular fluid, did not cause bradycardia in the cat, despite a prompt and marked fall in blood pressure [37–39,49]. For example, Kao et al. demonstrated that the carotid blood pressure in cats can be reduced by i.v. injection of 2–3 μg/kg from 140/110 to 80/50 within two to three minutes, without affecting heart rate [41]. In some studies, even lethal doses of 7–10 μg/kg did not significantly alter heart rate in cats [38]. When heart rate reduction was measured, it often occurred subsequent to the onset of hypotension [38,40,45,46]. Severely intoxicated guinea pigs (15 μg/kg, i.p.) showed no signs of ECG alterations shortly before respiratory failure and death, despite a substantial drop in blood pressure [45]. In pithed rats, heart rate fell about 10% after injection of 1–2 MLD, but also soon regained the pre-injection state, in contrast to the long-lasting blood pressure reduction [43]. In another study, a 30–40% reduction in heart rate was observed in intoxicated rats (20 μg/kg). A few minutes after artificial respiration was commenced heart rate increased by 20%, but blood pressure further declined [48]. Altogether, these data indicate that bradycardia is not the primary cause of hypotension, although it may contribute to some extent to a reduced arterial blood pressure, in particular in severe cases of TTX intoxication.

Concerning the mechanism behind the observed bradycardic effect, one can exclude a primary chronotropic effect via blockade of cardiac TTXs Na⁺ channels: Firstly, direct application of a highly concentrated pufferfish solution or a toxin powder on the sinus node had no effect on the heart rate [27]. Secondly, Tomlinson and James [47] injected a volume of 2 ml of a 310 nM solution rapidly into the sinus node artery of dogs in order to avoid profound extracardiac effects of systemically applied TTX. The authors noticed that heart rate and rhythm remained constant. Only a 10-fold higher concentration caused a negative chronotropic effect (see below). Thirdly, sympathetic denervation of the cat heart resulted in a fall in heart rate to ~42–71%, but the subsequent application of a relatively high toxin dose (15 μg/kg) did not further decrease the heart rate [37]. Fourthly, the same lethal TTX dose of 15 μg/kg was rapidly injected as a bolus intraperitoneally into guinea pigs [45]. The authors noticed a decline in blood pressure, but even several minutes after intoxication, a regular heart rate and no change of the ECG wave form. ECG alterations indicative of paroxysmal ventricular tachycardia, sinus bradycardia and atrioventricular (AV) block emerged only after a complete cessation of respiratory movement and the substantial drop in arterial O₂ (see also Section 4). Fifthly, TTXs Na⁺ currents do not contribute to normal automaticity in isolated adult sinus node cells, as recently shown by Protas and co-workers [34]. The authors demonstrated that Na⁺ current density in the canine sinus node decreases with age and that TTXs Na⁺ channels are not available at physiological potentials [34]. Application of 100 nM TTX did neither change cycle length nor action potential parameters of adult sinus node cells. Consequently, TTXs Na⁺ channels are present in the sinus node, but they do not exert a physiological function under normal conditions [34]. This conclusion is in agreement with previous data [50].

The transient or permanent reduction of the heart rate is most likely the result of a complex systemic reaction to TTX intoxication. There are several parameters that interfere with a normal heart beat in intoxicated mammals, like (a) a diminished pressoreflex despite hypotension, due to blockade of respective afferent and efferent nerve fibers, (b) a reduced venous return, due to massive vasodilatation and hypotension [39], (c) uncertain effects of respiratory irregularities on medullary cardiovascular centers normally involved in respiratory sinus arrhythmia, (d) direct effects of TTX on medullary neurons [26,35,40,45], or (e) block of sympathetic nerve fibers [37]. In particular the latter effect seems to play a predominant role in TTX-induced bradycardia [37], because in isolated sympathetic nerve-right atrium preparations, TTX at low concentrations (30–60 nM) abolished positive inotropic and chronotropic responses to nerve stimulation without any effect on spontaneous rate or force.

At very high TTX doses of at least 1 MLD, the toxin has a direct cardiac effect [26,27,43], most likely via blocking an increasingly number of Na_v1.5 channels, leading to sinus bradycardia and conduction disturbances (see Section 3.4). This conclusion is supported by the following observations. Firstly, bradycardia was induced only when injecting a TTX solution of 3 μM into the canine sinus node artery (see above) [47]. Similar data at 3 μM TTX were observed using isolated rabbit atrial muscle strips including the sinus node [51]. Notably, sinus node action potentials were retained even at a concentration of up to 30 μM, which is far in excess of what is necessary to block conduction of nerve and skeletal muscle [51]. Secondly, very similar symptoms, i.e., sinus bradycardia and/or sinoatrial block, are observed in familial SSS, a well-recognised loss-of-function SCN5A channelopathy [7]. Recently, Verkerk and co-workers even succeeded in demonstrating a large Na⁺ inward current in human sinoatrial node cells at resting potentials negative to −60 mV [52]. Although the molecular nature of this current could not be elucidated in detail, the pronounced hyperpolarized closed-state inactivation clearly points to Na_v1.5 channels. And thirdly, heterozygous Scn5a^+/− mice showed depressed heart rates and occasionally sinoatrial block [53], implicating an important role of the normal cardiac Na⁺ channel, Na_v1.5, for pacemaker rates and sinus node conduction.

A reduced contractile force at low TTX concentrations has been suggested for Langendorff-perfused mouse and guinea pig hearts [11]. Consequently, a resulting decline in stroke volume should be visible in intoxicated animals, and this decline should contribute to hypotension.

However, unchanged stroke volumes were observed in independent studies, when sub-lethal or, depending on the application method, even lethal TTX doses were applied. For example, an unchanged blood flow in the ascending aorta of rats was observed, when injecting a sub-lethal dose of 2.5 μg/kg into the right external jugular vein. At this dose, arterial blood pressure was reduced from 120–150 to 40–55 mmHg [43]. In a similar study, injection of 5 μg/kg caused a rapid fall in blood pressure at an initially unchanged heart rate and stroke volume [46]. Furthermore, slow intravenous TTX injections of 9.3 μg/kg, a lethal dose for dogs, did not alter the cardiac index until respiratory arrest [44]. At apnoea, the authors reported blood pressure reduction and decline in heart rate at an unchanged stroke volume. Using isolated perfused rat hearts, the TTX-containing blue-ringed octopus venom caused even a marked increase in ventricular contraction tension [48], although this effect could be caused by other compounds in the toxin extract.

In some studies, a reduced contractile force was observed upon TTX intoxication [35–37,43]. The apparent negative inotropic action of TTX was discussed as the result of three different mechanisms: (a) blockade of excitability of central and/or peripheral nerve fibres at nanomolar concentrations, preventing neurogenic catecholamine release [35–37,41,43], (b) a direct negative inotropic effect occurring at micromolar concentrations in association with the reduction of the maximum rate of depolarization of the ventricular action potential, an effect that was not seen at 30 to 120 nM TTX [43,54–56], and (c) reduced cardiac output when marked peripheral vasodilatation caused a reduction in venous return [39,46]. The latter mechanism is probably the most important one, because hypotension occurred often sooner than cardiac depression [38]. Moreover, compression of the abdominal aorta as well as volume expansion normalized arterial blood pressure and cardiac performance instantaneously [26,27,39,41].

ECG alterations indicative of cardiac conduction abnormalities were observed rarely at sub-lethal doses of TTX (Table 2). Only slight PR prolongations were observed in cats and dogs when injecting 2.5–10 μg/kg [37,38]. In other studies, ECG alterations were not seen in the same species at similar toxin concentrations [35,36,43,49]. In guinea pigs, the ECG waveform remained unchanged even minutes after injecting a high lethal toxin dose (15 μg/kg) [45].

Already in their early reports on the systemic effect of TTX, the Japanese researchers noticed conduction abnormalities in severely intoxicated animals, particularly after the cessation of respiration [26,27]. Severe TTX intoxication, which is typically characterized by cyanosis, areflexia, apnoea and hypotension, often triggered second or third degree AV block. In many intoxicated animals, ventricular contractions appeared regular, but at a lower frequency than those of the atria. Subsequent to complete AV block, ventricles stopped beating, but atria not. In one case, Takahashi and Inoko found atrial contractions that persisted even after the onset of rigor mortis [26]. Although it seems that these previous reports are a little antiquated, basically similar results were observed tens of years later using advanced techniques [37,43,45,57]. High lethal TTX doses (1–4 MLD) often caused conduction irregularities such as lengthening of the AV interval, true AV dissociation, bundle brunch block, ventricular flutter or fibrillation. Ventricular asystole was noticed when rapidly injecting a dose of 8 MLD in rats [43].

Conduction disturbances in severely intoxicated animals were most likely due to two mechanisms: (a) blockade of a significant portion of cardiac Na_v1.5 channels, and (b) insufficient oxygen supply. The first conclusion is supported by the observation that conduction abnormalities were often associated with SCN5A channelopathies, like isolated CCD or LQT3 [6]. Moreover, Scn5a^+/− mice showed impaired AV conduction [58]. The second conclusion is supported by the fact that AV conduction block or paroxysmal ventricular tachycardia was observed subsequent to respiratory arrest and to the onset of hypoxia [26,45].

According to a previous report [17], one should expect that the Purkinje fibers are the cardiac structures that respond most rapidly to TTX treatment with a diminished function. However, it is interesting to note that the AV node is obviously most susceptible to TTX. Yoo and colleagues precisely studied Na_v1.5 expression in different regions of the AV node [59]. Na_v1.5 labeling was absent in the open node, but present at a relatively low level in the pathways into the open node (inferior nodal extension and transitional zone). This suggests that the reduced expression of Na_v1.5 already diminishes the safety factor for conduction, and consequently, TTX selectively aggravates conduction in this AV nodal region.

Other ECG changes were rarely reported. Some authors noticed T-wave alterations, like a non-specific T wave reversal [38], the disappearance of the T wave [27], or an increase in amplitude [49]. Reasons for these phenomena are unknown. Kao and Fuhrman suggested an involvement of the adrenal medulla in intoxicated animals, and consequently, an indirect effect on the heart of circulating catecholamines [49]. In another study, a decrease in P wave at a relatively large sub-lethal dose of 5 μg/kg was found in dogs [38].

TTX is one of the most potent, non-protein poisons known to man. It has been detected not only in many pufferfish species, but also in a wide variety of other animals, including the blue-ringed octopus, newts, gobies, frogs, worms, starfish, horseshoe crab, gastropods, and in several marine microorganisms, that produce and accumulate the toxin [60,61]. In the pufferfish body, TTX concentration and distribution depend on the species. Ovary, liver and skin usually contain highest TTX concentrations [60].

Despite, or maybe even because of its severe toxicity, pufferfish is a speciality in Japan. The fish, also known as “fugu”, is normally prepared by licensed puffer cooks, making the (expensive!) meals a culinary adventure, rather than a hazardous Russian roulette. Pufferfish poisoning or tetrodotoxication is, however, still one of the most common food poisonings along the coast of Asia. Ingestion of the toxic fish can be intentional or inadvertent. In Japan, it may happen by eating homemade liver dishes prepared from self-caught pufferfishes. In Bangladesh, several outbreaks of TTX poisoning with dozens of victims were reported, which occurred after pufferfishes (called “potka” fish) were available at local markets at a very cheap price [62–64]. Emergency physicians in Southern Europe should also be aware of the typical intoxication symptoms. Probably due to climate change and rising water temperatures, the toxin has approached the Portuguese coast, and caused recently the first case of tetrodotoxication in Europe (intoxication by a trumpet shellfish species) [65,66].

There is a long history of TTX intoxication. The first Europeans severely intoxicated were Captain Cook and his naturalists, J.R. and G. Forster. They enjoyed a pufferfish dinner, survived, and precisely described their symptoms [67]. Many case reports were known until 1941, when Fukuda and Tani provided a clinical grading system for TTX poisoning [61,68]. This four-degree classification is based on symptoms and stages of progression, and is still of clinical value (Table 3). The degree of intoxication actually depends on three factors: the TTX amount ingested, the time until admission to the emergency department, and pre-existing diseases (see below). First-degree and second-degree cases are relatively mild cases of tetrodotoxication (Table 3). There is a diminution or loss of some neuromuscular and neurological functions, but reflexes are still intact. The third degree is characterized by more severe disturbances, like ataxia, widespread paralysis, pronounced hyporeflexia, drop in blood pressure, fixed/dilated pupils, cyanosis, and respiratory failure (e.g., dyspnoea, decreased vital capacity or lower forced expiratory volume). Hypothermia also develops, when skeletal muscle contractions and conduction in nerve fibers are gradually blocked. Fourth-degree cases are severely intoxicated victims presenting with cessation of respiration, decreased arterial O₂, unconsciousness, bradycardia, and hypotension. Severe cardiac manifestations, like conduction block or ventricular asystole, occurred when extremely high levels of TTX were ingested and as a late sign in fourthdegree intoxicated patients [68–70].

Tetrodotoxication can be rapidly fatal, and antidotes do not exist. Death may occur in as little as 17 minutes after ingesting the toxin [71,72]. The human killing dose is assumed to be 1–2 mg [61,72,73]. Treatment is entirely supportive and may involve mechanical ventilation for oxygen supply, normal saline infusion for distending the intravascular volume, gastric emptying procedures, application of activated charcoal, atropinization, or treatment with dopamine. Cholinesterase inhibitors and other drugs have been suggested, but not tested adequately [69,70,74]. Prognosis is good if the patient arrives at the emergency department conscious and prior to respiratory arrest, and if the patient survives the first 24 hours. Symptoms usually resolve over a period of 24 hours to five days [64,65,75–77]. Early diagnosis and prompt clinical management essentially contribute to low mortality rates. In Japan, the fatality rate fell from 80% at the beginning of the last century, with more than 100 deaths per year, to about 6% in the 90s [41,60,61]. In other Asian countries, mortality rates between 2–22% were reported recently [62–64,75].

In contrast to the in vitro assays (see 2) and in vivo animal experimentations (see 3), when crystalline TTX was used, human tetrodotoxication occurs by ingesting TTX-containing food. It is possible that this crude or combined TTX is more easily absorbed in the digestive tract. A respective crude TTX dose of as little as 20 μg/kg can be lethal for humans. However, feeding cats with a 10-fold higher dose of crystalline TTX (200 μg/kg) did not kill them, although an i.v. or i.p. injection of 10 μg/kg was lethal [35]. Furthermore, it is possible that the toxin-containing food exerts slightly different effects in the human body, when compared to those observed after injecting a highly purified TTX solution into animals. Some marine species may produce and accumulate also smaller quantities of TTX analogs, or possibly even other toxic compounds, so that the symptoms may not always be solely due to authentic TTX [66].

Electrophysiological recordings demonstrated an IC₅₀ of about 10 nM for TTXs Na⁺ channels [2]. In order to compare this in vitro TTX sensitivity with blood levels in patients, and to correlate the in vivo TTX concentrations with the symptoms, all currently available blood concentrations were plotted against the intoxication grade of the corresponding patients (Figure 1; for values see Table 4). In less severely intoxicated patients (first and second degree), blood levels were similar to or lower than the IC₅₀ of TTXs Na⁺ channels. In severe cases of intoxication (fourth degree in Figure 1), TTX concentrations of 40–164 nM were observed. The calculated average concentration of 88 ± 22 nM (n = 5) would be indeed high enough to block the vast majority of TTXs Na⁺ channels in the body.

Unfortunately, a precise correlation between the biological activity of an ingested TTX bolus and the clinical symptoms is currently difficult to establish, because blood concentrations in victims were rarely determined. It is likely that the effective TTX concentration in the heart is higher than the values reported. All TTX concentrations were determined from “spot” samples taken during the first day of hospitalization. Peak concentrations were probably missed in some cases. Moreover, serum levels may not necessarily indicate the effective concentration in an organ. TTX is rapidly absorbed in the human digestive tract, but it remains in the serum compartment only for a relatively short period (less than 24 h). From day two of hospitalization, TTX serum levels were below the detection limit in severe intoxicated patients [78,79], but TTX appeared in the urine until day four [78]. During this time, intoxication symptoms persisted and mechanical ventilation was still required [79]. It seems that TTX is rapidly eliminated from the serum not only by excretion in the kidney, but also via an efficient binding to the TTX receptors. Thus, the toxin may accumulate in an excitable tissue. As suggested by O’Leary and colleagues [80], elimination of TTX from the body requires unbinding from Na⁺ channels and redistribution to the serum. This could be the rate-limiting step in the elimination of TTX from the body. The idea that TTX accumulates in organs with TTX binding sites is supported by previous work of Ogura who detected high TTX levels in whole heart and kidney (excretion), and lowest concentrations in brain and blood [41,42]. Low levels in the brain suggest a limited ability of TTX to cross the human blood-brain barrier. In this context it is interesting to note that victims often remain conscious, but cannot speak, move or breathe.

Cardiac dysrhythmias, like AV node conduction abnormalities, bundle-brunch block, tachycardia, or even ventricular stoppage were previously reported to occur in some severely intoxicated patients [68,69]. Because a lethal dose of about 2 mg TTX is unlikely to result in serum concentrations significantly higher than 200 nM, blocking cardiac TTXs Na⁺ channels, rather than blocking Na_v1.5, could contribute to these cardiac dysrhythmias. However, cardiac excitation abnormalities may not causally depend on a direct action of TTX on cardiomyocytes, but mainly on oxygen limitation resulting from respiratory arrest. If the latter assumption is true, cardiac dysrhythmias in severely intoxicated victims should have been observed rarely during the last 25 years, because of the high standard of modern medicine and the prompt availability of a mechanical ventilation device when respiratory arrest is imminent.

To prove this assumption, a literature screening for reports on outbreaks and cases of tetrodotoxication between 1983 and 2009 was performed (Table 4). Particular attention was paid to cardiovascular effects in victims. Table 4 represents a summary of several representative reports, but not a complete catalog of all cases of tetrodotoxication. In those 20 studies, more than 500 cases of tetrodotoxication were reported. Notably, all patients were admitted to an emergency department or an intensive care unit. However, the cardiovascular status, including blood pressure or ECG, was often not explicitly mentioned, despite an accurate description of neurological and neuromuscular symptoms.

The overall mortality rate during this 27-year period was about 6.6% (34 fatal cases). Hypotension was reported in 22 cases (4.2%). Blood pressure reduction was usually accompanied by rather severe other symptoms, like respiratory arrest or block of nerve conduction. Among fourth-degree cases, only 20.6% of the patients developed hypotension, as documented in [75]. Sinus bradycardia was mentioned only in seven cases, and two of those patients had a pre-existing chronic disease (diabetes mellitus). Surprisingly, AV block was diagnosed only in one patient, who experienced cardiac arrest before arriving at the emergency department and who also had diabetes mellitus [76]. No other pathological ECG signs, like bundle brunch block or ventricular arrhythmias, were reported (Table 4). Even severely intoxicated patients, i.e., fourth-degree victims, presented with a stable cardiovascular status, when they were successfully resuscitated or when oxygen supply was rapidly provided by artificial ventilation [65,89]. In a recent case report, the patient’s serum contained nearly 80 nM TTX, which was shown to be high enough to completely block nerve conduction [65]. At the same time, heart rate and blood pressure were normal, and cardiac conduction disturbances were not observed [65]. The authors concluded that cardiac arrhythmias or cardiac arrest, as observed for example in [85], were due to hypoxemia secondary to respiratory muscle paralysis [65]. Higher blood concentrations of 114 nM were observed in another patient that developed bradycardia and hypotension, but no cardiac conduction abnormalities were noted [78]. More severe symptoms and poor prognosis were reported for victims with a pre-existing disease, in particular for diabetes mellitus patients [76,83]. The reasons are unknown, but there could be a synergistic effect of diabetic neuropathy and TTXs Na⁺ channel blockade on nerve and skeletal muscle excitability [76].

It is interesting to note that cardiovascular manifestations, like hypotension and sinus bradycardia, cannot be considered as general accompanying symptoms in severely intoxicated and mechanically ventilated patients [65,88,89]. Some patients even presented with hypertension [83]. If TTXs Na⁺ channels increase the spontaneous heart rate and improve the contractile force under normal physiological conditions, their blockade by TTX would result in a decreased cardiac output. In order to maintain (or increase) the arterial blood pressure, such a cardio-depressive effect must be counteracted by an increased vasomotor tone. This scenario is very unlikely to occur when conduction in sympathetic fibers is partially or completely blocked. The TPR in TTX-poisoned patients was not yet determined. However, TTX should produce vasodilatation instead of vasoconstriction, and hypotension can be sufficiently explained by a reduced vasomotor tone (see 3).

The heart beat was regular, also when sinus bradycardia was noted [70,87]. In another study, an intermittent sinus bradycardia was reported [86]. The occurrence of cardiac dysautonomia, i.e., transient increases and decreases in heart rate, did not correlate with the TTX levels [86]. As discussed by the authors, the phenomenon of intermittent sinus bradycardia is likely due to TTX-mediated imbalances between the parasympathetic and sympathetic nervous system [86].

In conclusion, severe neurological and neuromuscular symptoms in third-degree and fourth-degree intoxicated victims strongly suggest that a significant portion of functional TTXs Na⁺ channels was blocked. Except for one patient, who experienced cardiac arrest before admission to the emergency department, ECG abnormalities, indicative of cardiac arrhythmias like SA block, AV block, bundle brunch block or ventricular tachycardia/fibrillation, were not reported to occur during treatment of the patients in an emergency department or intensive care unit. This strongly suggests that cardiac excitation is not significantly impaired in intoxicated victims with relatively high TTX blood levels, as long as sufficient oxygen is provided.