Abstract: The present study is a trial to suppress or attenuate the cardiotoxicity of catecholamines, which have been developed synthetically for the treatment of several cardiovascular disorders and conditions such as asthma and nasal congestion. Epinephrine (Adrenaline) is the most commonly available inotrope and in many cases the most appropriate drug to maintain blood pressure. However, tachycardia, ventricular arrhythmias and hypertension are side effects of this drug. In this investigation, induction of catecholamines cardiotoxicity was experimentally achieved using epinephrine. In isolated toads’hearts, the cardiac arrhythmias produced by different concentrations of epinephrine (10, 50, 100, 200 and 300 ng ml-1) included extrasystoles, tachyarrhythmias and bradyarrhythmias as well as abnormalities of both P-wave and ST segment. Application of wild honey (0.5 g ml-1) nearly managed to counteract these effects. These results suggest that wild honey may be useful in the treatment of some cardiac arrhythmias, especially which are associated with hyper-adrenergic activity.
Introduction
Honey is a most remarkable substance. Revered by the ancients, it can be enjoyed today in exactly the condition in which it was discovered many thousands of years ago. It is the only sweatening material that requires no manipulation or processing to render it ready to eat. The first known written passages dealing with honey have been dated about 4000 years ago, and honey has been treasured ever since. An extensive essay on the history of honey is available (Crane, 1975). As will become evident, honey is an exceedingly variable and complex material, and we are far from knowing all about it (White, 1992).
Several studies on the honey bee products, including honey as the most familiar product, started a long time ago. Most of these studies have focused on their potential health benefits for human, but the influence of these products on the cardiovascular system needs more investigations. However, Rakha et al. (2003) studied the effect and mechanism of action of wild honey on the electrical activity of the heart. Chemical analysis of the wild honey was also performed to establish the relationship between the honey constituents and its mode of action. The results of the study revealed that wild honey has both negative chronotropic and dromotropic effects as well as positive inotropic effect on the isolated toads’hearts. Besides, substantial quantities of calcium, potassium, chlorine, sodium and magnesium were also estimated in wild honey. The conclusion emerged that effects of wild honey on the heart activity may be due to its direct action on the myocardium. Moreover, minerals in wild honey play a prime role in its cardiac activity.
A number of synthetic catecholamines have been developed for the treatment of cardiovascular disorders and conditions such as asthma and nasal congestion because of their ability to activate alpha- and beta-receptors, especially in the cardiovascular system. However, high circulating concentrations of epinephrine and norepinephrine and high doses of synthetic catecholamines such as isoproterenol may induce toxic effects on the heart, including myocardial necrosis. Because the oxidation of catecholamines may result in the formation of aminochromes and oxygen free radicals, oxidative stress may play a significant role in catecholamine-induced cardiotoxicity (Klaassen and Watkins, 1999).
Sympathetic stimulation or the presence of catecholamines is known to encourage arrhythmia formation. Experimentally induced arrhythmia may be substantially reduced by the thoracic sympathectomy and cardiac denervation, while stellate ganglion stimulation increases sensetivity to arrhythmias. Furthermore, there is a close association between palpitation, tachycardia and arrhythmia in patients with phaeochromocytoma, which is a tumor of the adrenal gland that secretes large amounts of epinephrine and norepinephrine. Paradoxically, however, at times of extreme stress or emotion vagal activity can predominate, leading to bradycardia rather than tachycardia (Brown and Kozlowski, 1997).
Typically, catecholamine cardiomyopathy is considered an experimentally induced phenomenon. Numerous hypotheses have been suggested to account for the mechanism of cardiac damage by isoproterenol, a synthetic derivative of the naturally occuring sympathomimetic amines epinephrine and norepinephrine, that has been shown to produce myocardial necrosis closely resembling infarction. In particular, increased plasma membrane permeability to Ca2+ and other ions results in ionic imbalances, followed by subsequent rupture of the sarcolemma that is coincident with cell death. The mechanism by which the sarcolemma becomes more permeable has not been defined (Klaassen and Watkins, 1999).
Accordingly, the present study is a trial to suppress or attenuate the cardiotoxicity of catecholamines using wild honey. It is worth noting that, we suggested in our previous paper (Rakha et al., 2003) that clinical trials with honey bee products, in particular honey, are needed on the level of the cardiovascular pathophysiology which can be used clinically in preventing or reducing some cardiac disorders arising sometimes from the ionic imbalances. So, this approach will gain more attention in the present investigation.
Materials and Methods
Honey collection
Pure natural wild honey was collected from an apiary in the area of Saint
Katherine protectorate, Southern Sinai, Egypt. Honey solution was freshly prepared
by dissolving honey in frog Ringer’s solution for the in vitro experiments
on the cardiac muscle of toads.
Catecholamines cardiotoxicity
Induction of catecholamines cardiotoxicity was experimentally achieved by
using epinephrine (adrenaline) purchased from Sigma Chemical Corporation and
prepared immediately before use by dissolving it in frog Ringer’s solution.
Animals
31 adult male toads (Bufo regularis) were used in this investigation
as an experimental model for in vitro study.
Experimental design
Experiments on cardiac muscle were applied on two groups of isolated toads’hearts.
The first group was directly perfused with different concentrations of epinephrine
(10, 50 ,100, 200 and 300 ng ml-1) to emphasize the cardiac disorders
induced by epinephrine (5 isolated hearts/concentration). The second group (6
isolated hearts) was pre-treated with epinephrine (200 ng ml-1) to
induce cardiac abnormalities, then wild honey (0.5 g ml-1) was added
to reveal the anti-arrhythmic effect of wild honey against epinephrine-induced
cardiotoxicity.
Electrocardiogram (ECG) recording
Electrocardiogram (ECG) was recorded directly from the surface of the isolated
heart according to the method described by Rakha et al. (2003). ECG was
taken before any application to serve as self control. After the direct perfusion
of the isolated toads’ hearts with epinephrine or honey, signals were recorded
each 5 minutes by the multipen rectilinear recorder DBE, UK with a paper speed
of 2 mm sec-1.
Results
Epinephrine-induced cardiotoxicity
Table 1 represents the percentage of incidence of ECGs
abnormalities recorded from isolated toads’hearts perfused with different
concentrations of epinephrine (10, 50, 100, 200 and 300 ng ml-1).
ECGs abnormalities included extrasystoles, tachyarrhythmias and bradyarrhythmias
as well as abnormalities of both P-wave and ST segment (n= 5 / each concentration).
| Table 1: | ECGs abnormalities recorded from isolated toads’hearts perfused with different concentrations of epinephrine ( n = 5 / concentration) |
| Fig. 1: | ECGs records showing examples of cardiac disorders induced
by direct perfusion of isolated toads hearts with different concentrations
of epinephrine A-Before treatment and B-After treatment, B1-Sinus bradycardia (10 mg ml-1) B2-Complete heart block with packed P-wave and ventricular extrasystole (50 ng ml-1) B3-First degree block (100 ng ml-1), B4-ST segment depression (200 ng ml-1) B5-Ventricular fibrillation (300 ng ml-1) |
Fig. 1 shows examples of the cardiac disorders induced by direct application of epinephrine on isolated toads’ hearts, such as sinus bradycardia (10 ng ml-1), complete heart block with peaked P-wave and ventricular extrasystole (50 ng ml-1), first degree block (100 ng ml-1) and ST segment depression (200 ng ml-1) as well as ventricular fibrillation (300 ng ml-1).
The direct perfusion of isolated toads’hearts with different concentrations of epinephrine revealed that the epinephrine-induced cardiotoxicity, in vitro, is not concentration-dependent.
Anti-arrhythmic effect of wild honey against epinephrine-induced cardiotoxicity
A group of 6 isolated hearts was pre-treated with epinephrine (200 ng ml-1)
to induce cardiac abnormalities, then wild honey (0.5 g ml-1) was
added to reveal the anti-arrhythmic effect of wild honey against epinephrine-induced
cardiotoxicity. Cardiac arrhythmias produced by epinephrine included extrasystoles,
tachyarrhythmias and bradyarrhythmias as well as abnormalities of both P-wave
and ST segment. Application of wild honey nearly abolished the previous cases
(100%).
| Fig. 2: | Anti-arrhythmic effect of wild honey (0.5 g ml-1)
against eardiotoxicity induced by direct application of epinephrine (200
ng ml-1) on isolated toads hearts A-Before tratment B-After epinephrine application (ease I shows ventricular extrasystole with peaked P-wave and case II shows ventricular paraystole), C-After honey application |
Fig. 2 demonstrates two cases (I and II) of epinephrine-induced cardiotoxicity (200 ng ml-1) for example. Case I shows ventricular extrasystole, reflecting a ventricular focus with enhanced automaticity of the ventricle, as well as peaked P-wave, reflecting right atrial hypertrophy (BI). The direct application of wild honey (0.5 g ml-1) on isolated toads’ hearts pre-treated with epinephrine (200 ng ml-1), nearly suppressed the arrhythmia and the abnormality of P-wave (CI). Case II shows ventricular parasystole, which refers to the presence of concurrent impulse formation by two pace makers, one in the SA node and the other in the ventricle (BII). Also, honey counteracted this arrhythmia (CII). The amazing thing is the ability of honey to enhance the positive inotropic effect of epinephrine, which was obviously noticed in both cases (I and II).
Discussion
The control of arrhythmia by anti-arrhythmic agents is one of the most complicated and challenging problems of medical practice. While the general approach to therapy still remains empirical, some novel rational drug treatments are being investigated. There remain, however, considrable problems and these were recently highlighted when an extensive clinical trial revealed potentially lethal side effects of anti-arrhythmic drugs in widespread use (Brown and Kozlowski, 1997 ).
Sympathetic transmitters interact with ß-adrenoceptors and via activation of adenylate cyclase raise intracellular cyclic adenosine monophosphate (cAMP). This in turn opens (through phosphorelation via protein kinase A) calcium channels enhancing the inward calcium current, iCa,L . The rates of sino-atrial (SA) node discharge and the rate of conduction in SA and atrioventricular (AV) nodal tissue are both increased when iCa,L increases. ß-Agonists can also make the ventricular cells more excitable, partly by the increase in iCa,L they cause and also by inducing an inward current carried by chloride ions. ß-Blockers are successfully used to reduce the occurrence of ventricular arrhythmias after myocardial infarctions, which are partly the result of increased sympathetic activity.They are also used to slow conduction within the AV node and so to reduce the ventricular rate in supraventricular tachycardias (Brown and Kozlowski, 1997).
A prominent hypothesis to explain the cardiotoxicity of catecholamines such as isoproterenol is derangement of electrolyte homeostasis of myocardial cells at the level of the sarcolemma and other subcellular membrane sites. Electrolyte shifts in magnesium and potassium have been suggested as possible factors in the myocardial dysfunction and necrosis associated with isoproterenol administration. However, there is a six- to sevenfold increase in the rate of calcium uptake and a doubling of the net myocardial calcium content in isoproterenol-induced cardiac necrosis. Accumulation of large amounts of calcium in myocardial cells may alter the integrity and function of several membrane systems and affect mitochondrial energy production. Oxidative degeneration of membrane lipids may result in increased sarcolemmal calcium permeability. Because isoproterenol may form oxidative by-products, these metabolites could interact with the lipid bilayer, cause sarcolemmal injury, and alter calcium regulatory mechanisms (Klaassen and Watkins, 1999).
In the present study, induction of catecholamines cardiotoxicity experimentally using epinephrine revealed that the arrhythmias produced by the direct perfusion of the isolated toads’hearts with different concentrations of epinephrine included extrasystoles, tachyarrhythmias and bradyarrhythmias as well as abnormalities of both P-wave and ST segment. Wild honey nearly managed to counteract these effects.
In a previous report (Rakha et al., 2003); we showed that the effects of wild honey on the electrical activity of the the heart may be due to its direct effect on the myocardium. Moreover, it contains quantities of mineral elements high enough to be considered responsible for its physiological action on the heart muscle.
Consequently, it can be suggested that the anti-arrhythmic properties of wild honey against catecholamines cardiotoxicity induced by epinephrine may be mediated, in large part, through the action of magnesium found in wild honey in a relatively high level as estimated by the chemical analysis (Rakha et al., 2003). Since, there is an evidence that magnesium by inhibiting the voltage-dependent calcium channels (Castillo and Engbaek, 1954; Vital Brazil et al., 1973) suppresses or attenuates the effects of catecholamines in the cardiovascular system (Vital Brazil et al., 1999).
Delayed after depolarization (DAD), also called oscillatory after potential and transient depolarization, is an oscillation of the membrane voltage after completion of the action potential in phase 4 (January and Fozzard, 1988; Fazekas et al., 1993; Ferrier, 1977). DAD is produced by catecholamines and it is the cause of their cardiac arrhythmias (Vital Brazil et al., 2002). DAD results from calcium overload of heart cells. Oscillatory releases of calcium from the sarcoplasmic reticulum into the cytoplasm give rise to an inward current (the cause of DAD), which is produced by either activation of a nonspecific cation channel or the Ca2+/ Na+ exchanger or by both mechanisms (January and Fozzard, 1988). Calcium injection into cardiomyocytes induces it (Matsuda et al., 1982). Blockade by ryanodine of calcium release from sarcoplasmic reticulum inhibits DAD production, while an increase in [Ca2+]0 enhances DAD (Vital Brazil et al., 2002). DAD is suppressed by magnesium (Fazekas et al., 1993). Suppression of DAD by magnesium is accounted for, at least in part, by its well known property of causing blockade of calcium channels (Vital Brazil et al., 2002). Also, the notable level of sodium in wild honey, which was estimated by its chemical analysis (Rakha et al., 2003); may be involved in its anti-arrhythmic potency against catecholamines cardiotoxicity since it is important for the process of Ca2+/Na+ exchange that the energy to move each calcium ion out of the cell is provided by the entry of three sodium ions which in turn prevents Ca2+ions accumulation within the cardiac cells (Brown and Kozlowski, 1997) and accordingly helps in establishing and maintaining the different ion gradients between the intracellular and extracellular space which can help in decreasing the excitability of ventricular cells and consequently reduces the occurrence of ventricular arrhythmias as ventricular extrasystole resulting from increased sympathetic activity under the influence of catecholamines.
Cardiac arrhythmias produced by the direct application of epinephrine on the isolated toads’hearts included tachycardia and ST segment depression. Attenuation of these effects by the wild honey may be due to its high potassium level, since Rakha et al. (2003) noticed that wild honey causes bradycardia and ST segment elevation to the isolated toads’hearts and attributed these effects to the high potassium level in wild honey after its chemical analysis. It also coincides with Heger et al. (1994) who proved that hyperkalemia leads to bradycardia as well as ST segment elevation.
At the same time, the pronounced level of calcium in wild honey can counteract the increased conduction velocity in the atrioventricular (AV) nodal tissue produced by catecholamines as ß-Agonists. Since, Rakha et al. (2003) stated that wild honey decreases the conduction velocity in the isolated toads’hearts and ascribed this effect to the pronounced level of calcium in wild honey after its chemical analysis. This also agrees with Heger et al. (1994) who mentioned that the P-R interval may be prolonged as a result of hypercalcemia.
On the other hand, the carrier molecules concerned with pH regulation in cardiac cells are first, the acid extruders which are activated when the internal pH falls. One of them is a sodium/bicarbonate symporter which moves Na+ and HCO3¯ ions into the cell together. The entry of Na+ ions raises internal sodium concentration and hence leads to less extrusion of Ca2+ ions by Ca2+/Na+ exchange leading to accumulation of Ca2+ ions inside the cardiac cells. It is becoming evident that ß-Agonists stimulate the activity of the Na+/HCO3¯ symporter (Brown and Kozlowski, 1997) and it can be considered that this activation may be responsible for the cardiac arrhythmias produced by catecholamines as a result of calcium overload.
The second type of membrane transporters concerned with pH regulation are acid loaders which are activated when the internal pH rises. A chloride / bicarbonate exchanger removes HCO3¯ ions from the cell in exchange for Cl¯ ions, leaving H+ ions inside. This is steeply activated in response to alkalosis. In this respect, honey is considered an alkaline food because it is extraordinarily rich in mineral elements, which have an alkaline potentiality ( Rakha et al., 2003); that in turn stimulates the Cl¯ / HCO3- exchanger and consequently can suppress or reduce the activity of the Na+/HCO3¯ symporter stimulated by the action of epinephrine as a ß-Agonist. Especially, honey contains chlorine in high level which is essential for Cl¯/HCO3¯ exchange process (Rakha et al., 2003). A new evidence which can assure the anti-arrhythmic property of wild honey against catecholamines cardiotoxicity.
The results of this study revealed that wild honey managed to suppress or attenuate the cardiotoxicity produced by epinephrine. At the same time, it potentiated the inotropic effect of epinephrine, since wild honey itself has a positive inotropic effect (Rakha et al., 2003).
In conclusion, the results of the current research introduced an evidence for the anti-arrhythmic effect of wild honey against catecholamines cardiotoxicity, in particular epinephrine (adrenaline). The mineral elements in wild honey can be considered the responsible for this anti-arrhythmic property.
Although no previous work described the anti-arrhythmic property of honey against catecholamines-induced cardiotoxicity, more concentrated clinical trials are needed to use honey together with epinephrine to minimize or reduce its side effects. Especially, honey potentiates the inotropic effect of epinephrine and synergism between their inotropic effects can be predicted. Also, trying to use wild honey itself as an inotrope.