J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 215 Original Article Cardiotoxic and Arrhythmogenic Effects of Hemiscorpius lepturus Scorpion Venom in Rats Khalil Pourkhalili 1, Euikyung Kim 2, Navid Reza Mashayekhy 3, Mostafa Kamyab 4, Seyed Mehdi Hoseiny 5, Reihane Evazy 6, Abbas Zare Mirakabadi 7, *Ramin Seyedian 8 1Department of Physiology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran 2College of Veterinary Medicine, Gyeongsang National University, Jinju, South Korea 3Department of Cardiology, Amir Kabir Hospital, Arak University of Medical Sciences, Arak, Iran 4Department of Biotechnology, Persian Gulf Research Institute, Persian Gulf University, Bushehr, Iran 5Department of Hematology and Oncology, Bushehr University of Medical Sciences, Bushehr, Iran 6Department of Biology, Jahrom Branch, Islamic Azad University, Jahrom, Iran 7Department of Venomous Animals and Antisera Produstion, Razi Vaccine and Serum Research Institute, Hesarak, Iran 8Department of Pharmacology and Toxicology, Bushehr University of Medical Sciences, Bushehr, Iran (Received 1 Feb 2014; accepted 22 June 2014) Abstract Background: Envenomation by Hemiscorpius lepturus is not painful and the clinical manifestations include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions,cardiac arrhythmia and in minority of cases acute renal failure which may lead to death following disseminated intravascular coagulation in infants. Cardiac effects of envenomation by H. lepturus venom including inotropic, chronotropic and arrhythmogenic properties are not studied as now in rat hearts with Langendorff apparatus. Methods: Rat hearts were allowed to equilibrate in its buffer and cardiotropic plus arrhythmogenic effects induced by injection of different doses of H. lepturus venom were detected and recorded by computer acquisition based data in Langendorff apparatus. The neutralizing effects of Razi Institute antivenom and autonomic drugs were assayed in parallel studies. Results: Hemiscorpius lepturus venom (25 µ g/100 l) treatment caused a negative inotropic (65.4 ± 3.2 versus 110.2 ± 3.4) and chronotropic effects (186.3 ± 4.2 versus 302 ± 6.3) in comparison to normal saline. Arrhythmogenic aspects including bradycardia, QRS widening and ST depression were induced by venom injection. Pre venom treatment (20 min) of Razi Institute antivenom (10 µ l) neutralized cardiotropic effects but post venom injection (15 min later) had no therapeutic role. Pre (10 min before) and post (15 min after) injection of adrenaline (10 µ l) neneutralized cardiotropic effects while pre venom injection (20 min) of propanolol (10 µ l) had aggravating effects. Conclusion: Our study paves the way for further in vivo investigation of cardiovascular effects of this venom for finding suitable treatments instead of its ordinary antivenom medication in cardiogenic shock induced by the venom. Keywords: Hemiscorpius lepturus, Contractility, Arrhythmogenicity, Envenomation Introduction Scorpion envenomation is common in tropical and subtropical areas of the world including southwestern part of Iran espe- cially in Khuzestan province (Prendini 2000, Shahbazzadeh et al. 2009). Envenomation by H. lepturus belonging to family Hemiscorpiidae is not painful and the clinical manifestations in stung patients include bloody urine due to hemoglobinuria or hematuria, dermonecrotic reactions and in minority of cases acute renal failure which may lead to death following dis- seminated intravascular coagulation in infants (Radmanesh 1990, Jalali et al. 2010). This crea- ture is one of the most dangerous scorpions, and responsible for 95 % of the scorpion- associated mortalities in Iran, that is proba- bly due to the various pathological enzymes in its venom like Hemitoxin, Hemicalcin and *Corresponding author: Dr Ramin Seyedian, E- mail: raminseyedian@gmail.com http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 216 Heminecrolysin (Shahbazzadeh et al. 2007, Srairi-Abid et al. 2008). Generally there is only a few case reports on direct cardiotoxic effects of scorpion en- venomation in human beings (Gueron et al. 1967, Bawaskar and Bawaskar 1998, Shapira et al. 1998, Cupo and Hering 2002). How- ever previous studies on the envenomation by H. lepturus suggest that there are in vivo arrhythmogenic and negative inotropic ef- fects in rabbits in spite of its small amount of accumulation of radiolabelled venom follow- ing subcutaneous injections in animal mod- els during biodistribution study (Mirakabadi et al. 2011, Seyedian et al. 2012). In this study, the direct effects of H. lepturus venom on rat hearts were investigated with Langendorff apparatus. First, inotropic and chronotrpic changes upon time were evaluated and then, neutralization properties of Iranian Razi Institute Antivenom and other drugs in- cluding epinephrine, propranolol and atropine were employed to analyze direct or indirect cardiotoxic effects of this venom thoroughly promoting further in vivo studies. Materials and Methods Drugs and chemicals The lyophilized venom was acquired by direct electrical stimulation of H. lepturus telsons (15 mV) in Iran. The venom was centrifuged, lyophilized and stored at -20 °C until reconstitution by addition of Normal Saline in our laboratory. The Iranian pepsin digested polyvalent antivenom from horse plasma against 6 common endemic scorpions (Odontobuthus doriae, Mesobuthus eupeus, Androctonus crassicauda, Buthotus saulcyi, Buthotus sach and Hemiscorpius lepturus) (Latifi and Tabatabai 1979, Dehghani and Fathi 2012) were purchased from Razi Vaccine and Serum Research Institute. The neutralizing ability of the used batches was 26 LD50/ml. Adrenaline, atropine and propranolol were purchased from Sigma (St Loius, MO). Animals Female Wistar rats (body weight 180–230 g) were obtained from Jundishapur Medical University breeding center (Ahvaz, Iran) and placed in groups of three in PVC cages with free access to water and hard food pellets in animal house of Bushehr University of Med- ical Sciences. They were kept at 20 ± 2 °C and maintained at 12 hours light-dark cycle starting at 7 AM. The experimental protocol used in this study conforms to the guidelines of the National Institute of Health (NIH). Langendorff perfused heart preparation Rats were heparinized (300–400 IU) and anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). After a bilateral thor- acotomy, hearts were rapidly excised and after insertion of an aortic cannula perfused retrogradely at a constant perfusion pressure of 90 cm H2O with gassed (5% CO2, 95% O2) Krebs-Henseleit buffer (pH 7.4) contain- ing NaCl (118.5 mM), NaHCO3 (25 mM), KCl (4.7 mM), KH2PO4 (1.2 mM), CaCl2. 2H2O (1.8 mM), MgSO47H2O (1.2 mM), D- glucose 11 mM) at 37 °C. An epicardial elec- trocardiogram (ECG) was continuously rec- orded using two fine stainless steel elec- trodes, one attached to the apex of the heart and the other placed on the right atrium and a metal clip attaching to the aortic cannula as the reference electrode. Left ventricular pres- sure was recorded throughout the experiment by a water filled latex balloon placed in the left ventricle and connected to a pressure transducer (MLT 844) through a fluid filled catheter. Volume of the balloon was adjusted to obtain end-diastolic pressure of 8–10 mm Hg at the end of stabilization period and un- changed for the remainder of the experiment. The electrical and hemodynamic functions of the heart were continuously monitored with a computer-based data acquisition sys- tem (PowerLab system with Chart 5 software, AD Instruments, Australia). Left ventricular developed pressure (LVDP), rate pressure http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 217 product (RPP), Max and Min dp/dt and heart rate were calculated. Coronary flow (CF) was measured by timed collections of the coro- nary effluent. Experimental protocol Hearts were allowed to equilibrate for 20 min with its buffer. After the stabilization period, different doses of H. lepturus venom (25, 50 and 100 µ g) dissolved in normal sa- line (100 µ l) were infused directly acquiring cardiotropic, inotropic and arrhythmogenic changes with Langendorff apparatus. Razi Institute multivalent antivenom (10 µ l) dis- solved in Normal Saline (100 µ l) was inject- ed pre (20 minutes) and post venom (15 min) treatment. In another set of experiments in- cubated venom (25 µ g) with Razi Institute antivenom (10 µ l) was titrated to 100 µ l with normal saline for 30 min at room tempera- ture to investigate its neutralizing effect against cardiac aspects of H. lepturus ven- om. In order to evaluate the neutralizing ef- fects of β adrenoceptors and cholinoceptors drugs, propranolol (10 µ l) in addition to adrenaline (10 µ l) and finally atropine (100 µ l) were used pre and post venom injection. Coronary blood flow, left ventricular devel- oped pressure, dp⁄ dtmax and heart rate were recorded and outflow samples were collected every 1 min initially and every 10 min se- quentially until the end of our experiments (60 min) to evaluate the coronary flow chang- es induced by the venom. Statistical analysis Results are expressed as mean ± SEM of three experiments. ANOVA plus post hoc test was carried out in cardiotropic exper- iments. Values of P< 0.05 were considered significant. Results Inotropic and chronotropic effects of Hemiscorpius lepturus venom Cardiotropic effects of various doses (25, 50 and 100 µ g ) of H. lepturus venom re- constituted in Normal Saline (100 µ l) are shown in Fig. 1–3 (n=3). Representative image of negative chrono- tropic effects of different doses from H. lepturus venom with the same volume (100 µ l) on isolated rat heart in Langendorrf ap- paratus upon time versus normal saline in- jection was depicted in Fig 1. Results present mean ± SEM of 3 independent experiments. Reducing effects on the contractile po- tency of the heart was induced shortly (two min later) following H. lepturus injection in langendorrf apparatus.Venom caused a sta- tistically significant decrease in heart contrac- tility (negative inotropic) and rate (negative chronotropism) for 40–60 min before ending the examination at 60 minutes. Coronary flow was significantly decreased (7.83 ± 0.21 ml to 2.81 ± 0.62 ml) post venom injec- tion (25 µ g) in our experiments. The neutralizing capacity of Razi Institiute antivenom against cardiogenic effects of Hemiscorpius lepturus Razi Institute antivenom (10 µ l) was used as pre (20 min) and post (15 min) venom (25 µg) injection and its neutralizing effect against the venom were depicted in Fig. 4 A, B. Razi Institute antivenom pretreatment (20 min before venom injection) or incubation with venom for 30 min at room temperature sig- nificantly neutralized negative inotropic ef- fects while post venom injection had no effect. In another study incubated venom and antivenom (25 µg in 10 µl) for 30 min at room temperature was titrated with normal saline (100 µ l) and injected to evaluate the neu- tralizing effects of this remedy on rat hearts (Fig. 4C). There was no bradycardia and neg- ative inotropic effects following H. lepturus http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 218 venom (25 µ g) injection pre incubated with the antivenom and the post treatment of antivenom failed to counteract the cardiotoxic effects of venom injection. Arrhythmogenic effects of Hemiscorpius lepturus venom Venom perfusion prominently induced bradycardia, with widened QRS complexes and ST depression after passing 50 min in all the envenomed hearts as shown in Fig. 5A. Normal heart rate and ECG parameter were induced by normal saline (100 µ l) infusion to serve as control (not shown). Antivenom ad- ministration (10 µ l), 20 min before venom injection had neutralized QRS widening and bradycardia induced by H. lepturus venom (Fig. 5B). Injection of adrenaline and atropine in our study counteracted the late arrhythmogenic effects induced by H. lepturus venom (data not shown). Protection and aggrevating experiments using adrenaline, atropine and propranolol Pre and post injection of adrenaline (10 µ l) following using of H. lepturus venom are represented in Fig. 6A, B. Adrenaline (10 µ l) treatment 10 min be- fore and 15 min after venom injection neu- tralized negative inotropic effects upon time. According to these diagrams negative inotropic effects were significantly neutral- ized by this drug since there was no decreas- ing of LVDP (left ventricular diastolic pres- sure) following venom injection upon time. In another experiment, pretreatment of pro- pranolol (10 µ l) resulted in additive negative effects on LVDP in isolated rat hearts (Fig. 7) and in our final examinations incubation of atropine (100 µl) as an anticholinergic drug with our venom (25 µg) prior to injection neu- tralized its inotropic effects showing the probability of its indirect mechanism (Fig. 8). Pretreatment of isolated rat hearts with propranolol, 20 min before venom injection had no neutralizing effect on negative inotropic aspects as represented in this figure. Incubation of venom and atropine for 30 min had no negative inotropic effects in isolated rat hearts upon time. Fig. 1. Heart rate changes by different doses of Hemiscorpius lepturus venom in rats in Langendorff apparatus # Significantly different from normal saline at P< 0.05 Heart rate changes induced by different doses of H. lepturus venom B ea ts ( b ea ts /m in u te ) Time (minutes) http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 219 Fig. 2. Contractility changes induced by Hemiscorpius lepturus venom in isolated rat hearts # Significantly different from normal saline at P< 0.05 ##Significantly different from normal saline at P< 0.01 Fig. 3. Inotropic effects induced by injection of Hemiscorpius lepturus venom (25µ g) in rat hearts with Langendorff apparatus A B Time (min) L V D P (L ef t V en tr ic u la r D ev el op ed P re ss u re ) (m m H g) 4 min 4 min 4 min Venom (25 µg) Venom (25 µg)Venom (25 µg) Antivenom (10 µg)Antivenom (10 µg) L V D P ( m m H g) L V D P ( m m H g) L V D P ( m m H g) Inotropic changes induced by doses of Hemiscorpius lepturus on isolated rat hearts (n= 3) http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 220 Fig. 4. Inotropic changes following pre and post venom (25 µ g) injection of Razi Institute antivenom (10 µ l) A B Fig. 5. Arrhythmogenic affects of Hemiscorpius lepturus venom injection (25 µ g) upon time (5A) versus normal saline (5B). Bradycardia, QRS widening and ST depression were induced with venom injection in Langendorff apparatus 15 minutes before venom injection 10 minutes following venom injection 30 minutes following venom injection 50 minutes following venom injection 50 minutes following venom injection 30 minutes following venom injection 10 minutes following venom injection 15 minutes before venom injection 1 Sec 1 Sec 1 Sec 1 Sec 1 Sec 1 Sec 1 Sec 1 Sec Time (minutes) v ol ta g e (m V ) Cardiac effects of scorpion venom (25 µg) incubated with antivenom (10 µl) for 30 minutes v ol ta g e (m V ) v ol ta g e (m V ) v ol ta g e (m V ) v ol ta g e (m V ) v ol ta g e (m V ) v ol ta g e (m V ) v ol ta g e (m V ) C http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 221 A B Fig. 6. Changes in inotropic effects upon the pre and post treatment of epineprine (10 µ l) added before and after venom (25 µ g) injection Fig. 7. LVDP changes following pretreatment with propranolol (10 µ l), added 20 min before venom (25 µ g) injection Fig. 8. LVDP chnages by the venom (25 µ g) preincubated with atropine (100 µ l) for 30 min Discussion In this study, in vitro negative inotropic, chronotropic and late arrhythmogenic effects of H. lepturus envenomation in rats were shown with Langendorff apparatus. The most interesting findings in envenomed patients by H. lepturus are hemoglobinuria, hematuria, cutaneous necrotic ulcers and in minority of cases disseminated intravascular coagulation leading to death (Radmanesh 1990, Shayesteh et al. 2011) as seen in Loxosceles envenomation (Bay et al. 1997, Tavares et al. 2004). Envenomation with H. lepturus which is not painful is commonly seen in South West Provinces of Iran and treated with in- tramuscular injection of Razi Institute poly valent antivenom and followed by close monitoring. In previous in vivo studies, bradycardia (decreasing from 227 beats/min to 94 beats /min) and ST elevation in lead II was in- 4 min 4 min 4 min 4 min Atropine (100 µl)+ Venom (25 µg) Venom (25 μg)Propranolol (10 µl) Venom (25 µg)Epi (10 µl) Venom (25 µg) Epinephrin (10 µl) L V D P ( m m H g) L V D P ( m m H g) L V D P ( m m H g) L V D P ( m m H g) http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 222 duced with intravenous injection of extreme doses (50 µ g/kg) of venom in rabbits with no significant changes of lactate dehydrogenase and Creatinine phosphokinase one hour after envenomation showing no myocardial ne- crosis (Mirakabadi et al. 2010). In this paper, a Langendorff model was assembled to an- alyze the direct and indirect chronotropic and inotropic effects of H. lepturus venom on rat hearts. Our venom induced a persistant negative dose response inotropic and chronotropic effects in rat hearts until up to 60 min, similar to loxosceles envenomation (Shahbazzadeh et al. 2007, Chatzaki et al. 2012). This study pru- dently approved that Hemiscorpius lepturus cardiotoxicity was highly dependent on dose and time of its administration. Coronary blood flow was significantly decreased (10.4 ± 2.21 to 3.16 ± 2.58 ) in envenomed hearts with no changes in LDH and CPK before and 60 min after envenomation (Data not shown) ruling out the myocardial necrosis. According to the nature of poisonous animals, cardiotoxic effects of scorpion venoms could be ex- plained with prevention or releasing of neu- rotransmitters including acetylcholine and epinephrine from nerve terminals inducing changes in heart rate and contractility (Tarasuik et al. 1994, Sauviat et al. 1995). Our data suggest that H. lepturus venom could be suppressing, at least partially on the adrenoceptors mimicking agent presenting in venom or from varicosities in the heart like other poisonous creatutres (Gomes et al. 2010) in addition to direct binding to its myocardial receptors that their shape and origin will be clarified in further studies. In our experiment, pre and post injection of epinephrine (10 µ l) counteracted negative inotropic and chronotropic effects induced with H. lepturus venom. It should be noted that coronary blood flow was not decreased by pre and post venom injection of adrena- line (data not shown) indicating H. lepturus indirect effect via supression of β receptors as one of the most autonomic determinants of cardiac contractility and coronary perfu- sion (Gordon et al. 1998). In a parallel study, preinjection of propranolol (10 µ l ) as a sym- pathetic antagonist had an additive role on cardiotropic effects of H. lepturus venom supporting our hypothesis (Fig. 7). Based on our study pre venom injection of atropine or incubated mixture of venom with this drug, had significant neutralizing effect in contractility following envenomation clearly showing that negative inotropic ef- fect of this venom possibly was also depend- ent on cholinergic system by direct stimula- tion or activation of muscarinic receptors in atria with releasing of acetylcholine from nerve terminals like Tityus cerrulatus scorpion ven- om (Teixeira et al. 2001). Post venom injec- tion of Atropine had no neutralizing effect on cardiotropic aspects possibly due to binding of the venom to its ion channel receptors strongly. There is a great debate about the efficacy of scorpion antivenom in neutraliza- tion of the detrimental effects induced by envenomation (Gueron et al. 1967, Abroug et al. 1999, Ismail 2003, Chatzaki et al. 2012) since clinical efficacy of treatment for en- venomed patients is affected deeply with the time between scorpion sting and administra- tion of antivenom. Generally H. lepturus venom like other poisonous animals have low molecular weight diffusing rapidly to target organs including kidney and heart while the heavy chain molecules of antivenom have not this ability making it unsuitable for treating envenomed patients (Ismail et al. 1983, Seyedian et al. 2010, Seyedian et al. 2012). In our experiment, prevenom injection of antivenom in addition to incubation of ven- om and antivenom completely antagonized all deterimental effects including negative inotropic, chronotropy, decreasing coronary blood flow and even late arrhythmogenic effects (bradycardia, QRS prolongation and ST depression after 60 min) in isolated rat http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 223 hearts while post venom injection had no effect. It seems that antivenom could bind firmly to venom and prevents its direct and indirect effects while injected before enven- omation but due to its high molecular weight structure and different pharmacokinetic and biodistribution profile versus H. lepturus ven- om had no treating effects in envenomed hearts even when injected 5 min after enven- omation (data not shown). Scorpion venoms generally act mainly on Na+ and to lesser de- grees on calcium and potassium channels pro- voking electrocardiographic changes (Gordon et al. 1998). Arrhythmogenic changes in our envenomed hearts could be induced by late ischemia or prolongation of repolarization phase with some purified toxins from H. lepturus as Hemicalcins and Heminecrolysin (Shahbazzadeh et al. 2007, Borchani et al. 2011) acting on ion channels. Those effects were neutralized by prevenom injection of Razi Institute multivalent antivenom. Arrythmogenic changes with venom including QRS prolon- gation and ST depression were not observed during treatments with other drugs (atropine, propranolol and adrenaline) and antivenom as control. Conclusion It seems that cardiotropic changes including negative inotropism and chronotropism of H. lepturus venom in addition to late arrhythmogenic manifestations has a detri- mental effect in heart failure induced by en- venomation in human beings. We believe that indirect and direct mechanisms are involved in this phenomenon since injection of adrenaline and atropine according to their high and low presence in the rat heart changed our results. Pre venom injection of Razi Institute antivenom as the customary treatment could neutralize its cardiotropic effects but unfor- tunately it had no effect even 5 min after envenomations making its beneficial role on detrimental cardiac consequences questionable. Acknowledgements The authors would like to thank the au- thorities of Bushehr University of Medical Sciences for providing facilities. References Abroug F, Elatrous S, Nouria S, Haguiga H, Touzi N, Bouchoucha S (1999) Serotherapy in scorpion Envenomation: a randomized controlled trial. Lancet. 354: 906–909. Bawaskar H, Bawaskar P (1998) Indian red scorpion envenoming. Ind J Pediatr. 65: 383–391. Bey TA, Walter FG, Lober W, Schmidt J, Spark R, Schlievert PM (1997) Lo- xosceles arizonica Bite Associated with Shock. Ann Emerg Med. 30: 701–703. Borchani L, Sassi A, Shahbazzadeh D, Strub JM, Tounsi-Guetteti H, Boubaker MS, Akbari A, Van Dorsselaer A, El ayeb M (2011) Heminecrolysin, the first hemolytic dermonecrotic toxin purified from scorpion venom. Toxicon. 58: 130– 139. Chatzaki M, Horta C, Almeida M, Pereira N, Mendes T, Dias-lopes C, Guimarães G, Moro L, Chávez-olórtegui C, Horta M (2012) Cutaneous loxoscelism caused by Loxosceles similis venom and neu- tralization capacity of its specific antivenom. Toxicon. 60: 21–30. Cupo P, Hering SE (2002) Cardiac troponin I release after severe scorpion enven- oming by Tityus serrulatus. Toxicon. 40: 823–830. Dehghani R, Fathi B (2012) Scorpion sting in Iran: A review. Toxicon. 60: 919– 933. Gomes, HL, Andrich F, Mauad H, Sampaio KN, De lima ME, FIgueiredo SG, Moyses MR (2010) Cardiovascular ef- fects of scorpionfish (Scorpaena plumieri) venom. Toxicon. 55: 580–589. http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 224 Gordon D, Savarin P, Gurevitz M, Zinn- justin S (1998) Functional anatomy of scorpion toxins affecting sodium chan- nels. Toxin Rev. 17: 131–159. Gueron M, Stern J, Cohen W (1967) Severe myocardial damage and heart failure in scorpion sting: Report of five cases. Am J Card. 19: 719–726. Ismail M (2003) Treatment of the scorpion envenoming syndrome: 12-years ex- perience with serotherapy. Int J Antimicrob Agents. 21: 170–174. Ismail M, Shibl A, Morad A, Abdullah M (1983) Pharmacokinetics of 125 I- labelled antivenin to the venom from the scorpion Androctonus amoreuxi. Toxicon. 21: 47–56. Jalali A, Pipelzadeh MH, Sayedian R, Rowan E (2010) A review of epide- miological, clinical and in vitro physi- ological studies of envenomation by the scorpion Hemiscorpius lepturus (Hemiscorpiidae) in Iran. Toxicon. 55: 173–179. Latifi M, Tabatabai M (1979) Immunolog- ical studies on Iranian scorpion venom and antiserum. Toxicon. 17: 617–620. Mirakabadi A, Khatoonabadi S, Teimoorzadeh S (2011) Antivenom injection time related effects of Hemiscorpius lepturus scorpion envenomation in rabbits. Arch Razi Inst. 66: 139–145. Mirakabadi A, Khatoonabadi S, Teimourzadeh S, Sabiri GH (2010) Serum enzymes studies in scorpion (Hemiscorpius lepturus) dose related envenomation in rabbits. Arch Razi Inst. 65: 83–89. Prendini L (2000) Phylogeny and classifica- tion of the superfamily Scorpionoidea Latreille 1802 (Chelicerata, Scorpiones): an exemplar approach. Cladistics. 16: 1–78. Radmanesh M (1990) Clinical study of Hemiscorpion lepturus in Iran. J Trop Med Hyg. 93: 327–332. Sauviat MP, Garnier P, Goudey-perriere F, Perriere C (1995) Does crude venom of the stonefish (Synanceia verrucosa) activate β-adrenoceptors in the frog heart muscle? Toxicon. 33: 1207–1213. Seyedian R, Jalali A, Babaee M, Pipelzadeh M, Rezaee S (2012) A biodistribution study of Hemiscorpius lepturus scor- pion venom and available polyclonal antivenom in rats. J Ven Anim Toxins Trop Dis. 18: 375–383. Seyedian R, Pipelzadeh MH, Jalali A, Kim E, Lee H, Kang C, Cha M, Sohn ET, Jung ES, RahmanI AH ,Mirakabady AZ (2010) Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin. Toxicon. 56: 521–525. Shahbazzadeh D, Amirkhani A, Djadid ND, Bigdeli S, Akbari A, Ahari H, Amini H, Dehghani R (2009) Epidemiologi- cal and clinical survey of scorpionism in Khuzestan province, Iran (2003). Toxicon.53: 454–459. Shahbazzadeh D, Srairi-abid N, Feng W, Ram N, Borchani L, Ronjat M, Akbari A, Pessah IN, De waard M, El ayeb M (2007) Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine- sensitive Ca2+ channels. Biochem J. 404: 89–94. Shapira MY, Haviv YS, Sviri S (1998) Second degree atrio-ventricular block and cardiotoxicity secondary to enven- omation by the scorpion Leiurus quinquestriatus (Yellow Scorpion')-an indication for serotherapy? Hum Exp Toxicol. 17: 541–543. Shayesteh AA, Zamiri N, Peymani P, Zargani FJ, Lankarani KB (2011) A novel management method for dis- seminated intravascular coagulation like syndrome after a sting of Hemis- corpius lepturus: a case series. Trop Biomed. 28: 518–523. Srairi-abid, N, Shahbazzadeh D, Chatti I, http://jad.tums.ac.ir Published Online: March 11, 2015 J Arthropod-Borne Dis, December 2015, 9(2): 215–225 K Pourkhalili et al.: Cardiotoxic and … 225 Mlayah-bellalouna S, Mejdoub H, Borchani L, Benkhalifa R, Akbari A, El ayeb M (2008) Hemitoxin, the first potassium channel toxin from the ven- om of the Iranian scorpion Hemis- corpius lepturus. FEBS J. 275: 4641– 4650. Tarasiuk A, Sofer S, Huberfeld SI, Scharf SM (1994) Hemodynamic effects fol- lowing injection of venom from the scorpion Leiurus quinquestriatus. J Crit Care. 9: 134–140. Tavares F L, Sousa-e-silva MCC, Santoro M L, Barbaro KC, Rebecchi IMM, Sano- martins IS (2004) Changes in hema- tological, hemostatic and biochemical parameters induced experimentally in rabbits by Loxosceles gaucho spider venom. Hum Exp Toxicol. 23: 477–486. Teixeira A, Fontoura B, Freire-maia L, Machado C, Camargos E, Teixeira M (2001) Evidence for a direct action of Tityus serrulatus scorpion venom on the cardiac muscle. Toxicon. 39: 703–709. http://jad.tums.ac.ir Published Online: March 11, 2015