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Dose dependent effects of standardized nose-horned viper (Vipera ammodytes ammodytes) venom on parameters of cardiac function in isolated rat heart
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Comparative Biochemistry and Physiology, Part C 147 (2008) 434 – 440 www.elsevier.com/locate/cbpc
Dose dependent effects of standardized nose-horned viper (Vipera ammodytes ammodytes) venom on parameters of cardiac function in isolated rat heart B. Luksic a , I. Brizic b , M. Lang Balija c , D. Modun b , V. Culic d , B. Halassy c , I. Salamunic e , M. Boban b,⁎ b
a Department of Infectious Diseases, University Hospital Split, Split, Croatia Department of Pharmacology, Split University School of Medicine, Split, Croatia c Institute of Immunology Inc., Zagreb, Croatia d Department of Internal Medicine, University Hospital Split, Split, Croatia e Department of Laboratory Diagnostic, University Hospital Split, Split, Croatia
Received 30 November 2007; received in revised form 22 January 2008; accepted 22 January 2008 Available online 5 February 2008
Abstract Direct, dose dependent effects of the nose-horned vipers (Vipera ammodytes ammodytes) venom on various parameters of cardiac action in isolated rat hearts were examined. Biochemical (protein content, SDS polyacrylamide gel electrophoresis) and biological (minimum haemorrhagic and necrotizing dose and lethal dose (LD50)) characterization of the venom was performed before testing. The hearts were infused with venom doses of 30, 90 and 150 µg/mL for 10 min followed by 30 min of wash out period. Left ventricular pressure, coronary flow, heart rate, atrioventricular conduction, myocardial oxygen consumption, incidence and duration of arrhythmias were measured and relative cardiac efficiency was calculated. Cardiac CPK, LDH, AST and troponin I were measured as biochemical markers of myocardial damage. The venom caused dose dependent electrophysiological instability and depression of contractility and coronary flow. Effects on the heart rate were biphasic; transient increase followed by significant slowing of the frequency. Relative cardiac efficiency decreased as oxygen consumption remained high relative to the heart rate-contractility product, indicating purposeless expenditure of oxygen and energy. Effects by the dose of 30 µg/mL were highly reversible while the dose of 90 μg/mL caused damages that were mostly irreversible. The dose of 150 μg/mL induced irreversible asystolic cardiac arrest. © 2008 Elsevier Inc. All rights reserved. Keywords: Cardiotoxicity; Isolated heart; Snake venom; Vipera ammodytes ammodytes
1. Introduction The nose-horned viper, Vipera ammodytes (V. a.) ammodytes, the most venomous snake in Europe, widespread in its southeastern part, including Croatia, belongs to the Viperidae family and Viperinae subfamily. The venoms of Viperinae cause severe local tissue damage and systemic symptoms, including cardiocirculatory disturbances (Maretic, 1982; Moore, 1988; Luksic et al., 2006; Frangides et al., 2006). Shock has been observed in 5.1% of patients after Viperinae ⁎ Corresponding author. Tel.: +385 21 557 904; fax: +385 21 465 073. E-mail address: [email protected] (M. Boban). 1532-0456/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.01.010
envenomation in southern Croatia (Luksic et al., 2006). Causal factor in shock may involve cardiac effects (Reid and Theakston, 1983). In animal experiments in vivo, V. a. ammodytes venom produced a rapid fall of the arterial blood pressure and induced changes in the electrocardiogram (ECG) (Valora et al., 1970a; Sket et al., 1973). However, studies on the direct heart effects of the venom are scarce and insufficient. Histological evaluation of the cardiac effect of V. a. ammodytes venom was generally discussed in in vitro studies by Petkovic and Unkovic-Cvetkovic (Unkovic-Cvetkovic et al., 1983; Petkovic et al., 1991). In the early work of the same group, only basic ECG and mechanical activity of the isolated rat heart exposed to venom and its fractions were described (Petkovic
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et al., 1979). The study, however, was conducted in a technically restricted and unstable system, and the venom used was not biologically standardized. Therefore, a more detailed examination of the V. a. ammodytes venom effects on various parameters of cardiac function, conducted under more controlled conditions, is warranted. In this study we present the effects of three different venom concentrations of standardized V. a. ammodytes venom on contraction function, coronary flow, heart rate, atrioventricular (AV) conduction time, type and duration of arrhythmias, myocardial oxygen consumption and relative cardiac efficiency. Myocyte enzymes and troponin I concentration were measured as biochemical markers of the venom induced heart damage. 2. Materials and methods 2.1. Chemicals The following chemicals used were: β-mercaptoethanol (BDH, United Kingdom), sodium-dodecyl sulphate (SDS) (Serva, Heidelberg, Germany), tris (hydroxymethyl-aminomethane) (Sigma-Aldrich, St. Louis, MO, USA), ethylendiaminotetraacetic acid (EDTA) (Fluka, Buchs, Switzerland), bovine serum albumin (BSA, Sigma), PhastGel (gradient, 8–25%) (Amersham Biotech, Sweden), low molecular weight protein standards (Pharmacia, Uppsala, Switzerland), NaCl, NaHCO3, sodium pyruvate, KH2PO4, KCl, MgSO4, CaCl2, glucose, mannitol (Kemika, Zagreb, Croatia) and insulin (Hospitalia, Zagreb, Croatia). 2.2. Venoms Crude venom of V. a. ammodytes was obtained from the Institute of Immunology Inc., Zagreb, Croatia. Venom was collected by “milking” snakes from Krapina and Karlovac regions and then air dried at ambient temperature. The venom was stored in crystalloid form at +4 °C. The experimental venom solutions were freshly prepared before each experiment, by dissolving the dried venom in the modified Krebs–Henseleit solution. V. berus venom was obtained from the Institute of Immunology Inc., V. aspis venom from Albert Stephan, Germany, V. lebetina and V. xantina venoms from Latoxan, France. 2.3. Protein content of the venom Protein content was determined according to the method described by Lowry et al. (1951). Venom was dissolved in saline (1 mg/mL). Protein content of the sample was calculated from the curve prepared with bovine serum albumin that was used as a standard. SDS polyacrylamide gel electrophoresis (PAGE) was used to separate protein venom components of different molecular masses, whose distribution is characteristic for the particular venom. Crude venom was dissolved in saline (10 mg/mL), diluted with buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) to final concentration of 3.3 mg/mL, and mixed with SDS (final concentration of SDS was 2.5%). Reduced
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sample of venom was prepared with 5% mercaptoethanol. Electrophoresis was carried out in 10–15% polyacrylamide gel (8–25% gradient gel) at pH 6.4 in PhastSystem following the manufacturer's instructions. The gel was stained with AgNO3. 2.4. Estimation of venom lethal toxicity, hemorrhagic and necrotizing doses The lethal toxicity, expressed as median lethal dose (LD50), i.e. the amount (in μg) of dry venom causing the death in half of the mice population used, was determined according to the method of Theakston and Reid (1983) and European Pharmacopoeia 5th ed. (2005). Adult male NIH/OlaHsd mice (Mus musculus) weighing 18– 20 g were used and the venom was injected intravenously (i.v.). Groups of six mice were injected with various concentrations of V. a. ammodytes venom ranking from 5.25 to 25 µg in a volume of 250 µL physiological saline. Mortality observation was carried out within 2, 24 and 48 h after the injection. LD50 was calculated from the number of mouse deaths by Probit analysis (Finney, 1971; Theakston and Reid, 1983). The minimum haemorrhagic dose (MHD) of venom is defined as the least amount (in µg) of dry venom which, when injected intradermally (i.d.) into rats, results in haemorrhagic lesion of 10 mm diameter 24 h later (Theakston and Reid, 1983). Male Wistar rats (Rattus norvegicus) weighing 300 ± 20 g were anaesthetized and shaved in the dorsal skin. Venom concentrations ranking from 5 to 50 µg in 100 µL physiological saline were injected i.d. After 24 h the animals were sacrificed and skin was cautiously, without stretching, stripped off from the dorsum. For each venom dose tested, the mean diameter of haemorrhage lesions in skin was measured. MHD was estimated by plotting the mean lesion diameter against the venom dose and by reading of the dose which was compatible with a 10 mm lesion diameter (Theakston and Reid, 1983; Lang Balija et al., 2005). The minimum necrotizing dose (MND) is defined as the least amount of venom (µg dry weight) that causes a necrotic skin lesion of 5 mm diameter, 72 h after injection i.d. in Wistar rats. The method used was the same as for the MHD, with an only difference that the skin was stripped off from the dorsum 72 h later (Theakston and Reid, 1983; Sells, 2003). As an orientational guide for dose selection in the isolated heart studies, groups of 4 rats were s.c. injected with 1–6 mg of the venom dissolved in 0.5 mL of saline. Observation on mortality was conducted during 48 h after injection. 2.5. Isolated heart preparation Thirty male Wistar rats weighing 300 ± 20 g were injected with urethane (1.2 g/kg) and 1000 units of heparin. After becoming unresponsive to noxious stimuli the animals were decapitated. After thoracotomy, the inferior and superior venae cavae were cut and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused retrogradly through the aorta excised. All hearts were perfused at constant perfusion pressure of 55 mmHg, measured at the aortic root. The perfusate,
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a modified Krebs–Henseleit solution, was filtered in-line (5 µm pore size; Whatman, Maidstone, England) and had following composition (in mmol/L): 120 NaCl; 23 NaHCO3; 2 sodium pyruvate; 1.2 KH2PO4; 5.9 KCl; 1.2 MgSO4; 1.4 CaCl2; 11.5 glucose; 16 mannitol and 5 units insulin/L. Perfusate and bath temperatures were maintained at 36.6 ± 0.1 °C using thermostatically controlled water circulator. The solution was equilibrated with gas mixture of 96% O2 and 4% CO2. Left ventricular pressure (LVP) was measured isovolumetrically with transducer (UFI type 1050, Morro Bay, CA, USA) connected to a thin, saline-filled latex balloon (Hugo Sachs Elektronik, KG, March-Hugstetten, Germany) inserted into the left ventricle through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a left ventricular diastolic pressure at 0 mmHg during the initial period. Developed LVP signified the difference between systolic and diastolic LVP. Two pairs of Teflon coated silver bipolar electrodes (125 µm in diameter) were placed subepicardially at the appendage of right atrium and at the right ventricular pulmonary conus, respectively, to monitor intracardic electrograms, from which spontaneous sinoatrial rate and AV conduction time were measured. The two electrode signals were amplified and displayed continuously on oscilloscope for monitoring. Atrial rate was determined from the right atrial beat-to-beat interval; AV conduction time was determined as the interval between the superior right atrial beat and the right ventricular conus beat. Ventricular tachycardia was defined as 4 or more consecutive uniform or multiform ventricular waveforms and a faster ventricular than atrial rate. Ventricular fibrillation was defined by the presence of erratic activity in the ventricular electrogram and by the absence of pressure generation by the left ventricle. The absence of electrical and mechanical activity of the heart was considered as an asystolic cardiac arrest. Electrogram intervals were recorded on-line by digital timer systems that allowed instantaneous interval and rate analyses. Coronary flow was measured at a constant temperature with an ultrasonic flow probe (Ultrasonic flow probe T 106, Transonic Systems Inc., Ithaca, NY, USA) placed into the aortic inflow line. Electrograms, heart rate (HR), AV conduction time, coronary flow (CF), LVP and perfusion pressure were digitalized (PowerLab/165, ADInstruments; Castle Hill, Australia) and recorded at 1000 Hz (CHART version 4.2, ADInstruments) on PC for later analysis. Coronary inflow and outflow (coronary sinus) O2 tensions (mmHg) were measured off line with an intermittently selfcalibrating analyzer system (IL 1610 Instrumentation Laboratory, Milan, Italy). Because oxygen supply to the heart depends solely on the crystalloid solution, O2 delivery (DO2) was calculated from the inflow O2 tension times O2 solubility (24 μL/mL saline/760 mmHg) times coronary flow per gram of wet heart tissue. Myocardial O2 consumption (MVO2) was calculated as O2 solubility times coronary flow per gram times (inflow O2–outflow O2 tension difference). Relative cardiac efficiency was calculated as the product of HR (beats per min) and developed isovolumetric LVP (systolic minus diastolic
LVP, in mmHg) divided by O2 consumption (µL·min− 1·g− 1) and expressed as (mmHg·beat)/(µLO2·g− 1). By this index, a relative decrease in the amount of oxygen consumed to perform an isovolumetric contraction indicates improved cardiac efficiency. 2.6. Enzymes and troponin I measurements Coronary sinus effluent was collected by cannula placed into the right ventricle through the pulmonary artery. Samples were collected, before, in fifth and tenth min of envenomation. Creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) concentration were measured with an Olympus (Cork, Ireland) commercial kit. All measurements were performed with automatic analyzer Olympus AU 600 (Olympus Mishima Co. Ltd., Shizuoka, Japan). Troponin I concentration were measured with Dade Behring (Wien, Austria) commercial kit. The measurements were performed with automatic analyzer Dimension Expand (Dade Behring, Newark, USA). 2.7. Experimental protocol Three doses of dried V. a. ammodytes venom of 3, 9 and 15 mg was dissolved in 1 mL of the modified Krebs–Henseleit solution. Dissolved venom was infused into the hearts for 10 min at the rate of 1% of coronary flow allowing final concentrations of approximately 30, 90 and 150 µg/mL. Observation after envenomation lasted for 30 min. The Ethics Committee of the Split University School of Medicine approved all procedures on animals. 2.8. Statistical analysis Data were analyzed using GraphPad Instat and GraphPad Prism version 4.00 for Windows, GraphPad Software (San Diego, CA USA, www.graphpad.-com). All data are expressed as means ± standard error of the means (SEM). Statistical analyses were performed using one-way ANOVA for repeated measures (intragroup statistical difference), and two-way
Fig. 1. SDS-PAGE (8–25% gradient gel) of V. a. ammodytes venom in comparison with several other Viperinae venoms. Lane 1 — V. a. ammodytes nonreduced; lane 2 — V. berus; lane 3 — V. aspis; lane 4 — V. lebetina; lane 5 — V. xantina; lane 6 — V. a. ammodytes reduced; lane 7 — molecular weight markers.
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ANOVA for repeated measures followed by Bonferroni test (intergroup statistical difference). The incidence of arrhythmias was evaluated by chi-square test and their duration was assessed by unpaired t-test. Mean values were considered significant at p b 0.05. 3. Results 3.1. Biochemical and biological characterization of the venom Protein content of V. a. ammodytes venom was 0.97 mg/mg of venom. SDS-PAGE of V. a. ammodytes venom showed a predominance of lower molecular weight bands (Fig. 1). The distribution pattern of protein bands was characteristic for V. a. ammodytes venom (Lang Balija et al., 2005) and obviously different from patterns obtained for venom of other tested Viperinae species. Biological properties of V. a. ammodytes venom were as follows: the i.v. LD50 of V. a. ammodytes venom was 12.48 µg/per mouse, MHD was 14 µg/per rat and MND 17 µg/per rat. The dose of 4 mg s.c. of V. a. ammodytes venom caused death to all rats in the course of 48 h. 3.2. The isolated heart studies 3.2.1. Contractile function The baseline values of developed LVP did not differ and were 104.30 ± 5.37, 102.63 ± 1.69 and 103.40 ± 6.05 mmHg in groups to be exposed to 30, 90 and 150 µg/mL of the venom,
Fig. 3. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on the heart rate (a) and AV conduction time (b) of the isolated rat heart (n = 10 per group).The dose of 150 μg/mL caused atrioventricular dissociation, before irreversible asystolic heart arrest. Results are shown as mean ± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, †p b 0.05 vs. 150 μg/mL, §p b 0.05 vs. initial control value.
respectively. All three doses of the venom induced a significant decrease of developed LVP. The 30 µg/mL dose caused the least effect and was 80% reversible after 30 min of wash out. The 90 µg/mL dose caused a more severe decrease of developed LVP and was poorly (20%) reversible. The 150 µg/mL dose caused irreversible arrest of the heart within 5–7 min of application (Fig. 2a). After exposure to all doses of the venom, diastolic LVP significantly increased from baseline values of 0 mmHg. The increase of diastolic LVP was dose dependent, and only partially reversible in hearts exposed to 30 and 90 µg/mL of the venom. The venom dose of 150 µg/mL caused strong permanent contracture in the arrested hearts (Fig. 2b).
Fig. 2. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on the developed LVP (a) and diastolic LVP (b) of the isolated rat heart (n = 10 per group). The dose of 150 μg/mL caused irreversible heart arrest in hypercontracture state. Results are shown as mean± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, †p b 0.05 vs. 150 μg/mL, §p b 0.05 vs. initial control value.
3.2.2. Electrophysiological effects All hearts exhibited normal sinus rhythm during the initial control period. The baseline HR values were 236.57 ± 3.76, 228.89 ± 4.94 and 233.14 ± 8.30 beats per min in groups to be exposed to 30, 90 and 150 µg/mL of the venom, respectively. All three doses of V. a. ammodytes venom induced significant changes in HR. For doses of 30 and 90 µg/mL the effect was biphasic; after approximately 2 min of envenomation the heart rate transiently accelerated, followed by significant slowing of HR. The slowing of HR was dose dependent. Depression of HR caused by a venom dose of 30 and 90 µg/mL was highly reversible and did not significantly differ from the initial control values. The venom dose of 150 µg/mL caused after 6–8 min irreversible asystolic heart arrest with no detectible electrophysiological activity (Fig. 3a).
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Fig. 5. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on the coronary flow of the isolated rat heart (n = 10 per group). In the hearts exposed to 150 μg/mL of the venom coronary flow could not be measured due to the hearts contracture after several min of envenomation. Results are shown as mean ± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, §p b 0.05 vs. initial control value.
mL group, coronary flow did not recover and stayed similar to the values at the end of envenomation. In the 150 µg/mL group, coronary flow could not be measured due to hearts contracture that precluded coronary circulation (Fig. 5).
Fig. 4. Incidence (a) and duration (b) of arrhythmias during 30 min of wash out period after envenomation. In the hearts exposed to 150 µg/mL of the venom, no electrical activity was recorded during the wash out period (n = 10 per group). Results are shown as mean ± SEM. ⁎p b 0.05 vs. 30 μg/mL.
3.2.4. Myocardial oxygen consumption (MVO2) and relative cardiac efficiency Baseline values of MVO2 in groups exposed to 30, 90 and 150 µg/mL were 153.38 ± 4.57, 150.03 ± 3.71 and 161.25 ± 4.90 µL/min·g, respectively. Increase in oxygen consumption
The baseline values of AV conduction time were 42.70 ± 0.73, 43.50 ± 1.03 and 42.00 ± 0.97 ms in groups to be exposed to 30, 90 and 150 µg/mL of the venom, respectively. All venom doses induced significant dose dependent prolongation of AV conduction time. The venom dose of 150 µg/mL induced atrioventricular dissociation after 5–6 min of envenomation, preceding asystolic cardiac arrest. The doses of 30 and 90 µg/ mL induced AV conduction prolongation that was only partially reversible upon wash out (Fig. 3b). During 30 min of washout period various disturbances of heart rhythm were recorded. Incidence and duration of ventricular tachycardia and fibrillation significantly differed between hearts exposed to 30 and 90 µg/mL of the venom. In the hearts exposed to 150 µg/mL of the venom, no electrical activity was recorded during the wash out period (Fig. 4). 3.2.3. Coronary flow (CF) Baseline values of CF in groups exposed to 30, 90 and 150 µg/mL of the venom were 11.44 ± 0.42, 10.98 ± 0.68 and 10.94 ± 0.42 mL/min, respectively, and did not significantly differ between each other. Different doses of the venom provoked different changes in CF: the dose of 30 µg/mL caused slight, but insignificant oscillations in CF during envenomation, while doses of 90 and 150 µg/mL caused a sharp decrease of CF after the few initial min of envenomation. During the wash out period, coronary flow remained at the initial baseline values in the hearts exposed to the venom dose of 30 µg/mL. In the 90 µg/
Fig. 6. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on myocardial oxygen consumption (MVO2) (a) and cardiac efficiency (b) of the isolated rat heart (n = 10 per group). In the hearts exposed to 150 μg/mL of the venom, MVO2 and cardiac efficiency could not be measured due to hearts contracture that precluded coronary circulation. Results are shown as mean ± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, †p b 0.05 vs. 150 μg/mL, §p b 0.05 vs. initial control value.
B. Luksic et al. / Comparative Biochemistry and Physiology, Part C 147 (2008) 434–440 Table 1 Effects of 30, 90 and 150 μg/mL of V. a. ammodytes venom on concentrations of CPK, LDH, AST and troponin I in sinus effluent of the heart during envenomation period (n = 10 per group) Enzymes and Venom dose Baseline troponin I µg/mL values
5th min of envenomation
10th min of envenomation
CPK (U/L)
12.00 ± 05.14⁎ 17.64 ± 05.76⁎ 56.29 ± 09.71⁎ 15.36 ± 07.38⁎ 34.00 ± 05.17⁎ 144.43 ± 14.46⁎ 100.55 ± 11.70⁎ 163.63 ± 18.96⁎ 358.14 ± 21.90⁎ 0.79 ± 0.54 1.25 ± 0.64⁎ 3.56 ± 0.02⁎
26.64 ± 06.24⁎ 93.73 ± 07.04⁎ S.N.A. 18.91 ± 07.13⁎ 93.45 ± 10.27⁎ S.N.A. 122.55 ± 11.30⁎ 335.24 ± 25.79⁎ S.N.A. 02.54 ± 0.84⁎ 06.18 ± 1.68⁎ S.N.A.
LDH (U/L)
AST (U/L)
Troponin I (µg/L)
30 90 150 30 90 150 30 90 150 30 90 150
2.27 ± 0.70 3.36 ± 0.96 2.14 ± 0.86 0.64 ± 0.47 0.27 ± 0.20 0.43 ± 0.29 5.36 ± 02.29 6.13 ± 02.97 3.29 ± 01.00 0.04 ± 0.03 0.01 ± 0.01 0.02 ± 0.01
Results are shown as mean ± SEM. *p b 0.05 vs. baseline values. CPK — creatine phosphokinase. LDH — lactate dehydrogenase. AST — aspartate aminotransferase. S.N.A. — sample not available.
appeared at the beginning of the envenomation period with the dose of 30 µg/mL. After that the venom induced a significant and dose dependent decrease of MVO2 (Fig. 6a). Baseline values for relative cardiac efficiency in groups exposed to 30, 90 and 150 µg/mL were 171.32 ± 3.85, 174.51 ± 3.60 and 169.35 ± 3.81 (mmHg·beat)/(µLO2·g−1), respectively. During the envenomation period, relative cardiac efficiency decreased by 65, 90 and 100% with 30, 90 and 150 µg/mL of the venom, respectively. During the wash out period it only partially recovered to a level significantly lower than initial control values (Fig. 6b). 3.2.5. Cardiac enzymes and troponin I Baseline values of cardiac CPK, LDH, AST enzymes and troponin I in the heart effluent were (2.27 ± 0.70, 0.64 ± 0.47, 5.36 ± 2.29 U/L and 0.04 ± 0.03 µg/L), (3.36 ± 0.96, 0.27 ± 0.20, 6.13 ± 2.97 U/L and 0.01 ± 0.01 µg/L) and (2.14 ± 0.86, 0.43 ± 0.29, 3.29 ± 1.00 U/L and 0.02 ± 0.01 µg/L) in groups to be exposed to 30, 90 and 150 µg/mL of venom, respectively. A statistically significant increase in concentration of enzymes and troponin I in the hearts effluent occurred with all three venom doses (Table 1). 4. Discussion The study presents a detailed description of dose dependent heart damaging effects of V. a. ammodytes venom on various indices of cardiac action. In order to make our results comparable we did basic biochemical and biological standardization of the venom used. As the mixture of venoms collected from snakes from Karlovac and Krapina regions in Croatia, its LD50, MHD and MND properties were in-between the values obtained in our previous study for each of these venoms separately (Lang Balija et al., 2005).
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In addition, the venom doses we used were selected to roughly correspond with the minimal venom dose that caused death in rats within 48 h of application (4 mg injected s.c.) or with the amount of venom injected by a snake in its natural environment, which is on average 20 mg of dry venom in a single bite (Maretic, 1985; Basoglu and Baran, 1997). Besides the use of the standardized venom, stable and controlled experimental conditions during continuous following of various parameters of cardiac action and indicators of myocardial tissue damage, are a prerequisite for conclusive results. Because of shortcomings in these premises, relevance of the results of many studies of this type is weakened and they cannot be easily compared with the results of others. The most prominent effect of all venom doses was depression of the hearts contractile function. An increase of diastolic and decrease of systolic left ventricular pressure occurred and in the case of the highest venom dose of 150 μg/mL, after several min of envenomation the hearts ended in irreversible asystolic cardiac arrest in the hypercontracture state (Fig. 2). These depressant effects on myocardial contractility were closely followed by significant, dose dependent increase of biochemical markers of heart tissue damage (Table 1). Our results indirectly support findings of histopathological studies with V. a. ammodytes venom on the isolated hearts (Petkovic et al., 1983;Unkovic-Cvetkovic et al., 1983) and of studies that used some other snake's venoms (Alloatti et al., 1986; El Saadani and El Sayed, 2003). The proposed underlying mechanism for the observed effects of venom on heart contractility was impairment of intracellular Ca2+ handling that leads to the elevation of cytosole Ca2+ which induce myocardial contracture. This accelerates energy expenditure and worsening of ventricular contracture resulting in damages of contractile elements and cellular disruption. Indeed, oxygen consumption remained relatively high in comparison to the greater depression of contractility, indicating purposeless expenditure of oxygen and energy in the envenomated hearts (Fig. 6). Related electrophysiological disturbances during the envenomation period were manifested by dual effects on the heart rate; transient increase during several min followed by dose dependent slowing of the frequency (Fig. 3a). Similar effects were observed with venoms of V. lebetina and B. nasicornis and by using propranolol and ranitidine, the positive chronotropic effects of these venoms were prevented (Alloatti et al., 1986, 1991a; Fatehi-Hassanabad and Fatehi, 2004). The authors suggested that observed effects were mediated by catecholamines and histamine released from the heart. In contrast to the changes in heart rate, prolongation of AV conduction time during the envenomation period was permanent and dose dependent (Fig. 3b). Severity of electrophysiological disturbances during the washout period correlated with damages of other parameters of cardiac function. Incidence and duration of ventricular tachycardia and fibrillation with the dose of 30 µg/mL were negligible in comparison with the effects of 90 µg/mL dose (Fig. 4). In an in vivo study by Valora et al. (1970a,b), after i.v. administration of 4–8 mg/kg of the V. a. ammodytes venom into
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rats, ECG recordings revealed an increase in heart rate and excitability in the early phase after envenomation. Under in vivo conditions, however, it is hard to distinguish to what extent the observed changes in heart rate are due to the direct effects of the venom on the heart and what is the consequence of compensatory homeostatic mechanisms in response to the effects of the venom on reduction of total peripheral resistance and hypotension as well as to the effects of the venom on other organ systems (Alloatti et al., 1991b). Effects of the lowest dose of venom on coronary flow were insignificant indicating mild and reversible damages to the vasculature. The doses of 90 and 150 µg/mL induced remarkable decrease of the flow (Fig. 5). However, observed decrease of the coronary flow should not be interpreted as the true direct effect of the venom on coronary vasculature as it is probably more affected by physical resistance to the flow due to venom induced contractures of the heart muscle. Separate analysis of the venom damaging effects on each abovementioned parameter of cardiac action may be united by calculation of relative cardiac efficiency, a parameter that interrelates product of the heart rate and contractility with the indicator of oxygen delivery and consumption in the hearts (Fig. 6b), thereby providing a more integrative insight into the direct heart effects of the V. a. ammodytes venom. In summary, our results indicate that direct heart effects of the venom may be a significant pathophysiological contributor to the general picture after envenomation in vivo. Presented description of dose dependent effects of the standardized venom on various parameters of cardiac action under controlled experimental conditions offer fundamental findings for researchers investigating specific cardiotoxic components of the V. a. ammodytes venom responsible for distinctive effects on different structures of the heart. Acknowledgements This work was supported in part by grants 021-02124322033 and 216-2160547-0537, from the Ministry of Science, Education and Sports of the Republic of Croatia. References Alloatti, G., Camino, E., Cedrini, L., Losano, G., Marsh, N.A., Whaler, B.C., 1986. The effects of Gaboon viper (Bitis gabonica) venom on the electrical and mechanical activity of the guinea-pig myocardium. Toxicon 24, 47–61. Alloatti, G., Gattullo, D., Losano, G., Marsh, N.A., Pagliaro, P., Vono, P., 1991a. The mechanical effects of rhinoceros horned viper (Bitis nasicornis) venom on the isolated perfused guinea-pig heart. Exp. Physiol. 76, 611–614. Alloatti, G., Gattullo, D., Marsh, N.A., Pagliaro, P., Vono, P., 1991b. The mechanical and electrical effects of rhinoceros viper (Bitis nasicornis) venom on the isolated perfused guinea pig heart and atrial preparations. Life Sci. 49, 1539–1548.
Basoglu, M., Baran, I., 1997. Türkiye Sürüngenleri Kisim II. Yilanlar (The Reptiles of Turkey Part II The Snakes). Ege University, Faculty of Science Book Series, Bornova-Izmir. El Saadani, M.A., El Sayed, M.F., 2003. A bradykinin potentiating peptide from Egyptian cobra venom strongly affects rat atrium contractile force and cellular calcium regulation. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 136, 387–395. Fatehi-Hassanabad, Z., Fatehi, M., 2004. Characterisation of some pharmacological effects of the venom from Vipera lebetina. Toxicon 43, 385–391. Finney, D., 1971. Probit Analysis. Cambridge University Press, Cambridge. Frangides, C.Y., Koulouras, V., Kouni, S.N., Tzortzatos, G.V., Nikolaou, A., Pneumaticos, J., Pierrakeas, C., Niarchos, C., Kounis, N.G., Koutsojannis, C.M., 2006. Snake venom poisoning in Greece. Experiences with 147 cases. Eur. J. Intern. Med. 17, 24–27. Lang Balija, M., Vrdoljak, A., Habjanec, L., Dojnovic, B., Halassy, B., Vranesic, B., Tomasic, J., 2005. The variability of Vipera ammodytes ammodytes venoms from Croatia — biochemical properties and biological activity. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 140, 257–263. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Luksic, B., Bradaric, N., Prgomet, S., 2006. Venomous snakebites in southern Croatia. Coll. Antropol. 30, 191–197. Maretic, Z., 1982. Electrocardiographic changes following bites and stings of venomous animals. Arh. Hig. Rada Toksikol. 33, 325–334. Maretic, Z., 1985. Nase otrovne zivotinje i bilje. Stvarnost Zagreb (in Croatian). Moore, R.S., 1988. Second-degree heart block associated with envenomation by Vipera berus. Arch. Emerg. Med. 5, 116–118. Petkovic, D., Jovanovic, T., Micevic, D., Unkovic-Cvetkovic, N., Cvetkovic, M., 1979. Action of Vipera ammodytes venom and its fractions on the isolated rat heart. Toxicon 17, 639–644. Petkovic, D., Pavlovic, M., Matejevic, D., Unkovic-Cvetkovic, N., Jovanovic, T., Aleksic, N., Cvetkovic, M., Colovic, J., Stamenovic, B., 1983. Influence of calcium on the action of Vipera ammodytes ammodytes snake venom on the myocardium. Toxicon 21, 887–892. Petkovic, D., Pavlovic, M., Jovanovic, T., Panic-Drzajic, V., Zdjelar, K., Unkovic, S., Matejevic, D., Alekisic, N., Cvetkovic, M., 1991. Neutralization of the activity of Vipera ammodytes ammodytes snake venom on myocardium of rats by antitoxinum viperinum: a histopathological study. J. Toxicol. Clin. Exp. 11, 343–347. Ph. Eur. 5th edition, 2005. Viper venom antiserum. European 0145, 806. Reid, H.A., Theakston, R.D., 1983. The management of snake bite. Bull. World Health Organ., Suppl. 61, 885–895. Sells, P.G., 2003. Animal experimentation in snake venom research and in vitro alternatives. Toxicon 42, 115–133. Sket, D., Gubensek, F., Adamic, S., Lebez, D., 1973. Action of a partially purified basic protein fraction from Vipera ammodytes venom. Toxicon 11, 47–53. Theakston, R.D.G., Reid, H.A., 1983. Development of simple standard assay procedures for the characterization of snake venom. Bull. World Health Organ., Suppl. 61, 949–956. Unkovic-Cvetkovic, N., Cvetkovic, M., Petkovic, D., Jovanovic, T., Unkovic, S., 1983. Histopathological changes in rat myocardium caused by Vipera ammodytes ammodytes (European viper) snake venom. Toxicon 21, 429–432. Valora, N., Bruno, C., Fidanza, A., 1970a. Action of Vipera ammodytes venom on the rat electrocardiogram. II. Boll. Soc. Ital. Biol. Sper. 46, 373–375. Valora, N., Bruno, C., Fidanza, A., 1970b. Effect of Vipera ammodytes venom on the rat electrocardiogram. I. Boll. Soc. Ital. Biol. Sper. 46, 67–71.
Comparative Biochemistry and Physiology, Part C 147 (2008) 434 – 440 www.elsevier.com/locate/cbpc
Dose dependent effects of standardized nose-horned viper (Vipera ammodytes ammodytes) venom on parameters of cardiac function in isolated rat heart B. Luksic a , I. Brizic b , M. Lang Balija c , D. Modun b , V. Culic d , B. Halassy c , I. Salamunic e , M. Boban b,⁎ b
a Department of Infectious Diseases, University Hospital Split, Split, Croatia Department of Pharmacology, Split University School of Medicine, Split, Croatia c Institute of Immunology Inc., Zagreb, Croatia d Department of Internal Medicine, University Hospital Split, Split, Croatia e Department of Laboratory Diagnostic, University Hospital Split, Split, Croatia
Received 30 November 2007; received in revised form 22 January 2008; accepted 22 January 2008 Available online 5 February 2008
Abstract Direct, dose dependent effects of the nose-horned vipers (Vipera ammodytes ammodytes) venom on various parameters of cardiac action in isolated rat hearts were examined. Biochemical (protein content, SDS polyacrylamide gel electrophoresis) and biological (minimum haemorrhagic and necrotizing dose and lethal dose (LD50)) characterization of the venom was performed before testing. The hearts were infused with venom doses of 30, 90 and 150 µg/mL for 10 min followed by 30 min of wash out period. Left ventricular pressure, coronary flow, heart rate, atrioventricular conduction, myocardial oxygen consumption, incidence and duration of arrhythmias were measured and relative cardiac efficiency was calculated. Cardiac CPK, LDH, AST and troponin I were measured as biochemical markers of myocardial damage. The venom caused dose dependent electrophysiological instability and depression of contractility and coronary flow. Effects on the heart rate were biphasic; transient increase followed by significant slowing of the frequency. Relative cardiac efficiency decreased as oxygen consumption remained high relative to the heart rate-contractility product, indicating purposeless expenditure of oxygen and energy. Effects by the dose of 30 µg/mL were highly reversible while the dose of 90 μg/mL caused damages that were mostly irreversible. The dose of 150 μg/mL induced irreversible asystolic cardiac arrest. © 2008 Elsevier Inc. All rights reserved. Keywords: Cardiotoxicity; Isolated heart; Snake venom; Vipera ammodytes ammodytes
1. Introduction The nose-horned viper, Vipera ammodytes (V. a.) ammodytes, the most venomous snake in Europe, widespread in its southeastern part, including Croatia, belongs to the Viperidae family and Viperinae subfamily. The venoms of Viperinae cause severe local tissue damage and systemic symptoms, including cardiocirculatory disturbances (Maretic, 1982; Moore, 1988; Luksic et al., 2006; Frangides et al., 2006). Shock has been observed in 5.1% of patients after Viperinae ⁎ Corresponding author. Tel.: +385 21 557 904; fax: +385 21 465 073. E-mail address: [email protected] (M. Boban). 1532-0456/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.01.010
envenomation in southern Croatia (Luksic et al., 2006). Causal factor in shock may involve cardiac effects (Reid and Theakston, 1983). In animal experiments in vivo, V. a. ammodytes venom produced a rapid fall of the arterial blood pressure and induced changes in the electrocardiogram (ECG) (Valora et al., 1970a; Sket et al., 1973). However, studies on the direct heart effects of the venom are scarce and insufficient. Histological evaluation of the cardiac effect of V. a. ammodytes venom was generally discussed in in vitro studies by Petkovic and Unkovic-Cvetkovic (Unkovic-Cvetkovic et al., 1983; Petkovic et al., 1991). In the early work of the same group, only basic ECG and mechanical activity of the isolated rat heart exposed to venom and its fractions were described (Petkovic
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et al., 1979). The study, however, was conducted in a technically restricted and unstable system, and the venom used was not biologically standardized. Therefore, a more detailed examination of the V. a. ammodytes venom effects on various parameters of cardiac function, conducted under more controlled conditions, is warranted. In this study we present the effects of three different venom concentrations of standardized V. a. ammodytes venom on contraction function, coronary flow, heart rate, atrioventricular (AV) conduction time, type and duration of arrhythmias, myocardial oxygen consumption and relative cardiac efficiency. Myocyte enzymes and troponin I concentration were measured as biochemical markers of the venom induced heart damage. 2. Materials and methods 2.1. Chemicals The following chemicals used were: β-mercaptoethanol (BDH, United Kingdom), sodium-dodecyl sulphate (SDS) (Serva, Heidelberg, Germany), tris (hydroxymethyl-aminomethane) (Sigma-Aldrich, St. Louis, MO, USA), ethylendiaminotetraacetic acid (EDTA) (Fluka, Buchs, Switzerland), bovine serum albumin (BSA, Sigma), PhastGel (gradient, 8–25%) (Amersham Biotech, Sweden), low molecular weight protein standards (Pharmacia, Uppsala, Switzerland), NaCl, NaHCO3, sodium pyruvate, KH2PO4, KCl, MgSO4, CaCl2, glucose, mannitol (Kemika, Zagreb, Croatia) and insulin (Hospitalia, Zagreb, Croatia). 2.2. Venoms Crude venom of V. a. ammodytes was obtained from the Institute of Immunology Inc., Zagreb, Croatia. Venom was collected by “milking” snakes from Krapina and Karlovac regions and then air dried at ambient temperature. The venom was stored in crystalloid form at +4 °C. The experimental venom solutions were freshly prepared before each experiment, by dissolving the dried venom in the modified Krebs–Henseleit solution. V. berus venom was obtained from the Institute of Immunology Inc., V. aspis venom from Albert Stephan, Germany, V. lebetina and V. xantina venoms from Latoxan, France. 2.3. Protein content of the venom Protein content was determined according to the method described by Lowry et al. (1951). Venom was dissolved in saline (1 mg/mL). Protein content of the sample was calculated from the curve prepared with bovine serum albumin that was used as a standard. SDS polyacrylamide gel electrophoresis (PAGE) was used to separate protein venom components of different molecular masses, whose distribution is characteristic for the particular venom. Crude venom was dissolved in saline (10 mg/mL), diluted with buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) to final concentration of 3.3 mg/mL, and mixed with SDS (final concentration of SDS was 2.5%). Reduced
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sample of venom was prepared with 5% mercaptoethanol. Electrophoresis was carried out in 10–15% polyacrylamide gel (8–25% gradient gel) at pH 6.4 in PhastSystem following the manufacturer's instructions. The gel was stained with AgNO3. 2.4. Estimation of venom lethal toxicity, hemorrhagic and necrotizing doses The lethal toxicity, expressed as median lethal dose (LD50), i.e. the amount (in μg) of dry venom causing the death in half of the mice population used, was determined according to the method of Theakston and Reid (1983) and European Pharmacopoeia 5th ed. (2005). Adult male NIH/OlaHsd mice (Mus musculus) weighing 18– 20 g were used and the venom was injected intravenously (i.v.). Groups of six mice were injected with various concentrations of V. a. ammodytes venom ranking from 5.25 to 25 µg in a volume of 250 µL physiological saline. Mortality observation was carried out within 2, 24 and 48 h after the injection. LD50 was calculated from the number of mouse deaths by Probit analysis (Finney, 1971; Theakston and Reid, 1983). The minimum haemorrhagic dose (MHD) of venom is defined as the least amount (in µg) of dry venom which, when injected intradermally (i.d.) into rats, results in haemorrhagic lesion of 10 mm diameter 24 h later (Theakston and Reid, 1983). Male Wistar rats (Rattus norvegicus) weighing 300 ± 20 g were anaesthetized and shaved in the dorsal skin. Venom concentrations ranking from 5 to 50 µg in 100 µL physiological saline were injected i.d. After 24 h the animals were sacrificed and skin was cautiously, without stretching, stripped off from the dorsum. For each venom dose tested, the mean diameter of haemorrhage lesions in skin was measured. MHD was estimated by plotting the mean lesion diameter against the venom dose and by reading of the dose which was compatible with a 10 mm lesion diameter (Theakston and Reid, 1983; Lang Balija et al., 2005). The minimum necrotizing dose (MND) is defined as the least amount of venom (µg dry weight) that causes a necrotic skin lesion of 5 mm diameter, 72 h after injection i.d. in Wistar rats. The method used was the same as for the MHD, with an only difference that the skin was stripped off from the dorsum 72 h later (Theakston and Reid, 1983; Sells, 2003). As an orientational guide for dose selection in the isolated heart studies, groups of 4 rats were s.c. injected with 1–6 mg of the venom dissolved in 0.5 mL of saline. Observation on mortality was conducted during 48 h after injection. 2.5. Isolated heart preparation Thirty male Wistar rats weighing 300 ± 20 g were injected with urethane (1.2 g/kg) and 1000 units of heparin. After becoming unresponsive to noxious stimuli the animals were decapitated. After thoracotomy, the inferior and superior venae cavae were cut and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused retrogradly through the aorta excised. All hearts were perfused at constant perfusion pressure of 55 mmHg, measured at the aortic root. The perfusate,
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a modified Krebs–Henseleit solution, was filtered in-line (5 µm pore size; Whatman, Maidstone, England) and had following composition (in mmol/L): 120 NaCl; 23 NaHCO3; 2 sodium pyruvate; 1.2 KH2PO4; 5.9 KCl; 1.2 MgSO4; 1.4 CaCl2; 11.5 glucose; 16 mannitol and 5 units insulin/L. Perfusate and bath temperatures were maintained at 36.6 ± 0.1 °C using thermostatically controlled water circulator. The solution was equilibrated with gas mixture of 96% O2 and 4% CO2. Left ventricular pressure (LVP) was measured isovolumetrically with transducer (UFI type 1050, Morro Bay, CA, USA) connected to a thin, saline-filled latex balloon (Hugo Sachs Elektronik, KG, March-Hugstetten, Germany) inserted into the left ventricle through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a left ventricular diastolic pressure at 0 mmHg during the initial period. Developed LVP signified the difference between systolic and diastolic LVP. Two pairs of Teflon coated silver bipolar electrodes (125 µm in diameter) were placed subepicardially at the appendage of right atrium and at the right ventricular pulmonary conus, respectively, to monitor intracardic electrograms, from which spontaneous sinoatrial rate and AV conduction time were measured. The two electrode signals were amplified and displayed continuously on oscilloscope for monitoring. Atrial rate was determined from the right atrial beat-to-beat interval; AV conduction time was determined as the interval between the superior right atrial beat and the right ventricular conus beat. Ventricular tachycardia was defined as 4 or more consecutive uniform or multiform ventricular waveforms and a faster ventricular than atrial rate. Ventricular fibrillation was defined by the presence of erratic activity in the ventricular electrogram and by the absence of pressure generation by the left ventricle. The absence of electrical and mechanical activity of the heart was considered as an asystolic cardiac arrest. Electrogram intervals were recorded on-line by digital timer systems that allowed instantaneous interval and rate analyses. Coronary flow was measured at a constant temperature with an ultrasonic flow probe (Ultrasonic flow probe T 106, Transonic Systems Inc., Ithaca, NY, USA) placed into the aortic inflow line. Electrograms, heart rate (HR), AV conduction time, coronary flow (CF), LVP and perfusion pressure were digitalized (PowerLab/165, ADInstruments; Castle Hill, Australia) and recorded at 1000 Hz (CHART version 4.2, ADInstruments) on PC for later analysis. Coronary inflow and outflow (coronary sinus) O2 tensions (mmHg) were measured off line with an intermittently selfcalibrating analyzer system (IL 1610 Instrumentation Laboratory, Milan, Italy). Because oxygen supply to the heart depends solely on the crystalloid solution, O2 delivery (DO2) was calculated from the inflow O2 tension times O2 solubility (24 μL/mL saline/760 mmHg) times coronary flow per gram of wet heart tissue. Myocardial O2 consumption (MVO2) was calculated as O2 solubility times coronary flow per gram times (inflow O2–outflow O2 tension difference). Relative cardiac efficiency was calculated as the product of HR (beats per min) and developed isovolumetric LVP (systolic minus diastolic
LVP, in mmHg) divided by O2 consumption (µL·min− 1·g− 1) and expressed as (mmHg·beat)/(µLO2·g− 1). By this index, a relative decrease in the amount of oxygen consumed to perform an isovolumetric contraction indicates improved cardiac efficiency. 2.6. Enzymes and troponin I measurements Coronary sinus effluent was collected by cannula placed into the right ventricle through the pulmonary artery. Samples were collected, before, in fifth and tenth min of envenomation. Creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) concentration were measured with an Olympus (Cork, Ireland) commercial kit. All measurements were performed with automatic analyzer Olympus AU 600 (Olympus Mishima Co. Ltd., Shizuoka, Japan). Troponin I concentration were measured with Dade Behring (Wien, Austria) commercial kit. The measurements were performed with automatic analyzer Dimension Expand (Dade Behring, Newark, USA). 2.7. Experimental protocol Three doses of dried V. a. ammodytes venom of 3, 9 and 15 mg was dissolved in 1 mL of the modified Krebs–Henseleit solution. Dissolved venom was infused into the hearts for 10 min at the rate of 1% of coronary flow allowing final concentrations of approximately 30, 90 and 150 µg/mL. Observation after envenomation lasted for 30 min. The Ethics Committee of the Split University School of Medicine approved all procedures on animals. 2.8. Statistical analysis Data were analyzed using GraphPad Instat and GraphPad Prism version 4.00 for Windows, GraphPad Software (San Diego, CA USA, www.graphpad.-com). All data are expressed as means ± standard error of the means (SEM). Statistical analyses were performed using one-way ANOVA for repeated measures (intragroup statistical difference), and two-way
Fig. 1. SDS-PAGE (8–25% gradient gel) of V. a. ammodytes venom in comparison with several other Viperinae venoms. Lane 1 — V. a. ammodytes nonreduced; lane 2 — V. berus; lane 3 — V. aspis; lane 4 — V. lebetina; lane 5 — V. xantina; lane 6 — V. a. ammodytes reduced; lane 7 — molecular weight markers.
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ANOVA for repeated measures followed by Bonferroni test (intergroup statistical difference). The incidence of arrhythmias was evaluated by chi-square test and their duration was assessed by unpaired t-test. Mean values were considered significant at p b 0.05. 3. Results 3.1. Biochemical and biological characterization of the venom Protein content of V. a. ammodytes venom was 0.97 mg/mg of venom. SDS-PAGE of V. a. ammodytes venom showed a predominance of lower molecular weight bands (Fig. 1). The distribution pattern of protein bands was characteristic for V. a. ammodytes venom (Lang Balija et al., 2005) and obviously different from patterns obtained for venom of other tested Viperinae species. Biological properties of V. a. ammodytes venom were as follows: the i.v. LD50 of V. a. ammodytes venom was 12.48 µg/per mouse, MHD was 14 µg/per rat and MND 17 µg/per rat. The dose of 4 mg s.c. of V. a. ammodytes venom caused death to all rats in the course of 48 h. 3.2. The isolated heart studies 3.2.1. Contractile function The baseline values of developed LVP did not differ and were 104.30 ± 5.37, 102.63 ± 1.69 and 103.40 ± 6.05 mmHg in groups to be exposed to 30, 90 and 150 µg/mL of the venom,
Fig. 3. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on the heart rate (a) and AV conduction time (b) of the isolated rat heart (n = 10 per group).The dose of 150 μg/mL caused atrioventricular dissociation, before irreversible asystolic heart arrest. Results are shown as mean ± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, †p b 0.05 vs. 150 μg/mL, §p b 0.05 vs. initial control value.
respectively. All three doses of the venom induced a significant decrease of developed LVP. The 30 µg/mL dose caused the least effect and was 80% reversible after 30 min of wash out. The 90 µg/mL dose caused a more severe decrease of developed LVP and was poorly (20%) reversible. The 150 µg/mL dose caused irreversible arrest of the heart within 5–7 min of application (Fig. 2a). After exposure to all doses of the venom, diastolic LVP significantly increased from baseline values of 0 mmHg. The increase of diastolic LVP was dose dependent, and only partially reversible in hearts exposed to 30 and 90 µg/mL of the venom. The venom dose of 150 µg/mL caused strong permanent contracture in the arrested hearts (Fig. 2b).
Fig. 2. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on the developed LVP (a) and diastolic LVP (b) of the isolated rat heart (n = 10 per group). The dose of 150 μg/mL caused irreversible heart arrest in hypercontracture state. Results are shown as mean± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, †p b 0.05 vs. 150 μg/mL, §p b 0.05 vs. initial control value.
3.2.2. Electrophysiological effects All hearts exhibited normal sinus rhythm during the initial control period. The baseline HR values were 236.57 ± 3.76, 228.89 ± 4.94 and 233.14 ± 8.30 beats per min in groups to be exposed to 30, 90 and 150 µg/mL of the venom, respectively. All three doses of V. a. ammodytes venom induced significant changes in HR. For doses of 30 and 90 µg/mL the effect was biphasic; after approximately 2 min of envenomation the heart rate transiently accelerated, followed by significant slowing of HR. The slowing of HR was dose dependent. Depression of HR caused by a venom dose of 30 and 90 µg/mL was highly reversible and did not significantly differ from the initial control values. The venom dose of 150 µg/mL caused after 6–8 min irreversible asystolic heart arrest with no detectible electrophysiological activity (Fig. 3a).
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Fig. 5. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on the coronary flow of the isolated rat heart (n = 10 per group). In the hearts exposed to 150 μg/mL of the venom coronary flow could not be measured due to the hearts contracture after several min of envenomation. Results are shown as mean ± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, §p b 0.05 vs. initial control value.
mL group, coronary flow did not recover and stayed similar to the values at the end of envenomation. In the 150 µg/mL group, coronary flow could not be measured due to hearts contracture that precluded coronary circulation (Fig. 5).
Fig. 4. Incidence (a) and duration (b) of arrhythmias during 30 min of wash out period after envenomation. In the hearts exposed to 150 µg/mL of the venom, no electrical activity was recorded during the wash out period (n = 10 per group). Results are shown as mean ± SEM. ⁎p b 0.05 vs. 30 μg/mL.
3.2.4. Myocardial oxygen consumption (MVO2) and relative cardiac efficiency Baseline values of MVO2 in groups exposed to 30, 90 and 150 µg/mL were 153.38 ± 4.57, 150.03 ± 3.71 and 161.25 ± 4.90 µL/min·g, respectively. Increase in oxygen consumption
The baseline values of AV conduction time were 42.70 ± 0.73, 43.50 ± 1.03 and 42.00 ± 0.97 ms in groups to be exposed to 30, 90 and 150 µg/mL of the venom, respectively. All venom doses induced significant dose dependent prolongation of AV conduction time. The venom dose of 150 µg/mL induced atrioventricular dissociation after 5–6 min of envenomation, preceding asystolic cardiac arrest. The doses of 30 and 90 µg/ mL induced AV conduction prolongation that was only partially reversible upon wash out (Fig. 3b). During 30 min of washout period various disturbances of heart rhythm were recorded. Incidence and duration of ventricular tachycardia and fibrillation significantly differed between hearts exposed to 30 and 90 µg/mL of the venom. In the hearts exposed to 150 µg/mL of the venom, no electrical activity was recorded during the wash out period (Fig. 4). 3.2.3. Coronary flow (CF) Baseline values of CF in groups exposed to 30, 90 and 150 µg/mL of the venom were 11.44 ± 0.42, 10.98 ± 0.68 and 10.94 ± 0.42 mL/min, respectively, and did not significantly differ between each other. Different doses of the venom provoked different changes in CF: the dose of 30 µg/mL caused slight, but insignificant oscillations in CF during envenomation, while doses of 90 and 150 µg/mL caused a sharp decrease of CF after the few initial min of envenomation. During the wash out period, coronary flow remained at the initial baseline values in the hearts exposed to the venom dose of 30 µg/mL. In the 90 µg/
Fig. 6. Effects of 30 (■), 90 (▲) and 150 μg/mL (○) of V. a. ammodytes venom on myocardial oxygen consumption (MVO2) (a) and cardiac efficiency (b) of the isolated rat heart (n = 10 per group). In the hearts exposed to 150 μg/mL of the venom, MVO2 and cardiac efficiency could not be measured due to hearts contracture that precluded coronary circulation. Results are shown as mean ± SEM. ⁎p b 0.05 vs. 90 and 150 μg/mL, †p b 0.05 vs. 150 μg/mL, §p b 0.05 vs. initial control value.
B. Luksic et al. / Comparative Biochemistry and Physiology, Part C 147 (2008) 434–440 Table 1 Effects of 30, 90 and 150 μg/mL of V. a. ammodytes venom on concentrations of CPK, LDH, AST and troponin I in sinus effluent of the heart during envenomation period (n = 10 per group) Enzymes and Venom dose Baseline troponin I µg/mL values
5th min of envenomation
10th min of envenomation
CPK (U/L)
12.00 ± 05.14⁎ 17.64 ± 05.76⁎ 56.29 ± 09.71⁎ 15.36 ± 07.38⁎ 34.00 ± 05.17⁎ 144.43 ± 14.46⁎ 100.55 ± 11.70⁎ 163.63 ± 18.96⁎ 358.14 ± 21.90⁎ 0.79 ± 0.54 1.25 ± 0.64⁎ 3.56 ± 0.02⁎
26.64 ± 06.24⁎ 93.73 ± 07.04⁎ S.N.A. 18.91 ± 07.13⁎ 93.45 ± 10.27⁎ S.N.A. 122.55 ± 11.30⁎ 335.24 ± 25.79⁎ S.N.A. 02.54 ± 0.84⁎ 06.18 ± 1.68⁎ S.N.A.
LDH (U/L)
AST (U/L)
Troponin I (µg/L)
30 90 150 30 90 150 30 90 150 30 90 150
2.27 ± 0.70 3.36 ± 0.96 2.14 ± 0.86 0.64 ± 0.47 0.27 ± 0.20 0.43 ± 0.29 5.36 ± 02.29 6.13 ± 02.97 3.29 ± 01.00 0.04 ± 0.03 0.01 ± 0.01 0.02 ± 0.01
Results are shown as mean ± SEM. *p b 0.05 vs. baseline values. CPK — creatine phosphokinase. LDH — lactate dehydrogenase. AST — aspartate aminotransferase. S.N.A. — sample not available.
appeared at the beginning of the envenomation period with the dose of 30 µg/mL. After that the venom induced a significant and dose dependent decrease of MVO2 (Fig. 6a). Baseline values for relative cardiac efficiency in groups exposed to 30, 90 and 150 µg/mL were 171.32 ± 3.85, 174.51 ± 3.60 and 169.35 ± 3.81 (mmHg·beat)/(µLO2·g−1), respectively. During the envenomation period, relative cardiac efficiency decreased by 65, 90 and 100% with 30, 90 and 150 µg/mL of the venom, respectively. During the wash out period it only partially recovered to a level significantly lower than initial control values (Fig. 6b). 3.2.5. Cardiac enzymes and troponin I Baseline values of cardiac CPK, LDH, AST enzymes and troponin I in the heart effluent were (2.27 ± 0.70, 0.64 ± 0.47, 5.36 ± 2.29 U/L and 0.04 ± 0.03 µg/L), (3.36 ± 0.96, 0.27 ± 0.20, 6.13 ± 2.97 U/L and 0.01 ± 0.01 µg/L) and (2.14 ± 0.86, 0.43 ± 0.29, 3.29 ± 1.00 U/L and 0.02 ± 0.01 µg/L) in groups to be exposed to 30, 90 and 150 µg/mL of venom, respectively. A statistically significant increase in concentration of enzymes and troponin I in the hearts effluent occurred with all three venom doses (Table 1). 4. Discussion The study presents a detailed description of dose dependent heart damaging effects of V. a. ammodytes venom on various indices of cardiac action. In order to make our results comparable we did basic biochemical and biological standardization of the venom used. As the mixture of venoms collected from snakes from Karlovac and Krapina regions in Croatia, its LD50, MHD and MND properties were in-between the values obtained in our previous study for each of these venoms separately (Lang Balija et al., 2005).
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In addition, the venom doses we used were selected to roughly correspond with the minimal venom dose that caused death in rats within 48 h of application (4 mg injected s.c.) or with the amount of venom injected by a snake in its natural environment, which is on average 20 mg of dry venom in a single bite (Maretic, 1985; Basoglu and Baran, 1997). Besides the use of the standardized venom, stable and controlled experimental conditions during continuous following of various parameters of cardiac action and indicators of myocardial tissue damage, are a prerequisite for conclusive results. Because of shortcomings in these premises, relevance of the results of many studies of this type is weakened and they cannot be easily compared with the results of others. The most prominent effect of all venom doses was depression of the hearts contractile function. An increase of diastolic and decrease of systolic left ventricular pressure occurred and in the case of the highest venom dose of 150 μg/mL, after several min of envenomation the hearts ended in irreversible asystolic cardiac arrest in the hypercontracture state (Fig. 2). These depressant effects on myocardial contractility were closely followed by significant, dose dependent increase of biochemical markers of heart tissue damage (Table 1). Our results indirectly support findings of histopathological studies with V. a. ammodytes venom on the isolated hearts (Petkovic et al., 1983;Unkovic-Cvetkovic et al., 1983) and of studies that used some other snake's venoms (Alloatti et al., 1986; El Saadani and El Sayed, 2003). The proposed underlying mechanism for the observed effects of venom on heart contractility was impairment of intracellular Ca2+ handling that leads to the elevation of cytosole Ca2+ which induce myocardial contracture. This accelerates energy expenditure and worsening of ventricular contracture resulting in damages of contractile elements and cellular disruption. Indeed, oxygen consumption remained relatively high in comparison to the greater depression of contractility, indicating purposeless expenditure of oxygen and energy in the envenomated hearts (Fig. 6). Related electrophysiological disturbances during the envenomation period were manifested by dual effects on the heart rate; transient increase during several min followed by dose dependent slowing of the frequency (Fig. 3a). Similar effects were observed with venoms of V. lebetina and B. nasicornis and by using propranolol and ranitidine, the positive chronotropic effects of these venoms were prevented (Alloatti et al., 1986, 1991a; Fatehi-Hassanabad and Fatehi, 2004). The authors suggested that observed effects were mediated by catecholamines and histamine released from the heart. In contrast to the changes in heart rate, prolongation of AV conduction time during the envenomation period was permanent and dose dependent (Fig. 3b). Severity of electrophysiological disturbances during the washout period correlated with damages of other parameters of cardiac function. Incidence and duration of ventricular tachycardia and fibrillation with the dose of 30 µg/mL were negligible in comparison with the effects of 90 µg/mL dose (Fig. 4). In an in vivo study by Valora et al. (1970a,b), after i.v. administration of 4–8 mg/kg of the V. a. ammodytes venom into
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rats, ECG recordings revealed an increase in heart rate and excitability in the early phase after envenomation. Under in vivo conditions, however, it is hard to distinguish to what extent the observed changes in heart rate are due to the direct effects of the venom on the heart and what is the consequence of compensatory homeostatic mechanisms in response to the effects of the venom on reduction of total peripheral resistance and hypotension as well as to the effects of the venom on other organ systems (Alloatti et al., 1991b). Effects of the lowest dose of venom on coronary flow were insignificant indicating mild and reversible damages to the vasculature. The doses of 90 and 150 µg/mL induced remarkable decrease of the flow (Fig. 5). However, observed decrease of the coronary flow should not be interpreted as the true direct effect of the venom on coronary vasculature as it is probably more affected by physical resistance to the flow due to venom induced contractures of the heart muscle. Separate analysis of the venom damaging effects on each abovementioned parameter of cardiac action may be united by calculation of relative cardiac efficiency, a parameter that interrelates product of the heart rate and contractility with the indicator of oxygen delivery and consumption in the hearts (Fig. 6b), thereby providing a more integrative insight into the direct heart effects of the V. a. ammodytes venom. In summary, our results indicate that direct heart effects of the venom may be a significant pathophysiological contributor to the general picture after envenomation in vivo. Presented description of dose dependent effects of the standardized venom on various parameters of cardiac action under controlled experimental conditions offer fundamental findings for researchers investigating specific cardiotoxic components of the V. a. ammodytes venom responsible for distinctive effects on different structures of the heart. Acknowledgements This work was supported in part by grants 021-02124322033 and 216-2160547-0537, from the Ministry of Science, Education and Sports of the Republic of Croatia. References Alloatti, G., Camino, E., Cedrini, L., Losano, G., Marsh, N.A., Whaler, B.C., 1986. The effects of Gaboon viper (Bitis gabonica) venom on the electrical and mechanical activity of the guinea-pig myocardium. Toxicon 24, 47–61. Alloatti, G., Gattullo, D., Losano, G., Marsh, N.A., Pagliaro, P., Vono, P., 1991a. The mechanical effects of rhinoceros horned viper (Bitis nasicornis) venom on the isolated perfused guinea-pig heart. Exp. Physiol. 76, 611–614. Alloatti, G., Gattullo, D., Marsh, N.A., Pagliaro, P., Vono, P., 1991b. The mechanical and electrical effects of rhinoceros viper (Bitis nasicornis) venom on the isolated perfused guinea pig heart and atrial preparations. Life Sci. 49, 1539–1548.
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