Cardiorespiratory alterations in rodents experimentally envenomed with Hadruroides lunatus scorpion venom

Abstract

Background: Hadruroides lunatus is the most abundant scorpion species in the Peruvian central coast, where most of the accidents involving humans are registered. In spite of its prevalence, there are only very few studies on H. lunatus envenomation. The aim of the present study was to analyze the cardiorespiratory alterations caused by H. lunatus envenomation in rodents.

Methods: Wistar rats injected with H. lunatus scorpion venom were submitted to electrocardiography. After euthanasia, rat lungs were collected and histopathologically analyzed. Mouse cardiomyocytes were used to perform immunofluorescence and calcium transient assays. Data were analyzed by ANOVA or Student's t-test. The significance level was set at p < 0.05.

Results: It was observed that H. lunatus venom increased heart rate and caused arrhythmia, thereby impairing the heart functioning. Lungs of envenomed animals showed significant alterations, such as diffuse hemorrhage. In addition, immunofluorescence showed that H. lunatus venom was capable of binding to cardiomyocytes. Furthermore, mouse ventricular cardiomyocytes incubated with H. lunatus venom showed a significant decrease in calcium transient, confirming that H. lunatus venom exerts a toxic effect on heart.

Conclusion: Our results showed that H. lunatus venom is capable of inducing cardiorespiratory alterations, a typical systemic effect of scorpionism, stressing the importance of medical monitoring in envenomation cases.

Keywords: Calcium transient; Cardiorespiratory alterations; Electrocardiography; Hadruroides lunatus venom; Immunofluorescence.

Fig. 1

Photo of a specimen of Hadruroides lunatus collected in Lima, Peru. This scorpion presents brown coloration and about 5 cm long

Fig. 2

SDS-PAGE 15 % of Hlsv. In lane 1, the low molecular weight marker. In the other lanes, 20, 10 and 5 μg of Hlsv under reducing conditions. The gel was silver-stained

Fig. 3

Electrocardiogram alterations in animals treated with Hlsv. ECG from two animals of the treated group showing (a) atrial premature complex and (b) ventricular premature complex 15 min after the envenomation. (c) At T0, all QRS complexes were normal. At T5, all complexes changed the morphology to rS wave. Velocity 50 mm/s, sensibility 2 N, lead II. (d) At T0, T wave was 0.05 mV. At T20, T wave more than doubled its amplitude, reaching 0.12 mV. At T30 T wave was 0.16 mV. (e) Increase on QT interval from 67 ms at T0, to 97 ms at T20 and 107 ms at T30. (f) Increase on PR interval from 43 ms at T0, to 50 ms at T10 and, then, to 63 ms at T30. Velocity 50 mm/s, sensibility 2 N, lead II

Fig. 4

Histopathology of the lungs. (a) Lungs of control rats showing no alterations and (b) lungs with diffuse hemorrhage of animals injected with 750 μg of H. lunatus venom. Magnification: 40x

Fig. 5

Immunolocalization of Hlsv in isolated mouse cardiomyocytes. a Localization of Hlsv detected with anti-Hlsv IgGs in ventricular myocytes. Cells were treated with Hlsv, incubated with anti-Hlsv IgGs antibodies, followed by anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen, USA). As control, cardiomyocytes were treated only with secondary antibodies (control 1) or with IgG anti-Hlsv + secondary antibodies (control 2). Seven cells were analyzed in each group. b Bar graph shows the concentration-dependency of venom binding to cardiac cells

Fig. 6

Cardiomyocytes exposed to Hlsv display reduced Ca²⁺ transient amplitude. a (Top) Representative confocal images of electrically stimulated intracellular Ca²⁺ transient recordings in ventricular myocytes. (Bottom) Ca²⁺ transient line-scan profile. b Significant reduction in peak Ca²⁺ transient amplitude was observed in freshly isolated adult ventricular myocytes incubated with 0.05 μg/mL of Hlsv venom for 5 to 20 min. Numbers inside the bars (34 in the control group and 32 in the in the treated group) represent the number of cells analyzed. Data are expressed as mean ± SEM, * p < 0.0001 compared to control