Smoking flies: testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster

Abstract

Studies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of 'smoking flies'. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.This article has an associated First Person interview with the first author of the paper.

Keywords: Downregulation; Drosophila; Heart; Nicotine; Receptors; Tobacco.

Fig. 1.

Device used for tobacco administration to Drosophila. This custom device consists of a tube that contain a three-way stopcock manifold. (A) Device is closed. Cigarette is attached to an extreme of the tube and the three-way stopcock manifold block the hole toward the vial containing flies. (B) The three-way stopcock manifold was rotated and the smoke from the manually burned cigarette is suctioned by a syringe. (C) After loading 10 cm³ of smoke, the three-way stopcock manifold is rotated again and the smoke contained in the syringe is sent to a vial with flies, through a tube connected to a vial with flies.

Fig. 2.

Commercial cigarette smoke affects cardiac performance of adult Drosophila. Cardiac performance was evaluated in GCaMP3 control flies not exposed (black dots) or exposed to two daily doses of 10 cc of cigarette smoke (red dots) for 7 days. Cigarette smoke exposure increased the heart rate (A) and reduced the Ca²⁺ transient amplitude (B). The maximal velocities of Ca²⁺ transient increase (dΔF/dtmax) and decay (−dΔF/dtmax) were augmented (C,D) in consistency with a shortening of the half-relaxation time (t1/2) (E,F). Diastolic periods and arrhythmia index remained unchanged with treatment (G,H). N=4–14 for control and N=6–9 for treated flies. All results were expressed as mean± s.e.m. **P<0.01 and ***P<0.001.

Fig. 3.

Acute administration of nicotine mimics some of the cigarette effects. Intracellular Ca²⁺ cycling parameters of GCaMP3 flies before and after a pulse of nicotine were evaluated. The presence of 7.5 mM of nicotine in the semi-intact preparation incremented the heart rate (A) without changes in Ca²⁺ transient amplitude (B). This was accompanied by an increased maximum rate of Ca²⁺ transient increase (dΔF/dtmax) and decay (−dΔF/dtmax) (C,D). Time to peak of Ca²⁺ amplitude (TTP) did not show significant changes, meanwhile half-relaxation time (t1/2) showed faster calcium transient relaxation (E,F). N=6–10 flies. All results were expressed as mean±s.e.m. *P<0.05 and **P<0.01.

Fig. 4.

Downregulation of Drosophila nicotinic receptor subunit 1 prevents part of the effects of cigarette smoke. Intracellular Ca²⁺ cycling parameters were evaluated in flies carrying a cardiac-specific iRNA targeted to the alpha-1 subunit of nAChR. Cigarette smoke increased the heart rate (A) and reduced the Ca⁺² transient amplitude (B) and the maximum rate of Ca²⁺ transient increase (+dΔF/dtmax) (C) in flies with alpha-1 downregulation. No changes in maximum rate of Ca²⁺ transient decay (-dΔF/dtmax), time to peak (TTP) and half-relaxation time (t1/2) of Ca²⁺ amplitude (D–F) occurred. Smoke reduced the diastolic periods and increased the arrhythmia index in flies harboring the iRNA against alpha-1 subunit (G,H). N=15–19 for control and N=8–9 for treated flies. All results were expressed as mean±s.e.m. *P<0.05 and **P<0.01.

Fig. 5.

Cardiac-specific downregulation of alpha 1 subunit of the AChR abolishes the response to nicotine in Drosophila’s semi-intact heart preparation. No changes in heart rate (A), amplitude of Ca²⁺ transient (B), maximum rate of Ca²⁺ transient increase (dΔF/dtmax) and decay (−dΔF/dtmax) and times to peak of Ca²⁺ amplitude (TTP) or to half-relaxation (t1/2) (C-F) were observed in alpha 1-downregulated flies after acute nicotine administration. N=14–18 flies. All results were expressed as mean±s.e.m. *P<0.05 and **P<0.01.

Fig. 6.

Downregulation of Drosophila nicotinic receptor subunit 7 prevents part of the effects of cigarette smoke. Flies expressing the iRNA targeted to the alpha 7 subunit of nAChR in cardiac tissue were exposed to tobacco smoke arising from a commercial cigarette, in comparison to non-exposed flies. No changes in heart rate (A) and maximum rate of Ca²⁺ transient increase (dΔF/dtmax) (C) were observed. Amplitude of Ca2+ transient showed and increase (B) in flies which were exposed to cigarette smoke. Increase of maximum rate of decay (−dΔF/dtmax) (D), shortening of TTP and t1/2 (E,F) were observed. Diastolic periods and arrhythmia index and remained unchanged with treatment (G,H). N=8–10 for control and N=7–11 for treated flies. All results were expressed as mean±s.e.m. *P<0.05 and **P<0.01.

Fig. 7.

Cardiac-specific downregulation of alpha 1 subunit of the AChR abolishes the response to nicotine in Drosophila’s semi-intact heart preparation. No changes in heart rate (A), amplitude of Ca²⁺ transient (B), maximum rate of Ca²⁺ transient increase (dΔF/dtmax) and decay (−dΔF/dtmax) and times to peak of Ca²⁺ amplitude (TTP) or to half- relaxation (t1/2) (C–F) were observed in alpha 7-downregulated flies after acute nicotine administration. N=5–10 flies. All results were expressed as mean±s.e.m. *P<0.05 and **P<0.01.