Arrhythmic Effects Evaluated on Caenorhabditis elegans: The Case of Polypyrrole Nanoparticles

Abstract

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes' pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs' biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs.

Keywords: C. elegans; Polypyrrole nanoparticles; arrhythmia models; cardiotoxicity; small animal models.

Figure 1

(a) Schematics of the cardiac contraction and action potential generation and propagation in humans. (b) C. elegans pharyngeal contraction and action potential graph showing a strong resemblance in ion transfer events as the human. (c) Scheme of the neuromuscular junction similar in humans and in C. elegans. The table reports the orthologous genes encoding cation channels and gap junctions at the neuromuscular junctions of the C. elegans pharynx and the human heart. Created with BioRender.com.

Figure 2

Stability of Ppy NPs (a) in Milli-Q (MQ) water, 50:50 mix of M9:MQ, and M9 buffer. (b) Stability of Ppy NPs at different pH values over time. (c) Schematic representation of the pH of the intestinal tract of worms, which is within the same range as humans but in the opposite direction.

Figure 3

(a) Ppy NPs dispersed in a bleach solution over time (top). (b) Hydrodynamic diameter of Ppy NPs in different concentrations of bleach solution. Optical microscopic images of the (c) untreated C. elegans (control), (d) C. elegans treated with Ppy NPs. TEM images of the (e) NPs after treatment at 0.3 M bleach + 5.0 M NaOH and 4 min vortex. (f) NPs recovered after bleach treatment of the treated worms.

Figure 4

Systemic effects of PL and RE on C. elegans. (a) Body length of first generation worms after 24 h treatment (N = 40). (b) Number of eggs and larvae produced by the worms in 72 h postexposure (N = 12). (c) Body length of fully developed second generation worms (N = 30). No statistical significance was found in any case. Top panel (scheme) created with BioRender.com.

Figure 5

(a) Pharynx pumping rate upon 24 h treatment with PL or RE at 100 μM concentration. (b) Impact of treatment and excretion duration of PL and RE in the change in pharynx pumping rate.

Figure 6

Systemic effects of Ppy NPs on C. elegans. (a) The percentage of survival of untreated worms and various concentrations of Ppy NPs treated worms (N = 60). (b) The measurement of the body length of 1st generation worms after 24 h of treatment (N = 40). (c) The number of eggs and larvae produced by the worms in 72 h post-exposure (N = 12). (d) The measurement of the body length of fully developed second generation worms (N = 30). No statistical significance was found in any case. Top panel (scheme) created with BioRender.com.

Figure 7

(a) Video snapshots of pharyngeal muscle contractions in C. elegans: pumping, isthmus peristalsis, and resting. The grinder is marked in red in the images, displaying contraction and relaxation, and the whole pharynx is localized with a green dotted line. (b) The pharynx pumping rate in C. elegans of N2, JD21, and DA464 strains, with and without Ppy NPs treatment (N ≈ 30).

Figure 8

Effect of the pumping rate change during exposure and after excretion of PPy NPs over time was evaluated in N2 (wild-type), JD21 (cca-1 calcium channel mutant), and DA464 (eat-5 gap junction mutant) (N ≈ 30).

Figure 9

(a) FT-IR spectra of untreated and treated C. elegans. (b) Fluorescent images of BODIPY stained control worms. (c) Fluorescent images of BODIPY stained Ppy treated worms. (d) Quantification of the lipid levels computed using BODIPY intensities, measurements of the (e) lipid oxidation ratio (A₁₇₄₀/A₂₉₂₀), (f) Lipid saturation ratio (A₂₉₂₀/A₂₉₆₀), and (g) lipid unsaturation ratio (A₃₀₁₂/A₂₉₂₀) of untreated (control) and treated worms (Ppy NP treated) using SR-μFTIR spectroscopy (N ≈ 50 worms).

Figure 10

Calcium transients in the pharynx are measured by fluorescent calcium imaging. (a) Calcium transient spikes in control and Ppy NP treated worms showing the frequency (F), width at half-maximum (WHM), width at baseline (Width), and peak height (H). (b) Quantification of the frequency of calcium transients, (c) width at half-maximum, (d) width at baseline, and (e) peak height of untreated and Ppy NP treated worms in N2 and JD21 strains.

Figure 11

Reproductive ability and intergenerational toxicity assays of C. elegans after exposure. Created with BioRender.com.

Figure 12

Experimental process of pharynx pumping rate measurements after exposure and excretion. Created with BioRender.com.