Comparative effects of parent and heated cinnamaldehyde on the function of human iPSC-derived cardiac myocytes

Highlights

• Cinnamaldehyde impairs contractile activity of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs).

• Exposure of hiPSC-CMs to cinnamaldehyde leads to depolarization of resting membrane potential.

• Heating cinnamaldehyde attenuates effects on hiPSC-CM function.

Abstract

Many e-cigarette products contain cinnamaldehyde as a primary constituent of cinnamon flavorings. When used as a food additive, cinnamaldehyde is generally regarded as safe for ingestion. However, little is known about the effects of cinnamaldehyde or its degradation products, generated after heating and inhalation, which may lead to elevated circulatory exposure to the heart. Hence, in this study, we tested the in vitro cardiac toxicity of cinnamaldehyde and its thermal degradation products generated by heating at low (200 ± 50 °C) and high temperatures (700 ± 50 °C) on the contractility, rhythmicity and electrical signaling properties of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs). Cellular impedance measurements on spontaneously beating hiPSC-CMs revealed that cinnamaldehyde significantly alters contraction-dependent signal amplitude, beating rate, and cell morphology. These effects were attenuated after cinnamaldehyde was subjected to heating at low or high temperatures. Current clamp analysis of hiPSC-CM action potentials (APs) showed only modest effects of acute application of 1–100 μM cinnamaldehyde on resting membrane potential, while prolonged (~20 min) application of 100 μM cinnamaldehyde resulted in progressive depolarization and loss of rhythmic AP spiking activity. Collectively, these results suggest that micromolar levels of cinnamaldehyde could alter cardiac excitability, in part by impairing the processes that regulate membrane potential and depolarization. Our results further suggest that heating cinnamaldehyde by itself does not directly lead to the formation of products with greater cardiotoxicity in vitro.

Introduction

Cinnamaldehyde is an aromatic aldehyde that confers the characteristic flavor and odor of cinnamon. As a primary constituent of cinnamon bark, cinnamaldehyde has been ingested by people for centuries and it is commonly heated at high temperatures in cooking and baking. More recently, with the increasing popularity of electronic cigarettes (e-cigarettes), cinnamaldehyde and its thermal degradation products are now routinely inhaled as this chemical is a major additive to commercially available e-liquid formulations (Behar et al., 2016). Chemical analyses of several of these e-liquids have measured cinnamaldehyde present at a range of concentrations, and as high as >1 M (Behar et al., 2016; Clapp et al., 2017). Although measurements of cinnamaldehyde and its metabolites in the plasma of e-cigarette users are lacking, pharmacokinetic analyses in rats suggest that cinnamaldehyde as well as products of cinnamaldehyde metabolism, cinnamyl alcohol and methyl cinnamate, persist in the plasma after oral and intravenous administration with a half-life of ~7 h (Zhao et al., 2014). These observations suggest that cinnamaldehyde enters the systemic circulation after these routes of exposure and can potentially affect the function of numerous cell types. Nevertheless, despite the increasing consumption of cinnamaldehyde via electronic nicotine delivery systems (ENDS), the cellular toxicity of cinnamaldehyde and its thermal degradation products remains unclear.

Previous work suggests that cardiovascular tissues may be particularly sensitive to chemical and xenobiotic exposure relative to other tissues, in part because cells of the heart and blood vessels have a low capacity for xenobiotic detoxification (Bhatnagar, 2004). This is consistent with studies showing that the heart may be highly vulnerable to cumulative injury subsequent to exposure to environmental toxins (Izzotti et al., 1999; Ping et al., 2003). Recently, it was reported that cinnamaldehyde reduces cardiac inflammation and fibrosis in fructose-fed rats (Kang et al., 2016) and that it attenuates LPS-induced cardiac dysfunction (Zhao et al., 2016), suggesting that anti-oxidant and anti-inflammatory actions of cinnamaldehyde can have a beneficial influence on cardiac function in the setting of induced inflammation. However, the direct effects of cinnamaldehyde on the function of cardiac myocytes have not been studied.

Despite potential beneficial effects of cinnamaldehyde on the heart, attributable to anti-inflammatory properties, cinnamaldehyde has been shown to affect ion channel activities (Alvarez-Collazo et al., 2014; Bandell et al., 2004) and the generation of reactive oxygen species (Ka et al., 2003; Noh et al., 2015). Hence, we tested the hypothesis that direct exposure of cardiac myocytes to cinnamaldehyde can acutely impact their function, independent of any potential chronic anti-inflammatory or anti-fibrotic effects. We also postulated that heating cinnamaldehyde, as during e-cigarette use and in cooking, could alter cinnamaldehyde toxicity and/or lead to the formation of new products that have their own unique cardiotoxicity profile. To test these hypotheses, we examined the effects of cinnamaldehyde and its thermal degradation products using spontaneously beating human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CM) as a model in vitro platform. These cells are stable and readily accessible for in vitro pharmacological and toxicological screening for potential cardiotoxic effects (Sharma et al., 2013). By using an integrated approach that combines cellular impedance measurements with electrophysiological evaluation of cellular action potential properties, we found that without heating, cinnamaldehyde impairs normal beating rhythmicity and contractility in hiPSC-CMs, before the onset of overt cytotoxicity, i.e., cell death. Significantly, we found that cells that were treated with an equivalent concentration of cinnamaldehyde, subjected to heating, did not exhibit the pattern of dysfunction seen with parent cinnamaldehyde, suggesting that the cardiotoxicity of cinnamaldehyde may depend upon heating conditions used prior to exposure.