Oxidative stress mediates cardiac electrophysiological injury in inhalation exposure to flavored vaping products

Abstract

BACKGROUND

Electronic nicotine delivery systems (ENDS) use “E-liquids” to generate “E-vapor,” an inhalable aerosolized mixture containing nicotine and flavors. Flavored ENDS are very popular among teens who vape, however, the possible cardiac electrophysiological harm of inhalation exposure to flavored ENDS are not fully understood.

OBJECTIVE

To test if inhalation exposure to flavoring carbonyls in E-liquids compromises mitochondrial integrity, increases oxidative stress, and leads to cardiac electrophysiological toxicity.

METHODS

Gas chromatography mass spectrometry (GC/MS), and flow cytometry, oxygen consumption rate measurement, in vivo programmed electrical stimulation (PES), and multielectrode array (MEA) were used in atrial like HL-1 myocytes, human induced pluripotent stem cells-derived cardiomyocytes, and in mice overexpressing the antioxidant mitochondrial catalase (mCAT) exposed to flavored E-vapor.

RESULTS

We compared the toxicity of E-vapor exposure from 30 differently flavored E-liquids (70% vegetable glycerin, 30% propylene glycol, with 6 mg/mL nicotine) in HL-1 cells using apoptotic annexin V flow cytometry. The toxicity of most E-liquids was significantly higher than air control. We subsequently determined the composition and quantified the concentrations of major flavoring carbonyls (cinnamaldehyde, vanillin, ethyl vanillin, maltol, and ethyl maltol) in the E-liquids using GC/MS. Linear regression analysis showed that toxicity correlated with carbonyl concentration. We then measured in HL-1 cells exposed to vanilla flavored E-vapor reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ) with flow cytometry, respectively. ROS increased in exposed cells, but this was prevented by N-acetylcysteine antioxidant pretreatment, and ΔΨ was depolarized in exposed cells. In vivo inhalation exposure to vanilla flavored E-vapor increased the duration of PES-induced ventricular tachycardia in wild type but not in mCAT mice, compared with air controls. Finally, MEA recordings in hiPSC-derived cardiomyocytes monolayers exposed to vanilla flavored E-vapor, with or without nicotine, resulted in decreased activation time, and increased corrected field potential duration.

CONCLUSION

Inhalation exposure to flavored ENDS negatively affects ventricular electrophysiology, in part via adverse mitochondrial remodeling, and increased oxidative stress.

Vaping, or the use of flavored electronic nicotine delivery systems (ENDS), remains a serious public health issue for school-aged children and young adults. ^1–4 Recently, the Centers for Disease Control and Prevention estimated that around 2.2 million middle and high school students reported vaping. ⁵ It is also estimated that about 88% of these students specifically prefer to use flavored ENDS. ⁶

Vaping is a form of electronic nicotine delivery where a coil is heated to aerosolize an “E-liquid,” a nicotine-containing solution. E-liquids are a mixture of base humectants—propylene glycol and vegetable glycerin—with varying concentrations of flavoring constituents and nicotine. The generated “E-vapor” is an inhalable smoke-like aerosolized mixture of nicotine, flavorings, humectant particles, and their resulting aldehyde byproducts.

It is becoming increasingly clear that vaping harms the cardiovascular system.^7–10 For example, endothelial dysfunction due to ENDS use has been examined. The findings congruently report that vaping exposure causes vascular stiffness, increased endothelial microparticles, decreased levels of nitric oxide, and elevated levels of reactive oxygen species (ROS). ^4,11–14 Also, animal studies have suggested cardiac structural and electrophysiological changes such as ventricular remodeling, sympathetic predominance in heart rate variability, and increased arrhythmogenesis.^7,15–19 In vaped mice, Oakes et al,¹⁹ reported right ventricular (RV) hypertrophy with increased RV free wall thickness, alongside increased RV systolic blood pressure. The authors,¹⁵ and others,^18,20–22 have shown increased sympathetic predominance in heart rate variability, increased arrhythmogenesis, action potential alternans, and electrocardiogram (ECG) changes in mice with inhalation exposure to ENDS.

Studies in human users have shown that vaping increases the Tp-e/QT, an ECG index of ventricular repolarization, which is associated with increased risk of sudden death. ^16,23 Furthermore, a case report and a retrospective analysis are pointing toward a possible association between recent vaping and sudden cardiac arrest. ^24,25 The retrospective analysis by Bains et al,²⁴ reported that in 6 patients (1% of the cases reviewed) sudden cardiac arrest or sudden death were temporally associated with vaping use. 4 patients experienced sudden cardiac arrest with ventricular fibrillation within minutes to hours of vaping, and 2 patients experience sudden death associated with vaping-induced respiratory distress. Interestingly, the mean age of these patients was 23 ± 5 years old, further raising concerns about the possible electrophysiological harm of vaping in young users.

Most studies have focused on the role of nicotine in mediating cardiac harm, but it has been suggested that other components of the E-liquids such as the humectants may negatively affect cardiac electrophysiology.²² Although the harm of flavorings in other organ systems such as the liver, lungs, vascular, and immune systems^26–34 have been investigated, the possible cardiac harm of flavoring carbonyls has not been extensively studied.

Although flavored ENDS remain popular among young users, and preclinical and clinical studies are pointing to possible adverse cardiac effects associated with vaping, more mechanistic insights into how vaping impacts the electrophysiology of the heart are needed. Thus, our objective in this study is to investigate if flavorings contribute to cardiac harm, and if inhalation exposure to flavored ENDS leads to ventricular arrhythmogenesis through increased oxidative stress.

Methods

All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of South Florida Morsani College of Medicine and conformed to the Guide for the Care and Use of Laboratory Animals.

HL-1 cell culture

HL-1 cells (mouse atrial myocytes) were obtained from the laboratory of Dr. Claycomb (Louisiana State University, Baton Rouge, Louisiana) and cultured following the recommended protocol.³⁵ Briefly, cells were grown in Claycomb medium (Sigma, St. Louis, Missouri) and supplemented with 10% fetal bovine serum (Sigma), 0.1 mM norepinephrine (Sigma), 2 mM L-glutamine (Sigma), and penicillin/streptomycin (100 U/mL/100 m/mL) on tissue culture plates (Corning, Corning, New York), coated with fibronectin/gelatin (Sigma).

E-liquids and E-vapor extracts

The flavored E-liquids used in this study are manufactured by USA Vape Labs (Huntington Beach, California). The 30 differently flavored E-liquids were Menthol, American Patriot Tobacco, Cuban, Euro Gold, Macchiato, Vanilla Custard (VC), Triple Strawberry, Straw Lime, Berry Pom, Guava, Peach Lemon, Amazing Mango, Azul Berries, Naked Unicorn, Berry Lush, GoNanas, Green Blast, Maui Sun, Lava Flow, All Melon, Hawaiian POG (POG), Peachy Peach, Mango Ice, Lava Flow Ice, Very Cool, Brain Freeze, Apple Cooler, Hawaiian POG Ice, Polar Freeze, and Apple Jax (APJ, Epic Juice, Santa Ana, California). These E-liquids are 70% vegetable glycerin/30% propylene glycol (70VG/30PG) and are stated by the manufacturer to contain 6 mg/mL free-base nicotine. Base only (70VG/30PG) (Sigma-Aldrich), and base plus 6 mg/mL free-base nicotine (Sigma-Aldrich) solutions were prepared in-house. E-vapor extracts were generated as we did previously,¹⁵ and following the same puff topography as we have done previously.¹⁵ In short, E-vapor extracts were prepared fresh before each experiment by bubbling 10 mL of fresh, untreated, culture media with 15 puffs of aerosol generated from the different E-liquids, or base, or base plus nicotine. The resulting E-vapor extracts had a concentration of 1.5 puffs/mL, as we have done previously.¹⁵ For the air control media, the same protocol was followed with the vape device powered off, thereby bubbling 10 mL of fresh, untreated, culture media with room air for an equivalent amount of time.

Apoptosis flow cytometry

Apoptosis was measured using the fluorescein isothiocyanate (FITC) annexin V staining assay (BD). HL-1 cells were plated in 12-well plates and cultured to ~70% confluency before being treated with either control media, or with one of the E-vapor extracts at 0.75 puffs/mL, as we have done previously.¹⁵ After 24 hours exposure to E-vapor extracts, cells in each well were detached with Accutase (approximately 8 mins), pelleted by centrifugation, washed with phosphate buffered saline (PBS), and stained with FITC annexin V according to the manufacturer’s recommendation. Cells were then pelleted by centrifugation, and washed and resuspended in PBS. 4′, 6-diamidino-2-phenylindole (DAPI) was added immediately before reading the samples on a BD LSRII Cytometer (using 488 nm and 405 nm lasers for the respective excitation of FITC annexin V and DAPI fluorescence, and 530/30 nm and 450/50 nm for their respective emissions). Data analysis was carried out using FlowJo software. As we did previously, the toxicity index was calculated as $\mathit{\text{TI}}=\frac{\%\mathit{\text{DC}}+\%\mathit{\text{AC}}}{\%\mathit{\text{LC}}}$ where $\%\text{DC}$ is the percentage of dead cells, $\%\text{AC}$ is the percentage of apoptotic cells, and $\%\text{LC}$ is the percentage of live cells per well. ¹⁵

Mass spectrometry analysis of E-liquids

E-liquids were diluted 1:40 in pesticide-grade methanol and ran through the Aglient 7890B gas chromatograph, mass spectrometer 5977B (GC/MS). The GC/MS parameters were optimized based on previous studies^15,25 and the samples were run as we have done in our previously published work.¹⁵ MassHunter Workstation Qualitative Analysis Software (Version B.07.00 SP2) in conjunction with National Institute of Standards and Technology mass spectrometry Search 2017 Library was used for analysis. We then quantified the concentrations of the most abundant flavoring aldehydes (cinnamaldehyde, vanillin, and ethyl vanillin), and ketones (maltol and ethyl maltol), that we found in the screened E-liquids (19 out of 30 E-liquids). To quantify the concentrations of these carbonyls, we generated standard curves using 20, 50, 100, 150, and 200 mM of each flavoring carbonyl in 70VG/30PG base. The total concentration of the flavoring carbonyls present in each of the E-liquid was then calculated. Figures were generated with OriginPro Lab Software 2018.

Reactive oxygen species flow cytometry

Oxidative stress was measured with the CellROX^™ Green Flow Cytometry Assay Kit (Invitrogen; Catalog # C10492). HL-1 cells were cultured to ~70% confluency before being treated with either control media or 0.75 puffs/mL VC E-vapor extract for 2 hours. Cells were then detached with Accutase (approximately 8 min), pelleted by centrifugation, washed and resuspended in complete Claycomb medium. Control cells were pre-incubated with 5 mM NAC (N-acetylcysteine) for 30 mins, or with 200 μM TBHP (tert-butyl hydroperoxide) for 30 minutes. 500 nM CellROX Green was then added to all samples and incubated for 30 minutes at 37°C. Cells were then pelleted by centrifugation, and washed and resuspended in PBS. Then, 5 μM SYTOX Red Dead Cell stain was added, and samples were read immediately on a BD LSRII Cytometer (using 488 nm and 639 nm lasers for the respective excitation of CellROX Green and SYTOX Red fluorescence, and 530/30 nm and 665/40 nm filters for their respective emissions).

Mitochondria membrane potential (ΔΨ) flow cytometry

Mitochondria membrane potential was measured in HL-1 cells using tetramethylrhodamine ethyl ester (TMRE, Thermo Fisher; Catalog # T669). HL-1 cells were cultured to ~70% confluency before being treated with either control media, 1 mM tert-butyl hydroperoxide (TBHP) as a positive control, or 0.75 puffs/mL VC E-vapor extract for 24 hours. Cells were then detached using 0.005% trypsin, pelleted by centrifugation, washed with PBS, stained with 50 nM TMRE, and incubated for 20 minutes at 37°C. Cells were then again pelleted by centrifugation, then washed and resuspended in PBS, and DAPI (0.1 μg/mL) was added immediately before reading the samples on a BD FACSCanto^™ II Cytometer (using 488 nm and 405 nm lasers for the respective excitation of TMRE and DAPI fluorescence, and 585/42 nm and 450/50 nm for their respective emissions).

Oxygen consumption rate

The oxygen consumption rate (OCR) of HL-1 cells was measured with the XFp Cell Mito Stress Test by Agilent Seahorse. Adenosine triphosphate (ATP)-linked respiration was quantified with 1.5 μM oligomycin, maximal respiration with 1.5 μM phenylhydrazone (FCCP), and nonmitochondrial respiration driven by processes outside the mitochondria with 0.5 μM rotenone and antimycin. 2 days prior to the experiment, 40,000 cells/well were plated in a gelatin/fibronectin precoated XFp mini cell culture plate (11.4 mm² area) at 37 °C. Cells were treated with either control media, APJ, or Hawaiian POG E-vapor extracts for 24 hours. Immediately before measuring, culture medium was replaced with the prewarmed assay medium (Agilent) containing 10 mM glucose, 1 mM pyruvate, and 200 mM L-Glutamate. Oligomycin, FCCP, and rotenone/antimycin were added to the cells in 20-minute intervals, according to the manufacturer’s instructions. At the end of the protocol, the culture media was gently removed, and 40 μl of RIPA lysis buffer was added, and cells were lysed using a 1 mL syringe. Protein concentrations were then quantified by BCA protein assay (Thermo Fisher Scientific) for normalization of the respiration parameters.

Isolation of mouse ventricular myocytes

Mouse ventricular myocytes were enzymatically isolated from 4 to 6 months old mitochondrial catalase (mCAT) overexpressing mice (Jackson Laboratory, Bar Harbor, Maine), and wild type (WT) littermates of both sexes. In short, the heart was quickly removed via thoracotomy from deeply anesthetized mice (2% isoflurane) after loss of all reflexes. The heart was then perfused retrogradely with calcium-free Tyrode solution (137 mM NaCl, 5.4 mM KCl, 10 mM HEPES, 1 mM MgCl₂, and 10 mM glucose, pH 7.3) using a peristaltic pump at a rate of 2 mL/min for 3 minutes at 36°C, until the outflow was free of blood. This was then followed by retrograde perfusion with the same calcium-free Tyrode solution supplemented with 1 mg/mL collagenase Type A (Roche, Cat. #9036–06-0) and 0.08 mg/mL protease Type XIV (Sigma-Aldrich, Cat. #48655321) for 8–11 minutes. Next, the heart was perfused with 0.2 mM CaCl₂ supplemented Tyrode solution for 5 minutes. Ventricular tissue was then separated and dissociated by gentle agitation to isolate individual ventricular myocytes. The resulting cell suspension was passed through a nylon mesh to remove debris, and the isolated myocytes were then plated in 35 mm dishes, stained, and incubated with E-vapor extract for subsequent cell viability and ROS imaging with fluorescence microscopy.

Confocal microscopy

For cell-viability imaging, plated ventricular myocytes were treated with either control media or media with different concentrations of VC E-vapor (0.015, 0.075, 0.15, 0.375, and 0.75 puffs/mL) and incubated at 37°C for 24 hours. Afterwards, myocytes were incubated with 5 μM calcein-AM ester (Biolegend, Cat. # 425201) for 30 minutes, followed by 2 washes with Tyrode solution containing 2 mM CaCl₂. ³⁶ Fluorescence imaging was then immediately performed using a Leica Stellaris microscope at 40x magnification. Green fluorescence intensity was quantified using FIJI (ImageJ) software, and the mean fluorescence intensity was plotted using Prism 10 software (GraphPad). Additionally, ROS imaging was performed using the mitochondrial ROS indicator MitoSOX Red (Thermo Fisher, Cat. # M36008).^37,38 Isolated ventricular myocytes from WT and mCAT transgenic mice were stained via incubation for 15 minutes with 5 μM MitoSOX^™ Red at room temperature then washed twice with 2 mM CaCl₂ Tyrode solution.^39,40 Preparations were treated with 0.375 puffs/mL VC E-vapor extract. Fluorescence imaging was performed on a Leica Stellaris microscope at 40X magnification before and then after incubation with the extract. The fluorescence intensity of MitoSOX Red was quantified using FIJI (ImageJ), and mean intensities were plotted using Prism 10 software (GraphPad).

Exposure of animals to E-vapor

Exposure of mice to E-vapor was performed as we previously described,¹⁵ using the Smok Species Baby V2 (SMOKTech, Shenzhen, China) vaping device with the Baby V2 A2 dual sub coils (SMOKTech) with a total resistance of 0.2 Ω, at 85 W. mCAT overexpressing and WT littermates of both sexes were exposed to either room air or VC E-vapor containing 6 mg/mL nicotine. For inhalation exposure, the animals were loaded into a custom-made chamber and were exposed to 4.7-second puffs of VC E-vapor at 1.4 L/minute, every 2 minutes for a total of 60 puffs in a 2-hour period. After exposure, the animals were returned to their cages. Puff topography used a total puff volume of 110 mL,¹⁵ mimicking what has been described in human use.³⁷ Mice were exposed 5 days a week, for a period of 5 weeks. Control mice experienced the same handling as the vaped animals, were placed in a similarly modified chamber, in the same environment, where the only difference was that they were exposed to normal room air. Mice were individually housed in ventilated racks, with ad libitum access to food and water.

In vivo ventricular tachycardia inducibility

Mice were anesthetized with 2% isoflurane, and a 1.2-French octapolar catheter (Millar) was placed trans-venously into the right jugular vein and advanced into the right ventricle. Electrograms were recorded using the PowerLab platform (AD Instruments). Programmed electrical stimulation (PES) for ventricular tachycardia (VT) induction was performed by pacing the right ventricle at twice the diastolic threshold with 1-second bursts from 20 to 50 Hz, in 2-Hz increments as we did before.^15,38,39 ECG intervals were determined in 20 averaged beats using LabChart (AD Instruments), and then manually verified.

hiPSC-derived cardiomyocytes culture and extracellular potential recording

Human-induced pluripotent stem cell-derived cardiomyocytes, iCell^® Cardiomyocytes, 01434 (Fujifilm; Catalog # R1220), were thawed, plated, and maintained according to MED64 Presto protocol. These iCell cardiomyocytes, which are reported to be enriched for cells having ventricular-like action potential,⁴⁰ were plated on the 24-well multiple electrode array (MEA) plates (24-well plate-eco, MED64; Catalog # MED-Q2430M), where each well had 4×4 electrodes, and 300 mm interelectrode distance. The monolayers of cells begin to beat spontaneously, periodically, and synchronously around 7 days after plating. Monolayers remained beating robustly in culture for at least 14 days after thawing. Monolayers were treated with either control media, or with 0.15 puffs/mL of unflavored 70VG/30PG base without nicotine E-vapor extract, or 0.15 puffs/mL reconstituted VC (RVC) without nicotine E-vapor extract, or 0.15 puffs/mL VC with nicotine E-vapor extract. RVC E-liquid without nicotine was made in-house after GC/MS analysis of the VC E-liquid (USA Vape Labs, 6mg/mL nicotine) (Table 1). The reconstituted E-liquid was done by supplementing unflavored 70VG/30PG base with 79.2 mM vanillin, 48.8 mM ethyl vanillin, and 79.0 mM ethyl maltol as determined by GC/MS in Table 2. This resulted in a reconstituted E-liquid with total carbonyls concentration of 207 mM, similarly to the original VC E-liquid. The Presto Multielectrode Array (MED64, Osaka, Japan) was used to simultaneously record the 16 unipolar extracellular potentials (digitized at 20 KHz) in each well of spontaneously beating hiPSC-derived cardiomyocytes monolayers at 37°C and 95% O₂/5% CO₂. Wells that showed spontaneous, rhythmic activity before treatment were used.

Table 1.

E-liquids grouped by type.

Type	E-liquid names Flavors listed on the label
MENTHOL	Crisp Menthol Menthol mint
TOBACCO	American Patriot Classic tobacco	Euro Gold Caribbean tobacco	Cuban Blend Smooth tobacco
VANILLA	Appl Jax Apple Cinnamon	Macchiato Vanilla Custard Coffee	Vanilla Custard Vanilla Custard
SIMPLE FRUITS	Triple Strawberry Sweet Strawberry	Straw Lime Strawberry Lime	Berry Pom Blueberry Pomegranate	Guava Strawberry Guava	Peach Lemon Peach Lemonade
CREAMY FRUITS	Amazing Mango Mango Peach Cream	Azul Berries Blueberry Raspberry Cream	Naked Unicorn Strawberry Cream	Berry Lush Strawberry Pineapple Cream	Go Nanas Banana Cream
MIXED FRUITS	Green Blast Honeydew Green apple Kiwi	Maui Sun Pineapple Orange Tangerine	Lava Flow Strawberry Coconut Pineapple	All Melon Watermelon Honeydew Cantaloupe	Hawaiian POG Passion Fruit Orange Guava	Peachy Peach Peach Apricot Nectarine
CREAMY FRUITS AND MENTHOL	Mango Ice Mango Peach Cream Menthol	Lava Flow Ice Strawberry Pineapple Coconut Menthol	Very Cool Blueberry Blackberry Raspberry Menthol	Brain Freeze Strawberry Kiwi Pomegranate Menthol	Apple Cooler Apple Green grape Watermelon Menthol	Hawaiian POG Ice Passion Fruit Orange Guava Menthol	Polar Breeze Honeydew Pineapple Cantaloupe Menthol

E-liquids grouped by type, with the E-liquids name (bold) and description of the flavor profile (italics) as printed on the manufacturer’s label.

Table 2.

Toxicity index and carbonyl concentrations.

Flavor	Toxicity index	Vanillin	Ethyl vanillin	Maltol	Ethyl maltol	Cinnamaldehyde	Total carbonyls
Vanilla Custard	7.9	79.2	48.8		79		207.0
Macchiato	6.0	83.9	57.1		33.6		174.6
Naked Unicorn	2.2	46.7	57.4	2.2	28.5		134.8
Apple Jax	5.3	48.1	17.1	1.5	19.2	40.4	126.3
Azul Berries	1.4	33.3	44.2		23.3		100.8
GoNanas	3.3	34.2	22.1		40.8		97.1
Cuban	2.9				83.3		83.3
Berry Lush	2.2	17.2	28.6	2.4	8.2		56.4
American Tobacco	1.3	15.3	5.1		22.2		42.6
Peachy Peach	2.8		32.8				32.8
GreenBlast	1.5	17.5		1.5	2.6		21.6
Strawlime	1.5		20.1				20.1
Mango	1.3	12.6	2.6				15.2
Mango ICE	1.1	11.9	2.5				14.4
BrainFreeze	1.7			2.3			2.3
LavaFlow ICE	1.6			2			2.0
LavaFlow	1.9			2			2.0
AllMelon	2.1			1.6			1.6
Applecooler	1.7			1			1.0

The table lists the flavored E-liquid’s name, toxicity index, carbonyl concentration in mM for each of the 5 flavorings (vanillin, ethyl vanillin, maltol, ethyl maltol, and cinnamaldehyde), and the total carbonyl concentration.

Five-minute recordings were obtained and quantified after treatment for 24 hours with the different E-vapor extracts or control media. Beating rate and corrected field potential duration (cFPD) were calculated in the MEA Symphony analysis software (MED64). Activation maps were generated using the propagation map feature of the Mobius Offline Toolkit software (MED64) and are displayed after bilinear interpolation for visualization.

Statistics

Data are presented as average ± standard deviation. Student t test and one-way analysis of variance (ANOVA) with post hoc Fisher’s LSD test were used as appropriate, and significance was taken at P < 0.05.

Results

We measured the toxicity of E-liquids in HL-1 mouse atrial cardiomyocytes³⁵ using flow cytometry of FITC conjugated annexin V, an apoptotic marker, and DAPI for cell viability. Cells were cultured for 24 hours with control media or E-vapor extract for each of the 30 E-liquids, containing 6 mg/mL nicotine-free base as per the label statement. Cells were then dissociated and stained with annexin V and DAPI. Flow cytometry analysis was performed to quantify the dead (high DAPI staining), apoptotic (high annexin V staining), and live (low annexin V and DAPI staining) cell populations, as we have previously done.¹⁵ The toxicity index of each of the E-liquid treated wells were calculated and normalized to air controls. Figure 1A shows a flow cytometry analysis of a control sample where most cells are in the live quadrant. Panel B shows the flow cytometry analysis of cells treated with Macchiato E-vapor extract which caused a shift of cells to the apoptotic and necrotic quadrants. Figure 1C compares the normalized toxicity index after 24 hours treatment of HL-1 cells with 0.75 puffs/mL of the different E-vapor extracts. In Table 1, we grouped the E-liquids into 7 types according to the description of the flavor profile as stated on the label. They were: 1- menthol, 2- tobacco, 3- vanilla, 4-simple fruits, 5- creamy fruits, 6- mixed fruits, and 7- creamy fruits and menthol. We also tested the unflavored E-liquid base containing 70VG/30PG and 6mg/mL nicotine. Although some flavored E-liquids were more toxic than others, as seen in Figure 1C, the toxicities of all E-liquids, including the unflavored base, were significantly higher than that of air, except for Mango Ice and Amazing Mango.

Figure 1.

Toxicity of 30 differently flavored E-liquids in HL-1 cells cultured with 0.75 puffs/mL E-vapor extracts for 24 hours. Flow cytometry of annexin V and DAPI staining in HL-1 cells (Air control, A, and Macchiato, B). Live, apoptotic, and necrotic quadrants are indicated. C: Toxicity index of the E-liquids organized into 7 groups: 1-menthol (purple), 2-tobacco (silver), 3-vanilla (red), 4-simple fruits (blue), 5-creamy fruits (yellow), 6-mixed fruits (orange), and 7-creamy fruits and menthol (green). Unflavored base with nicotine (Base Nic) (black) and Air (black and white) were also included. All samples, expect Amazing Mango and Mango Ice E-vapor, extracts were found to be significantly toxic (P < 0.05, each flavor vs its own air control, t-test, N = 4 to 6 each). DAPI = 4′, 6-diamidino-2-phenylindole.

We then used GC/MS to analyze the chemical composition of the E-liquids listed in Table 1. Figure 2A shows the chromatograms of Macchiato (high toxicity index, Figure 1) E-liquid, and American Patriot Tobacco (low toxicity index, Figure 1) E-liquid. The propylene glycol peak was trimmed to enhance visibility of the smaller peaks of flavorings as customarily done.^41,42 The glycerin and nicotine peaks are evident, in addition to other flavorings peaks corresponding to flavoring carbonyl products, such as the aldehydes vanillin and ethyl vanillin, and the ketone ethyl maltol. The peaks of vanillin, ethyl vanillin, and ethyl maltol are larger in the more toxic Macchiato E-liquid compared with the less toxic American Patriot Tobacco E-liquid (Figure 1). GC/MS analysis revealed that 19 of the 30 E-liquids contained vanilla (vanillin, and ethyl vanillin), cinnamon (cinnamaldehyde), or sweet (maltol, and ethyl maltol) flavoring carbonyls. In the rest of the E-liquids, other types of flavorings were present, but in small quantities. Since these carbonyls were the most abundant, we decided to focus on them. We then quantified using GC/MS the concentrations of the most abundant flavoring aldehydes. To quantify the concentrations, we constructed standard curves for each of the carbonyl compounds. Figure 2B is an example showing the GC/MS chromatograms of unflavored 70VG/30PG base that we spiked with known amounts of ethyl vanillin. Standard curves were constructed using the area under the curve. The concentration of the 5 carbonyls in the 19 E-liquids were then calculated. Some E-liquids contained 1 of the carbonyls (eg, Strawberry Lime: 20.1mM ethyl vanillin, [total carbonyls] = 20.1mM, toxicity index = 1.48), whereas others contained a combination (eg, VC: 79.2 mM vanillin, 48.8 mM ethyl vanillin, 79mM ethyl maltol, [total carbonyls] = 207 mM, toxicity index = 7.85). Table 2 lists the 19 E-liquids, with their corresponding toxicity indexes from Figure 1, and the concentration of the different carbonyls in mM. Figure 2C is a plot of the total carbonyl concentration vs the toxicity index of the 19 E-liquids. Linear regression analysis revealed that toxicity correlated significantly with carbonyl concentration, where the coefficient of correlation was 0.84, P < 0.001.

Figure 2.

GC-MS analysis of flavoring carbonyls in the flavored E-liquids. A: GC-MS chromatograms of Macchiato (left) and American Patriot (right) flavored E-liquids. Glycerin, propylene glycol, and nicotine peaks are apparent in addition to peaks denoting flavoring carbonyls such as ethyl maltol, vanillin, and ethyl vanillin. B: Chromatograms of ethyl vanillin at different concentrations (20mM, 50mM, 100mM, 150mM, and 200mM) used for constructing the standard curve, which was used to quantify ethyl vanillin concentrations in the different E-liquids. The same was done for vanillin, maltol, ethyl maltol, and cinnamaldehyde. C: Linear regression analysis of total carbonyl concentration vs toxicity index (R = 0.84, P < 0.01). GC-MS = Gas chromatography-mass spectrometry.

Next, we measured ROS in HL-1 cells treated for 2 hours with 0.75 puffs/mL VC E-vapor extract to test whether vaping increases oxidative stress in cardiac myocytes. Figure 3A is the flow cytometry histogram analysis where VC treatment caused the cell population to shift toward higher ROS staining (red) compared with control (green). The black histogram shows that antioxidant pretreatment with 5 mM N-acetylcysteine (NAC) for 30 minutes blunted the effects of VC E-vapor extract on ROS generation. Blue represents control cells treated only with 5 mM NAC for 30 minutes. Panel B is a quantification of the percentage of cells with high ROS staining. A significant increase in ROS positive cells were observed in the VC treatment, and the NAC pretreatment significant reduced ROS generation.

Figure 3.

Quantification of CellRox staining in HL-1 cells with flow cytometry. A: Histogram of normalized cell counts for air control (green curve), 5 mM N-acetylcysteine pretreatment (NAC, blue curve), 0.75 puffs/mL Vanilla Custard (VC) E-vapor extract (red curve), and 0.75 puffs/mL VC E-vapor extract with 5 mM N-acetylcysteine pretreatment (VC + NAC, black curve). B: Quantification of the % of cells with high ROS staining (**P < 0.01, VC vs all other groups, one-way ANOVA with Fisher’s LSD test).

We also measured the mitochondrial membrane potential (ΔΨ) in HL-1 cells treated with either control media, TBHP, or VC E-vapor extract. Figure 4 panel A shows the flow cytometry histogram analysis where VC treatment (red) caused the cell population to shift toward lower TMRE fluorescence, suggesting a depolarization of the ΔΨ compared with control (green). Similarly, TBHP treatment (positive control, black) caused a depolarization of the ΔΨ. Figure 4B is the quantification of the percentage of cells with depolarized ΔΨ, which was significantly lower in control vs TBHP and VC treatments.

Figure 4.

Quantification of TMRE staining in HL-1 cells with flow cytometry. A: Histogram of normalized cell counts for air control (green curve), 0.75 puffs/mL Vanilla Custard (VC) E-vapor extract (red curve), and 1 mM tert-butyl hydroperoxide (TBHP, black curve). B: Quantification of the % of cells with low TMRE staining (**P < 0.01, one-way ANOVA with Fisher’s LSD test). ANOVA = analysis of variance; TMRE = tetramethylrhodamine, ethyl ester.

We then compared cellular respiration in HL-1 cells after 24-hour treatment with 0.375 puffs/mL E-vapor extract from an E-liquid with a high toxicity (APJ, toxicity index = 5.3) or lower toxicity (POG, toxicity index = 2.3) as reported in Figure 1. Figure 5A is a graph of OCR vs time for control, APJ, or POG E-vapor extract treated cells. Figure 5B quantifies basal OCR (OCR before addition of Oligo), ATP-linked OCR (OCR before Oligo minus OCR before FCCP), and maximal OCR (OCR before A/R minus OCR after A/R). Basal and ATP-linked OCR were significantly depressed with APJ and POG treatment vs air, but maximal OCR was significantly lower in the air vs APJ only. The results suggest that the high toxicity APJ E-liquid caused a greater decrease in OCR than the POG E-liquid, further supporting the results of Figures 1 and 2 that E-liquids with higher toxicity lead to more severe dysfunction.

Figure 5.

Oxygen consumption rate in HL-1 cells cultured with APJ and POG E-vapor extracts. A: Normalized OCR vs time in cells cultured for 24 hours with control media, or 0.375 puffs/mL APJ, or POG E-vapor extracts. The lines denote injections of Oligo (oligomycin), FCCP (phenylhydrazone), and A/R (antimycin and rotenone). B: Quantification of basal, ATP-linked, and maximal OCR (*P < 0.05, **P < 0.01, one-way ANOVA with Fisher’s LSD test). APJ = Apple Jax; ATP = adenosine triphosphate; OCR = oxygen consumption rate.

To study the impact of VC E-vapor extract on primary ventricular myocyte viability in a dose-dependent manner, isolated ventricular myocytes were treated with increasing concentrations (0.015–0.75 puffs/mL) for 24 hours and stained with calcein-AM, a fluorescent marker of viable cells. Fluorescence imaging revealed a progressive reduction in calcein signal with increasing E-vapor concentration (Figure 6 A). Figure 6B shows a dose-dependent decrease in green fluorescence intensity, indicating reduced cell viability with increasing concentration of the E-vapor extract.

Figure 6.

Dose-dependent effects of Vanilla Custard E-vapor extract on mouse ventricular myocytes viability. A: Representative brightfield and green fluorescence images of ventricular myocytes treated with control or increasing concentrations of Vanilla Custard E-vapor extract (0.015, 0.075, 0.15 puffs/mL) for 24 hours. Cells were stained with 5 μM calcein-AM. Green fluorescence intensity, indicating viable cells, decreased with higher E-vapor concentrations. B: Quantification of fluorescence intensity at the different doses. N = 2 animals, and n = 32 to 60 cells per concentration (*P < 0.05, Ctr vs all other groups, one way ANOVA, with Fisher’s LSD test).

To investigate the role of inhalation exposure to flavored E-vapor on cardiac electrophysiology and examine the possible involvement of ROS, we used mCAT overexpressing mice and their WT littermates. mCAT overexpressing and WT littermate mice were vaped with VC E-liquid or with room air for 5 weeks. In vivo PES was performed. Figure 7A shows example ECG traces of inducible VT in mice from the different groups. In panel B, we quantify the duration of all inducible VT episodes having more that 3 extra beats. Although most animals were inducible, the VT duration was significantly longer in the vaped WT mice compared with all other groups, where 4 out of 6 WT air, 5 out of 6 WT vaped, 4 out of 5 mCAT air, and 4 out of 5 mCAT vaped animals were inducible. In Figure 7C, we also quantify sustained VT (episodes lasting more than 250 ms), where the duration of inducible sustained VT was significantly longer in WT vaped mice, and where 2 out of 6 WT air, 5 out of 6 WT vaped, 2 out of 5 mCAT air, and 1 out of 5 mCAT vaped animals were inducible for sustained VT. Importantly, overexpression of mCAT abrogated the effects of inhalation exposure to VC E-vapor on VT duration. We also performed analysis of ECG intervals recorded during PES in the anesthetized mice (Figure 7C). Mice in the mCAT air group displayed reduction of RR and PR intervals. In contrast, the QRS interval was reduced in the WT vaped compared with WT air group, and the QTc interval was prolonged in the WT vaped group compared with all other groups.

Figure 7.

In vivo ventricular tachycardia (VT) inducibility in mCAT overexpressing and WT littermates exposed to room air or Vanilla Custard E-vapor. A: ECG traces of inducible VT in WT air, WT vape, mCAT air, and mCAT vape mice. B: Quantification of VT duration (more than 3 extra beats) in the different groups. C Quantification of sustained VT duration in the different groups. N = 6 mice for WT air, 6 for WT vape, 5 for mCAT air, and 5 for mCAT vape. (* P < 0.05. **P < 0.01, WT vape vs all other groups one-way ANOVA with Fisher’s LSD test). Stim: burst pacing stimulation. D: quantification of ECG intervals in anesthetized animals. N = 6 mice for WT air, 6 for WT vape, 5 for mCAT air, and 5 for mCAT vape. (* P < 0.05. **P < 0.01, one-way ANOVA with Fisher’s LSD test). ECG = electrocardiogram; mCAT = mitochondrial catalase; WT = wild type.

We assessed ROS in mouse ventricular myocytes following E-vapor exposure, where we performed fluorescence imaging using the mitochondrial specific ROS probe MitoSOX Red. WT and mCAT ventricular myocytes were stained with 5 μM MitoSOX Red and treated with 0.375 puffs/mL VC E-vapor extract for 1 hour. Confocal imaging of MitoSOX Red fluorescence was then done pre- and post-incubation with the E-vapor extract in stained cells. In WT myocytes, E-vapor exposure led to a marked increase in mitochondrial ROS, as indicated by significantly elevated MitoSOX fluorescence intensity compared with pretreatment controls (Figure 8). In contrast, mCAT myocytes, which overexpress mitochondrial-targeted catalase, showed diminished changes in ROS levels following exposure.

Figure 8.

Mitochondrial ROS accumulation in ventricular myocytes following exposure to Vanilla Custard E-vapor extract. A: Representative brightfield and fluorescence images of ventricular myocytes isolated from wild-type (WT) and mCAT transgenic mice before (Pre) and after (Post) exposure to Vanilla Custard E-vapor extract (0.375 puffs/mL for 1 hour). Cells were loaded with 5 μM of the mitochondrial reactive oxygen species (ROS) indicator MitoSOX Red. B: Quantification of MitoSOX Red fluorescence intensity in WT and mCAT ventricular myocytes before (pre), and after (post) incubation Vanilla Custard E-vapor extract. N = 2 animals, and n = 42 to 71 cells per group. (*P < 0.05, one way ANOVA, with Fisher’s LSD test).

We then investigated whether exposure to flavorings alone, without nicotine, has an impact on cardiomyocytes. We tested in spontaneously beating hiPSC-derived cardiomyocytes, the effects on beating rate of: 1- control media, 2- E-vapor extract of PG/VG base without nicotine, 3- E-vapor extract of RVC 4-E-vapor extract of USA Vape Lab’s VC with 6mg/mL nicotine. We reconstituted VC E-liquid based on the concentrations of flavoring carbonyls in the VC E-liquid from USA Vape Labs, as determined by GC/MS and reported in Table 2. To do so, we used 70VG/30PG base without nicotine, supplemented with 79.2 mM vanillin, 48.8 mM ethyl vanillin, and 79 mM ethyl maltol to make the RVC E-liquid with a total carbonyl concentration of 207 mM. Figure 9A shows extracellular electrograms after 24 hours treatment for each group. RVC accelerated the beating rate, whereas VC caused a deceleration. Panel B shows activation maps from the different conditions where RVC and VC treatments increased the activation time of the preparation. In Panel C, the average spontaneous beating rate (beats per minute [BPM]) significantly increased with RVC, and decreased with VC treatments compared with controls. The average activation time also significantly decreased in RVC and VC groups, and cFPD significantly increased in VC treatment. This is consistent with the results of Figure 7C where exposure to vaping decreased the QRS and increased the cQT intervals in mice.

Figure 9.

Electrophysiological analysis of hiPSC-derived cardiomyocytes monolayers treated with either control media, or with 0.15 puffs/mL unflavored 70VG/30PG base without nicotine, or Reconstituted Vanilla Custard E-vapor extract (RVC), or Vanilla Custard with nicotine E-vapor extract (VC) for 24 hours. A: Extracellular electrogram recordings after the different treatments. B: Activation maps of the spontaneously beating monolayers. C: Quantification of beating rate (BPM), activation time, and cFPD after 24 hours treatment with control (N = 6), base (N = 3), RVC (N = 8), and VC (N = 6). (**P < 0.01, One-way ANOVA with Fisher’s LSD test, and *P < 0.05, VC vs all other conditions, One-way ANOVA with Fisher’s LSD test).

Discussion

Our study suggests that inhalation exposure to flavored ENDS negatively affects ventricular electrophysiology, in part via mitochondrial dysfunction and increased oxidative stress. Additionally, our results indicate that exposure to flavoring carbonyls may exert toxicity on cardiac myocytes. Furthermore, oxidative stress and mitochondrial dysfunction may mediate increased ventricular arrhythmogenicity in inhalation exposure to flavored ENDS.

In this paper, we investigated the toxicity of 30 differently flavored E-liquids on HL-1 cardiomyocytes. Most of the E-liquids tested (28 out of 30) were significantly toxic with some E-liquids being more toxic than others (Figure 1). We surmised that this was independent of nicotine content since all tested E-liquids contained 6 mg/mL nicotine as stated by the manufacturer. We used HL-1 like myocytes in the current study as in in vitro model to gain general insights into the stress response of myocytes to exposure. In line with our study, reduced cellular viability as a result of exposure to flavored ENDS has been reported by others, however the tests were conducted in non-cardiac cells. ^41,43–45 The exposure related increased apoptosis in HL-1 cells were also confirmed to be present in exposed primary ventricular myocytes (Figure 6). Additionally, the higher ROS we observed in exposed WT but not in mCAT overexpressing mouse ventricular myocytes (Figure 8) are in line with our results in HL-1 myocytes where E-vapor exposure also induced an increase in ROS and depolarization of the ΔΨ (Figures 3 and 4), further supporting the role of oxidative stress as a mediator of E-vapor induced cardiotoxicity.

Our results also suggest that the flavoring constituents in the 30 different E-liquids drive the variation in toxicity. GC/MS quantification of carbonyls in the flavored E-liquids identified 5 major flavoring carbonyls (vanillin, ethyl vanillin, cinnamaldehyde, maltol, and ethyl maltol), and that toxicity correlated with the concentration of the total flavoring carbonyls (Figure 2). Sassano et al⁴¹ performed a high throughput assay of 148 E-liquids and found that vanillin and cinnamaldehyde correlated with toxicity in non-cardiac cells. Interestingly, E-liquids in the vanilla group were found to be more toxic than any other group and were found to have the highest concentrations of vanillin and cinnamaldehyde.

Since the accumulation of toxic carbonyls has been shown to cause an increase in oxidative stress,^46–48 we investigated whether exposure to E-liquids increases ROS in cardiac myocytes. In HL-1 cardiomyocytes exposed to flavored E-vapor extracts, ROS significantly increased, but was attenuated by pre-treatment with antioxidant NAC^49,50 (Figure 3). We also assessed the effects of exposure on mitochondrial function by measuring ΔΨ (Figure 4) and OCR (Figure 5). VC E-vapor extract depolarized the ΔΨ, and APJ (a high toxicity E-liquid, Figure 1) impaired the cellular respiratory process more than POG (a low toxicity E-liquid, Figure 1) or air control. ROS is regularly produced as a consequence of the mitochondria’s normal cellular respiratory processes, and the high ROS accumulation that we observed may be in part due to flavored ENDS exposure mediated mitochondrial dysfunction. To further corroborate this contention, we tested in vivo the potential arrhythmogenicity of inhalation exposure to a flavored E-liquid and the possible role of mitochondria therein. mCAT overexpressing mice and their WT littermates were exposed to VC E-vapor or room air. In PES studies, WT mice exposed to VC E-vapor had significantly more sustained VT compared with the mCAT overexpressing animals (Figure 6). This suggests that the overexpression of mCAT attenuated the effect of inhalation exposure on ventricular arrhythmogenesis and further points to the involvement of mitochondrial pathways in cardiac electrophysiological injury due to flavored ENDS exposure. Furthermore, it is possible that impaired energy metabolism due to exposure leads to the depolarization of the mitochondrial membrane potential and to the generation of ROS. ⁵¹ ROS can affect the depolarizing sodium and calcium currents, as well as the intracellular calcium cycling process and contractility. It may also result in alterations in calcium handling which has been implicated in the genesis of action potential or T-wave alternans which precede ventricular tachyarrhythmias, or triggered action potentials due to delayed or early afterdepolarizations which can initiate tachyarrhythmias.^51–53

Although nicotine’s harmful effects on the human heart have been known and well-documented^54–57 it remains unclear whether the other components of flavored ENDS can also harm the heart. In our hiPSC-derived cardiomyocytes experiments, the presence of nicotine decelerated the spontaneous beating rate. It is possible that the prolongation of the cFPD contributed to this effect possibly through cholinergic nicotinic receptors that have been recently described in ventricular myocytes.⁵⁸ The increased activation time that we observed in the hiPSC-derived cardiomyocytes RVC and VC groups, and the increased cFPD are consistent with ECG changes observed in human subjects who vape or smoke tobacco cigarettes and with the mouse ECG data in Figure 7D, which showed decreased QRS and increased cQT interval durations in vaped WT mice. For instance, it has been reported that QRS duration is decreased, and the QT interval is prolonged in individuals who vape.⁵⁹ Similarly, shortened QRS duration and prolonged QTc intervals have been observed in individuals who smoke combustible cigarettes, as shown in the Third National Health and Nutrition Examination Survey.⁶⁰ This study found that ECG abnormalities, including prolonged QTc and shortened QRS durations, were predictive of cardiovascular mortality in smokers.⁶⁰

Limitations

Our in vitro exposure studies do not accurately represent the actual plasma concentrations of the flavoring constituents, base humectants, nicotine, their thermal degradation byproducts, or blood metabolites. This is an important limitation that warrants improvements driven by a better understanding of these byproducts and metabolites concentration in the blood of ENDS users. It remains unclear if the ROS byproducts that have been identified in E-vapor initiate cellular oxidative stress or whether the components and thermal degradation byproducts in E-vapor directly cause oxidative stress. Additionally, although our results point out an important contribution of mitochondrial ROS to arrhythmogenesis in vaping, our study does not address the possible role of non-mitochondrial ROS sources such as peroxisomes, endoplasmic reticulum, and nicotinamide adenine dinucleotide phosphate oxidases. It is also possible that a combination of these scenarios results in ROS begetting ROS, leading to a cascade of amplification. In our prior work using a similar flavored E-vapor inhalation model, we reported significant alterations in heart rate variability (HRV), indicative of autonomic imbalance and consistent with possible atrial electrophysiological effects. Although in vivo atrial PES was not performed here, the combination of our prior HRV data and current HL-1 cell findings may point out that vaping exposure can influence atrial electrophysiology in part, through oxidative stress mediated harm. Future studies focused on the atria are needed to gain better insights into the effects of vaping exposure on atrial electrophysiology. We also acknowledge that HL-1 cells, being atrial-derived, do not fully replicate ventricular myocyte electrophysiology. Although HL-1 cells have been used to gain insights into ventricular electrophysiology and function, ^61–65 our results should not be overinterpreted in the context of ventricular arrhythmogenesis. To mitigate this limitation, we confirmed key findings in primary ventricular myocytes (Figures 6 and 8), in vivo mouse inhalation models (Figure 7), and iPSC-derived cardiac myocytes (Figure 9). Pre-clinical and clinical studies are pointing to harmful consequences of ENDS exposure on the heart.^16,23–25 However, our results remain limited by the translatability of findings from mouse models to humans. Although the findings in iPSC-derived cardiomyocytes may raise additional mechanistic questions that warrant future investigation, they provide a translational value to this work. Nevertheless, direct extrapolation of our findings to human physiology should be done with caution.

Abbreviations

APJ

Apple Jax E-liquid

ENDS

electronic nicotine delivery systems

GC/MS

gas chromatography/mass spectroscopy

mCAT

mitochondrial catalase overexpressing mice

NAC

N-acetylcysteine

OCR

oxygen consumption rate

POG

Hawaiian POG E-liquid

RVC

reconstituted Vanilla Custard E-liquid

VC

Vanilla Custard E-liquid