Tobacco and Marijuana Induce Adverse Effects on Cardiac Function and Platelet Activity and Reduce Tissue Preservation Following Myocardial Infarction

Abstract

Smoking disrupts cardiac autonomic regulation, vascular biology, and platelet function; however, the acute and longer-term cardiovascular effects of resurging and emerging tobacco and marijuana products remain incompletely characterized. We investigated the impact of exposure to smoke and aerosol from several tobacco and marijuana products on cardiac function, platelet activity, and myocardial susceptibility to ischemia/reperfusion myocardial infarction (MI). Male/female Sprague-Dawley rats underwent single or daily 5-min pulsatile exposure sessions to emissions from tobacco cigarettes, e-cigarettes (JUUL), heated tobacco products (IQOS), marijuana cigarettes, or cannabinoid-depleted (“placebo”) marijuana cigarettes; with an air negative control. Cardiac function was assessed immediately after single exposure using echocardiography and intraventricular hemodynamics. Platelet aggregation was measured immediately after single exposure and one day after repeated (2-week) exposure. Myocardial infarct size, normalized to area-at-risk, was assessed following MI after repeated (4-week) exposure. Single exposure to tobacco smoke or JUUL aerosol reduced left ventricular ejection fraction relative to pre-exposure. Acute marijuana smoke exposure similarly impaired left ventricular function assessed by hemodynamics. Single exposure to tobacco, marijuana, and placebo marijuana smoke increased collagen-induced platelet aggregation, with more pronounced responses in females, whereas repeated (2-week) exposure to smoke/aerosol from tobacco cigarettes, JUUL, IQOS, and placebo marijuana increased platelet aggregation across groups. Notably, repeated (4-week) exposure to tobacco smoke/aerosol or marijuana smoke prior to MI reduced myocardial tissue preservation, resulting in greater infarct size post-MI. These findings indicate that individual tobacco and marijuana smoking and vaping similarly impair cardiac function, enhance platelet reactivity, and reduce myocardial tolerance to ischemia/reperfusion injury.

NEW & NOTEWORTHY

Inhalable products marketed as reduced-harm alternatives to combustible cigarettes, including e-cigarettes, heated tobacco products, and marijuana, produce rapid and sustained adverse cardiovascular effects. Even brief exposure impairs cardiac function and enhances platelet aggregation, while prior exposure increases myocardial susceptibility to ischemia/reperfusion injury, resulting in larger infarcts following myocardial infarction. These findings challenge the perception that noncombustible or “reduced-harm” products are cardiovascularly benign.

INTRODUCTION

Smoking-induced cardiovascular disease (CVD) remains a major global health burden and is the leading cause of death worldwide (1, 2). CVD accounts for approximately 800,000 deaths annually in the United States (3), of which nearly 20% are directly attributable to cigarette smoking (1). Although cigarette smoking rates have declined over recent years (1), this reduction has been accompanied by a concomitant increase in the use of alternative inhalational tobacco products, including electronic cigarettes (e-cigs; e.g., JUUL) and “heated tobacco products” (HTPs; e.g., IQOS). Concurrently, the rapid expansion of marijuana legalization in the United States and internationally has further broadened the landscape of inhalational exposures, extending beyond traditional combustible cigarettes to include marijuana and a range of tobacco products that all may increase the risk of CVD. E-cigs, IQOS, and marijuana are often viewed as less harmful than conventional tobacco cigarettes. However, accumulating evidence from our group and others demonstrates that aerosols from IQOS and several types of e-cigs including ultrasonic models lacking heating coils, as well as secondhand and mainstream marijuana smoke and marijuana dry-leaf vaporizers, adversely affect cardiovascular function and potentiate blood platelet activation (4–23). Consistent with this, we have recently shown that long-term daily smoking or vaping of tobacco, e-cigs, and marijuana progressively increases systolic blood pressure, impairs cardiac function, promotes cardiac fibrosis, and enhances susceptibility to atrial and ventricular arrhythmias in rats (21). Collectively, these findings suggest that smoking or vaping, regardless of product type, exerts deleterious effects on the cardiovascular system and circulating blood elements to varying degrees.

Despite the growing popularity of these products and their potential public health implications, important knowledge gaps remain regarding their acute and longer-term effects on cardiac performance, platelet reactivity, and smoking-induced myocardial injury. Epidemiologic studies indicate that cigarette smoking increases the incidence of myocardial infarction (MI) in both men and women (24) and that MI rates, including first MI, are rising among younger populations of tobacco smokers (25, 26), whereas emerging population studies report that both e-cig and marijuana use are associated with increased odds of MI (27–31). Cigarette smoking produces myocardial injury through both direct toxic effects on cardiac myocytes and indirect cardiovascular alterations, demonstrated clinically and experimentally (32). Although nicotine administration has been reported to increase infarct size in animal models (33), it remains unclear whether prior exposure to tobacco smoke or aerosol and marijuana smoke exacerbates myocardial injury during a subsequent ischemia/reperfusion (I/R) MI. Given the increasing prevalence of smoking or vaping products other than tobacco cigarettes, and the rising incidence of MI among exposed populations, further investigation is warranted to define the cardiovascular consequences of these inhalational products.

In the present study, we exposed rats to a single session or repeated smoking/vaping sessions of combustible tobacco cigarettes, JUUL, IQOS, marijuana cigarettes, or placebo marijuana cigarettes. Cardiac function was evaluated following a single exposure, platelet aggregation was assessed following both single and repeated (2-week) exposures, and the impact of prior repeated (4-week) exposure on susceptibility to I/R-induced myocardial injury was also determined. Our findings indicate that use of these inhalational products, despite their difference in physical state (smoke vs. multiple types of aerosol) and main bioactive component (nicotine or cannabinoids), is associated with acute impairments in cardiac function and enhanced platelet aggregation. Moreover, repeated inhalational exposures to tobacco smoke/aerosol or marijuana smoke before an MI reduced myocardial tissue survival in the ischemic region, resulting in greater myocardial injury following I/R MI.

METHODS

Rats

Male and female Sprague-Dawley rats (10-11 weeks of age; average body weight ~250 g) were randomized to experimental groups, with equal numbers of each sex (n = 6-16 per group, depending on the experiment). Rats were obtained from Charles River Laboratories (Wilmington, MA) and housed in a temperature-controlled facility under a 12:12-h light-dark cycle with ad libitum access to standard chow (LabDiet 5053; LabDiet). For euthanasia, rats were anaesthetized with 5% isoflurane for 5 min, and hearts were excised after confirmation of the absence of a paw withdrawal reflex. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (UCSF) and were conducted in accordance with UCSF guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Tobacco Products

Regular tobacco cigarettes (Marlboro Red; batch no. V284 Y81B5) were used as combustible tobacco products. Before each experiment, cigarettes were humidified overnight at room temperature by placement in an airtight container over 16% glycerol in water, as in our previous tobacco studies (34, 35). Cigarettes were used within 5 min after humidification. The e-cig product used was JUUL (Virginia Tobacco flavor) containing a 30:70 propylene glycol/vegetable glycerin (PG/VG) e-liquid with 5% nicotine (batch no. G0811CG-1). Two commercially available heated tobacco products were used: Marlboro-branded American HeatSticks (IQOS-USA; batch no. GA0021) and Parliament-branded Russian HeatSticks (IQOS-Rus; batch no. zz0362/718). HeatSticks were used with an IQOS 3.0 Multi device obtained commercially in Germany.

Marijuana and Placebo Marijuana Cigarettes

Marijuana (~10% Δ-9-tetrahydrocannabinol (THC)) was obtained in bulk from the University of Mississippi via RTI International under contract with the National Drug Supply Program of the National Institute on Drug Abuse and was rolled into cigarettes in our laboratory. “Placebo” marijuana (<0.01% THC), depleted of all cannabinoids, was supplied as pre-rolled cigarettes from the University of Mississippi via RTI. All required federal, state, and institutional approvals for the acquisition and possession of marijuana, as well as for rodent exposure, were obtained (13). Before each experiment, marijuana and placebo-marijuana cigarettes were humidified overnight at room temperature by placing them in an airtight container over 50 mL of saturated sodium chloride solution in a locked desk drawer. Cigarettes were used within 5 min after humidification.

Exposure Conditions

To imitate active smoking/vaping, conscious rats were individually placed in restrainers (DecapiCones, DC 200, Braintree Scientific, Inc., Braintree, MA) and exposed to pulsatile smoke or aerosol generated from a Universal Vaping Machine (version 5.0; Gram Research, Oakland, CA), via a nosecone after three daily acclimation sessions to the restrainers (21). The simple modular design of the Gram system, which enables swapping of all parts of the pathway (i.e., separate syringe pump components, valves, tubing, and nosecones), ensures that one type of smoke/aerosol does not contaminate other exposure groups. Nosecone delivery of undiluted smoke, rather than chamber exposure, better mimics human smoking, prevents continued post-exposure absorption from material adhered to fur, and allows immediate switching between smoke/aerosol and room air.

Exposure conditions for each product were based on standard conditions (e.g., ISO Standard 3308:2012 for tobacco smoke) but were modified to maximize consistency across products. The tobacco products were exposed using a common puffing protocol to facilitate comparisons among nicotine-containing products. A modified puff regimen was used for marijuana cigarettes because of differences in combustion characteristics and smoke generation, allowing consistent smoke production and reproducible animal exposure. Accordingly, the exposure protocols were designed to achieve reliable product-specific inhalation rather than equivalent delivered doses across all products. For tobacco smoking/vaping, a single session consisted of 10 cycles of consecutive 30 sec over 5 min. Each cycle included a 5-second exposure generated from a 55-ml draw of smoke or aerosol, followed by 25 sec of room air, as described previously (4). For marijuana and placebo marijuana smoking, a single session consisted of 5 cycles of consecutive 60 sec over 5 min. Each cycle included a 5-second exposure from a 55-ml draw of smoke, followed by 55 sec of room air, as described previously (21). The clean air control group underwent the identical pulsatile exposure protocol consisting of a single session of 10 cycles of consecutive 30 sec over 5 min, except that no tobacco or marijuana product was connected to the exposure apparatus, and only room air was delivered.

For repeated (2-week) exposure (platelet aggregation study), four conscious rats were simultaneously exposed to smoke or aerosol via a four-branch adapter connected to four nosecones, as previously described (21). Animals received one pulsatile session per day (as described above for tobacco and marijuana exposures), 5 days per week for 2 weeks. As with the single session exposures, each exposure group used a separate syringe set, valve, tubing, and nosecones to prevent cross-contamination of different aerosols and smoke. A clean air group underwent the same 10-cycle pulsatile protocol for 2 weeks.

For repeated (4-week) exposure followed by surgical I/R MI, two conscious rats were simultaneously exposed to smoke or aerosol via a two-branch adapter connected to two nosecones. Animals received one pulsatile session daily, 7 days per week for 4 weeks. A clean air group underwent the same daily pulsatile session for 4 weeks.

Echocardiography

Echocardiography was performed in rats anesthetized with 1.5% isoflurane at baseline (pre-exposure) and immediately after a single exposure, using a Vevo 3100LT micro-ultrasound system (VisualSonics Inc., Toronto). Two-dimensional (B-mode) images were acquired in parasternal long-axis and short-axis views. Left ventricular (LV) end-systolic volume (ESV) and end-diastolic volume (EDV) were measured using Simpson’s method with VevoLab software (VisualSonics Inc.), and ejection fraction (EF) was calculated accordingly (36). The investigator performing echocardiographic measurements was blinded to group allocation.

Intraventricular Hemodynamic Data Acquisition and Analysis

In the terminal procedure following single exposure, rats underwent cardiac catheterization after echocardiography. Animals were anesthetized with isoflurane (1.5~2%) and received preoperative analgesia (buprenorphine, 0.1 mg/kg). Hemodynamic variables were continuously recorded using a PowerLab data acquisition system (ADInstruments) with LabChart Pro7 software and a catheter transducer (ADV500 PV System, Transonic Systems Inc.). During catheterization, arterial pressure and LV pressure-volume (PV) signals were acquired and recorded in real time. Briefly (36), for intraventricular PV loop recording, the ADV500 PV System was configured to the normal heart model prior to zero calibration of the pressure channel and data acquisition. A 1.6F rat PV catheter (Transonic Systems Inc.) was inserted into the right carotid artery, advanced through the aorta into the LV chamber, and positioned centrally within the chamber by monitoring pressure-conductance (magnitude) loops and phase signals. Once optimal signal quality was achieved and conductance magnitude and pressure signals were stable and maximal, pressure and volume data were recorded for analysis using the LabChart PV Loop Module. The LabChart Blood Pressure Module was used for offline analysis of arterial pressure signals.

Platelet Aggregation Rate Measurement in Vitro

Rats were exposed to smoke or aerosol from tobacco products and both kinds of marijuana by single and repeated (2-week) exposure protocols. During the terminal procedure, animals were anesthetized with 2% isoflurane, and whole blood was collected from the abdominal aorta using a 21G Safety-Lok^™ Butterfly Blood Collection Needle (BD, Franklin Lakes, NJ) into sodium citrate Vacutainer tubes (BD). Blood was obtained immediately after single exposure or one day after the final session of the 2-week exposure. Collected blood was incubated at room temperature for 30 min prior to analysis. Platelet aggregation was assessed using a Whole Blood Impedance Aggregometer (Model 810; Chrono-Log Corporation, Havertown, PA) according to manufacturer’s instructions. Briefly, 500 μl of whole blood was diluted with 500 μl of prewarmed (37°C) 0.9% physiological saline in a test cuvette. Prewarmed electrodes were immersed in the diluted sample, and electrical calibration was performed. Platelet aggregation was induced by adding 2 μl of collagen (2 μg/ml (37); Chrono-Log Corporation, P/N385) to the cuvette with diluted blood. Changes in electrical impedance between the two electrodes were recorded for 6 min. The area under the curve (AUC) of the aggregation tracing was calculated by AGGRO/LINK^® for Windows software (version 5.1; Chrono-Log Corporation). Each sample was analyzed in duplicate, and mean values were used for statistical analysis.

Surgical Procedure for I/R MI and Evans Blue Injection

As described previously (36), an I/R MI model was used. Rats were anesthetized with 2% isoflurane and subjected to temporary occlusion of the left anterior descending (LAD) coronary artery for 25 min to induce regional myocardial ischemia, followed by 40 min of reperfusion. Briefly, animals were intubated and mechanically ventilated using a small-animal volume-controlled ventilator (Harvard Rodent Ventilator, Model 683, South Natick, MA). Following left thoracotomy, the heart was exteriorized. A 6-0 nonabsorbable surgical suture was passed beneath the LAD approximately 2 mm from the tip of the normally positioned left auricle and tied to achieve temporary occlusion. After 25 min of ischemia, the ligature was released to allow reperfusion of the formerly ischemic myocardium. After 40 min of reperfusion, the LAD was reoccluded, and 1 ml of 1.5% Evans blue solution (Sigma-Aldrich, E2129) prepared in phosphate-buffered saline (PBS) was injected via the right jugular vein, or via the LV apex if venous injection was unsuccessful, to delineate the nonischemic myocardium. The heart was excised immediately following Evans blue perfusion. Mortality during the ischemic period was 8%.

Assessment of Ischemic Risk Area and Infarct Size

Hearts were excised, rinsed with PBS to remove excess Evans blue dye, and weighed. The right ventricle and atrial tissues were trimmed away. The LV tissue was weighed and stored at −20°C for 30 min to facilitate sectioning. Frozen LV tissue was sliced manually with a razor blade transversely from apex to base into 2-mm-thick slices and immediately imaged using a digital microscope (246S, 249S; ANDONSTAR, Shenzhen, China). Following imaging, slices were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC; Ward’s Science, NY, 470300-012) prepared in PBS for 30 min at room temperature to stain viable myocardium (red). Slices were then fixed overnight at 4°C in 10% PBS-buffered formalin and reimaged. The ischemic area at risk (AAR) was defined as myocardium not stained by Evans blue. The infarct area was defined as TTC-negative (pale) regions within AAR. AAR and infarct size were quantified using ImageJ.

Statistical Analysis

Based on standard deviations of EF measurements from our previous experiments, power calculations using a power of 0.8 and significance level of 0.05 determined that sample sizes of n = 12/group (for the single-exposure experiment) and n = 6/group (for the repeated-exposure experiment) were sufficient to detect within-group changes in EF from pre- to post-exposure of 2.54 and 3.78 percentage units, respectively. Within-group comparisons of pre- and post-exposure measurements were performed using two-tailed paired t tests (Prism 10). For multiple groups at a single terminal time point, comparisons of each smoking or vaping group with the air control group were analyzed by nonparametric one-way ANOVA followed by Dunnett’s multiple comparisons test or Tukey’s post-hoc test (Prism 10). Data are presented as mean ± standard deviation (SD).

RESULTS

Single Exposure to Smoke/Aerosol from Tobacco Products or Marijuana Induced Acute Cardiac Functional Changes

To determine the acute effects of smoke or aerosol exposure on cardiac function, conscious rats underwent a single smoking/vaping session with conventional tobacco cigarettes (Marlboro Red), JUUL, IQOS, marijuana, or clean air (Fig. 1). Cardiac function was evaluated by echocardiography before and immediately after single exposure.

Figure 1. Experimental timeline for single and repeated exposures to smoke or aerosol from tobacco and marijuana products.

Cardiac function was assessed by echocardiography immediately after a single exposure, followed by measurement of intraventricular hemodynamics. Platelet aggregation was measured either immediately after a single exposure or 1 day after the final session of a 2-wk repeated exposure. Acute myocardial infarction (MI) was induced by surgical ischemia-reperfusion 1 day after the final session of a 4-wk repeated exposure.

Single exposure to tobacco cigarette smoke or JUUL aerosol significantly reduced EF in the overall cohort relative to pre-exposure (P = 0.006 and P = 0.016, respectively; Fig. 2A; Table S1), although all groups except for air showed a downward trend. In sex-stratified analyses, reductions narrowly missed significance in males for both exposures (P = 0.092 and P = 0.066; Fig. 2B) and in females for tobacco cigarette smoke (P = 0.052; Fig. 2C). However, the group sizes for sex-stratified analysis were smaller, and thus under-powered. Tobacco cigarette smoke significantly decreased heart rate (HR; P = 0.02), whereas JUUL aerosol modestly increased HR (P = 0.053; Fig. S1). Stroke volume (SV) and cardiac output (CO) were not significantly altered in these groups (Table S1). In both IQOS groups, EF showed a modest but non-significant reduction relative to pre-exposure (P = 0.083 and P = 0.073; Fig. 2A), with a similar trend in males exposed to IQOS-Rus (P = 0.074; Fig. 2B). However, SV and CO were significantly decreased relative to pre-exposure without changes in HR (Table S1; Fig. S1), suggesting impaired LV function independent of HR effects. Interestingly, single marijuana exposure resulted in a small, non-significant reduction in EF relative to pre-exposure (P = 0.565), but was associated with apparent decreases in SV, CO, and HR (Table S1; Fig. S1). EF remained unchanged in the air control group (P = 0.898). ESV was largely unchanged across tobacco product groups, except for a modest increase relative to pre-exposure (considered an adverse change) in the tobacco cigarette group (P = 0.02; Fig. 2A). In contrast, ESV was decreased relative to pre-exposure in the marijuana group, barely missing significance in the overall cohort (P = 0.065; Fig. 2A) but reaching significance in females (P = 0.016; Fig. 2C). For different changes in ESV, see Discussion for product-specific differences in LV geometry. EDV was significantly decreased relative to pre-exposure in both IQOS groups and in the marijuana group (P = 0.003 for both IQOS groups; P = 0.0004 for marijuana group; Fig. 2A), with consistent effects in both sexes (Fig. 2, B and C). These reductions in EDV potentially indicate impaired ventricular relaxation or reduced preload following single exposure.

Figure 2. Changes in cardiac function after a single exposure to smoke or aerosol from tobacco products or marijuana.

Cardiac function was assessed by echocardiography after smoking or vaping. A: changes in ejection fraction (EF), end-systolic volume (ESV), and end-diastolic volume (EDV) in the overall cohort. B: changes in EF, ESV, and EDV in males. C: changes in EF, ESV, and EDV in females. Numerical data are summarized in Supplemental Table S1. Bars represent SD. Statistical analyses were performed with 2-tailed paired t tests comparing preexposure and postexposure values within each group. Significance is set at P ≤ 0.05, but all P values < 0.1 are shown.

To minimize the potential influence of restraint-related effects, inter-animal variability, and differences in body and heart size, echocardiographic parameters (EF, ESV, and EDV) were analyzed by normalizing each animal’s post-exposure values to its own pre-exposure (baseline) measurements. The percentage changes among exposure groups were then compared with the air control group. Analysis of the overall cohort showed significant reductions in EDV relative to the air control group following single exposure to IQOS-Rus and marijuana (P = 0.016 and P = 0.019, respectively; Fig. S2). No significant differences in baseline echocardiographic parameters were observed among groups before single exposure (Table S1).

Overall, a single smoking or vaping session acutely impaired cardiac function, with tobacco cigarette smoke reducing EF and increasing ESV, JUUL aerosol reducing EF, and IQOS or marijuana mainly lowering SV, CO, and EDV, indicating impaired ventricular performance and filling.

Single Exposure to Smoke/Aerosol from Tobacco Products or Marijuana Altered Intraventricular Hemodynamics

Approximately 30 min following the non-invasive echocardiographic assessment, rats underwent terminal cardiac catheterization for invasive hemodynamic measurements. Compared with the air control group, EF measured by PV analysis was significantly reduced (an adverse change) in the tobacco cigarette, JUUL, and marijuana groups in the overall cohort (P = 0.042, P = 0.025, and P = 0.031, respectively, vs. air; Fig. 3; Table S2). A similar pattern was observed in males (P = 0.02, P = 0.022, and P = 0.059, respectively). The EF reductions induced by tobacco smoke and JUUL aerosol in males was consistent with the echocardiographic findings (Fig. 2B). Marijuana smoke exposure was additionally associated with significantly lower mean arterial pressure (MAP; P = 0.0008) and LV end-systolic pressure (ESP; P = 0.006), as well as sustainably reduced HR (P = 0.019) at 30 min after exposure, compared with clean air (Fig. 3; Fig. S1). Hemodynamic parameters, including SV, CO, cardiac index (CI; CO normalized to body weight), peak rates of pressure rise and decline (dP/dt_max and dP/dt_min), the contractility index P@dP/dt_max (dP/dt_max normalized to instantaneous pressure), and the time constant of isovolumic relaxation (Tau), were not significantly altered across groups (Table S2). Given the complexity of relationships among these parameters, see Discussion for a broader assessment of adverse cardiac effects.

Figure 3. Intraventricular hemodynamic changes after a single exposure to smoke or aerosol from tobacco products or marijuana.

Intraventricular hemodynamics were measured after echocardiographic assessment after smoking or vaping. Numerical data are summarized in Supplemental Table S2. Bars represent SD. Statistical analyses were performed with nonparametric 1-way ANOVA followed by Dunnett’s multiple-comparisons test to compare each smoking or vaping group with the air control group. Removal of the potential outlier in the Marlboro Red end-diastolic pressure (EDP) group did not change the overall result. CI, cardiac index; dP/dt_max, peak rates of pressure rise; dP/dt_min, peak rates of pressure decline; EDV, end- diastolic volume; EF, ejection fraction; ESP, end-systolic pressure; ESV, end-systolic volume; MAP, mean arterial pressure; Tau, the time constant of isovolumic relaxation.

Invasive PV analysis 30 min after a single exposure showed that EF remained reduced in the tobacco cigarette, JUUL, and marijuana groups, supporting the echocardiographic findings. Marijuana smoke also caused decreases in MAP, ESP, and HR, suggesting sustained acute hemodynamic alterations.

Single and Repeated Exposures Enhanced Platelet Aggregation with Exploratory Sex-Stratified Differences

Given that tobacco smoking and e-cigarette vaping exert both acute and long-term pro-thrombotic effects on platelet reactivity (38, 39), we exposed conscious rats to smoke or aerosol from tobacco cigarettes, JUUL, IQOS-USA, marijuana, and placebo marijuana. Collagen-induced platelet aggregation in whole blood was assessed immediately after single exposure and one day after the final session of repeated exposure (5 days/week for 2 weeks; Fig. 1). Platelet aggregation was quantified by the area under the curve (AUC) of the aggregation tracing and by impedance. Single exposure to tobacco cigarette smoke and both types of marijuana smoke (marijuana and placebo marijuana) increased platelet aggregation (AUC) relative to clean air, but only in females (for tobacco and marijuana smoke, P = 0.024 and P = 0.018, respectively; with a trend toward significance after placebo marijuana smoke, P = 0.057; Fig. 4A). Treatment × sex interaction analyses did not reach statistical significance (P = 0.072), although there was a trend toward a sex-dependent effect. Similar patterns were observed for impedance (P = 0.009, P = 0.017, and P = 0.078, respectively; Fig. S3A). After 2 weeks of repeated exposure, platelet aggregation (AUC) was significantly increased compared with clean air following exposure to JUUL, IQOS, and placebo marijuana (P = 0.002, P = 0.0004, and P = 0.015, respectively; Fig. 4B). Tobacco cigarette exposure showed a trend toward increased platelet aggregation (P = 0.063), whereas marijuana exposure did not significantly affect platelet aggregation (P = 0.12; Fig. 4B). In contrast, impedance was significantly increased only after repeated IQOS exposure in the overall cohort (P = 0.003; Fig. S3B). Intriguingly, repeated IQOS exposure induced a male-specific increase in platelet aggregation (P = 0.018 for AUC and P = 0.026 for impedance; Fig. 4B; Fig. S3B), whereas repeated JUUL exposure induced a female-specific increase (P = 0.01 for AUC and P = 0.072 for impedance; Fig. 4B; Fig. S3B). Exploratory sex-stratified analyses suggested that certain responses following single exposure appeared more pronounced in females; however, treatment × sex interaction analyses did not reach statistical significance. Therefore, these findings do not provide sufficient statistical evidence for a sex-specific effect. Taken together, these findings indicate that smoking and vaping induce sustained increases in platelet aggregability, thereby increasing susceptibility to thrombosis and adverse cardiovascular events (40, 41).

Figure 4. Blood platelet aggregation was enhanced after single and repeated exposures, with exploratory sex-stratified differences.

A: single exposure. B: repeated exposure for 2 wk. Platelet aggregation was quantified by the area under the curve (AUC) of the aggregation tracing and by impedance (Supplemental Fig. S3). Sample size in A and B: n = 7 or 8 per group. Bars represent SD. Statistical analyses in A and B were performed with nonparametric 1-way ANOVA followed by Dunnett’s multiple-comparisons test to compare each smoking or vaping group with the air control group.

Repeated Exposure to Tobacco Smoke/Aerosol or Marijuana Smoke Increased Infarct Size Following I/R MI

Intravenous administration of nicotine has been reported to increase infarct size in a rat model of I/R MI; that is, the percent of the myocardium within the transiently ischemic region (area at risk; AAR) that dies (33). We therefore explored whether prior repeated exposure to tobacco smoke or other inhalational products similarly exacerbates MI. To address this question, we exposed conscious rats once daily to tobacco cigarette smoke, JUUL e-cigarette aerosol (i.e., nicotine without smoke), marijuana smoke (i.e., smoke without nicotine), or air negative control for 4 weeks (7 days/week; see schedule in Fig. 1). One day after the final exposure, all animals underwent surgical I/R MI. As described in the Methods section, Evans blue was injected to delineate the AAR in the LV, and hearts were harvested for quantification of the AAR and of infarct size within the AAR (Fig. 5, A and B). As expected, the AAR itself did not differ significantly among groups (Fig. 5C; Fig. S4), indicating consistent surgical occlusion conditions leading to comparable ischemic burden. However, infarct size normalized to AAR in the tobacco cigarette, JUUL, and marijuana groups was more than double that of the air group (Fig. 5C). Consistent with the overall findings, sex-stratified analyses showed that repeated exposure to tobacco smoke/aerosol or marijuana smoke significantly increased infarct size in both male and female animals (Fig. S5). Heart weight, LV weight, and the LV-to-heart weight ratio were not significantly different across groups (Fig. S6). These findings demonstrate that repeated exposure to tobacco cigarette smoke, nicotine-containing aerosol, and marijuana smoke each increased infarct size following I/R injury. These results indicate that the adverse effect is shared across multiple inhaled products with different chemical compositions, although the specific mediators and underlying mechanisms remain to be determined.

Figure 5. Repeated exposure to tobacco smoke or aerosol or marijuana smoke increased infarct size following ischemia-reperfusion (I/R) myocardial infarction (MI).

Conscious rats were exposed daily to smoke or aerosol from tobacco cigarettes, JUUL, or marijuana or to clean air for 4 wk before surgical ischemia (25 min) followed by reperfusion (40 min). A: schematic illustration of I/R and dye staining. One day after the final exposure, the left anterior descending (LAD) coronary artery was occluded for 25 min to induce ischemia, followed by 40 min of reperfusion. The LAD was then reoccluded, and dye was injected into the left ventricular (LV) chamber to delineate the area at risk (AAR). The left ventricle was subsequently sliced and stained with 2,3,5- triphenyltetrazolium chloride (TTC) to identify the infarct region. B: representative LV images showing Evans blue staining (left) and TTC staining (right) for assessment of the ischemic AAR (left, yellow outline) and infarct area within the AAR (right, pale region within yellow outline). C: quantification of ischemic AAR (left) and infarct size expressed as a percentage of AAR (right). Sample size in B: n = 9–17 per group. Bars represent SD. Statistical analyses were performed with nonparametric 1-way ANOVA followed by Tukey’s post hoc test to compare each smoking or vaping group with the air control group.

DISCUSSION

In this study, we demonstrate that both combustible and non-combustible tobacco products, as well as marijuana smoke, induce acute impairments in LV function and enhance platelet aggregation. Repeated exposure further augments platelet reactivity and, importantly, reduces myocardial tissue preservation following I/R MI, resulting in larger infarct size. Our study is notable in its conservative modeling. For example, unlike some frequently used smoking/vaping systems that switch between product inhalation and air gradually, the Gram exposure system ensured that rats received truly pulsatile delivery of smoke or aerosol with complete replacement of smoke/aerosol by air between each puff, like human smoking or vaping. Moreover, this entire study was based on just one 5-min smoking/vaping session per day, making the profound increase in post-MI tissue death after a month of daily smoking/vaping all the more impressive, not to mention rather distressing.

The changes in LVEF after acute smoke and aerosol exposure involved modest declines from pre-exposure that did not significantly differ from the air group (either in raw values or changes normalized to pre-exposure), and remained within the normal physiological range for healthy rats. Importantly, these acute functional changes occurred along with enhanced platelet aggregation following both acute and repeated exposures, as well as the increased myocardial infarct size following I/R MI after repeated exposure, suggesting that subtle functional disturbances may represent early manifestations of cardiovascular stress that precede more pronounced cardiovascular injury with continued exposure.

Collectively, these findings indicate that even short-term exposure to smoking or vaping products can acutely disrupt cardiovascular physiology and promote a prothrombotic state, while repeated exposure persistently heightens platelet aggregation and increases myocardial vulnerability to I/R injury.

Single exposure to tobacco smoke and JUUL aerosol significantly reduced LV function, reflected by decreased EF as assessed by both echocardiography and invasive hemodynamics, most prominently in males and independent of heart rate. Acute marijuana smoke similarly impaired LV function as assessed by invasive hemodynamics. Although the significance of EF reduction varied across products and sex subgroups, measurable functional effects were observed after all single-exposure conditions except clean air. Interestingly, these reductions in EF reached statistical significance in males despite smaller subgroup size. Although mean EF values were also lower than clean air in other male subgroups and across female subgroups, the study was not powered to determine whether these differences would reach significance with larger sample sizes. Therefore, we refrain from definitively concluding that the effect is sex-specific. Nonetheless, the robustness of the effect in males is intriguing and hypothesis-generating.

Product-specific differences in LV geometry were notable. For example, assessed by echocardiography, ESV was increased significantly (i.e., contractility was reduced) by tobacco cigarette smoke but tended to decrease with marijuana smoke. EDV was decreased (ventricular relaxation was impaired) in the IQOS and marijuana groups. These heterogeneous chamber responses suggest that different smoking and vaping products exert distinct acute physiological effects. The non-intuitive variation in EDV across products remains unexplained but likely reflects differences in loading conditions and autonomic regulation.

In addition, the discordant echocardiographic and hemodynamic findings following marijuana smoke exposure, along with temporal changes in HR and loading indices, suggest a dynamic and time-dependent cardiac response. Single marijuana exposure reduced hemodynamic EF and was accompanied by decreases in MAP and ESP, as well as significant reductions in echocardiographic EDV, SV, and CO. These findings indicate acute impairment in LV function after marijuana smoke exposure, despite no change in echocardiographic EF. Taken together, these results indicate that the differential changes in LV function and dimensions across exposure groups may reflect varying degrees of cardiac autonomic activation induced by different smoking and vaping products. Previous studies report that acute and chronic smoking are associated with impairments in LV systolic and diastolic function in humans and rodents (21, 42–47). Smoking-induced acute toxic effects, together with heightened sympathetic activation, may contribute to the mostly adverse cardiac functional changes observed in echocardiographic and hemodynamic parameters.

Several exclusive mechanisms may underlie the acute cardiac dysfunction following exposure. First, tobacco smoking and nicotine-containing aerosols have been reported to alter autonomic balance, including increased sympathetic activity and changes in catecholamine release (19, 21, 46, 48–55). In the present study, the heterogeneous changes in ESV and EDV across products suggest acute alterations in cardiovascular loading conditions and autonomic regulation; however, the study was not designed to determine the specific mechanisms responsible for the observed changes in chamber volumes and systolic function. Second, oxidative stress and endothelial dysfunction are plausible contributors to the acute effects (4–8, 13, 34, 35, 49, 50, 56–62). In our previous human and rodent studies (4–8, 13, 14, 18, 20, 34), we demonstrated that exposure to tobacco and marijuana smoke/aerosol induces endothelial dysfunction. Specifically, we found that nitric oxide (NO) production was significantly reduced in the blood from exposed subjects, indicating impaired endothelial NO bioavailability, a well-established marker of endothelial dysfunction that provides a plausible mechanistic link between inhaled tobacco and marijuana product exposure and cardiovascular injury. Combustion-derived particles and aerosolized constituents generate reactive oxygen species and reduce nitric oxide bioavailability, potentially increasing afterload and impairing myocardial relaxation. The reduction in EDV observed in the IQOS and marijuana groups may reflect transient ventricular underfilling, possibly secondary to altered ventricular compliance or acute changes in loading conditions. Although these interpretations remain speculative, they are consistent with prior literature on inhalational particulate exposure and cardiovascular dysfunction (50, 59–61).

Moreover, the observation that most of the adverse cardiac effects described here result similarly from exposure to products with disparate emissions that differ considerably in their physical nature (i.e., combustion smoke, partially pyrolyzed IQOS aerosol, and droplet-based e-cigarette aerosols) and in their main active chemicals (THC/cannabinoids vs. nicotine) is reminiscent of our earlier report (7) showing that vascular endothelial function is impaired by all of the smoke/aerosol exposures in the current study. Comparable effects were also caused by inert carbon particles and by acrolein in the absence of a solid component, suggesting that these acute effects were likely driven by a common airway-irritation response mediated by the vagus nerve. A potential mechanistic connection of those findings to our current results is that reduced myocardial survival from MI in rats after having smoked or vaped for one month may result from smoke/aerosol-induced impairment of coronary vascular endothelial function. However, the mechanisms underlying the acute changes in LV function and hemodynamics observed after a single exposure remain uncertain. Although endothelial dysfunction may contribute to the overall cardiovascular effects of smoke and aerosol exposure, the present study did not assess coronary vascular function or myocardial perfusion and therefore cannot establish endothelial dysfunction as the cause of the acute declines in EF and EDV or the observed hemodynamic changes. These acute cardiovascular responses may instead reflect multiple interacting mechanisms that warrant further investigation.

In parallel with acute cardiac functional changes, single exposure to tobacco cigarettes, marijuana, and placebo marijuana increased collagen-induced platelet aggregation, with more pronounced in females. After two weeks of repeated exposure, enhanced platelet aggregation was observed across tobacco product JUUL and IQOS groups and the placebo (cannabinoid-depleted) marijuana group (combined sexes). Interestingly, repeated exposure to placebo marijuana significantly increased platelet aggregation, whereas THC-containing marijuana did not. This finding suggests that combustion-derived constituents or other non-THC components of marijuana smoke may contribute to platelet activation. Because the present study was not designed to directly compare these two marijuana products or identify the responsible constituents, the basis for this difference remains uncertain and warrants further investigation. Regular marijuana containing cannabinoids was an interesting exception, in that while the effect size relative to air was similar to that of the other groups, the p value was quite high (P = 0.12), possibly reflecting confounding effects of cannabinoids acting through their receptors. Together, these findings indicate that both acute and repeated exposure promote a prothrombotic phenotype, with evidence of sex-modified susceptibility.

Platelet hyperreactivity provides a plausible mechanistic link between inhalational exposure and increased susceptibility to cardiac ischemic injury. Enhanced aggregation may result from direct particulate-platelet interactions, oxidative modification of plasma proteins, endothelial activation with reduced nitric oxide bioavailability, and catecholamine-mediated platelet priming following sympathetic activation (8, 49, 50, 59–61, 63). The persistence of enhanced aggregation after repeated exposure aligns mechanistically with the observed increase in infarct size following I/R MI. Prior repeated exposure may induce a sustained prothrombotic and vascularly compromised state characterized by enhanced platelet aggregation, which likely contributes to impaired microvascular perfusion and reduced myocardial salvage during reperfusion. This effect occurs without differences in ischemic risk area, indicating that prior exposure conditions the myocardium toward increased vulnerability to ischemic injury rather than altering ischemic severity. Smoking-mediated myocardial injury likely reflects both direct and indirect cardiovascular effects. Direct effects include exposure to toxic chemical constituents, oxidative damage, myocardial inflammation, and cumulative endothelial dysfunction. Indirect effects arise from secondary cardiovascular alterations including heightened sympathetic tone, microvascular impairment, hemodynamic stress, a prothrombotic state driven by increased platelet aggregation, and hypertension associated with vascular stiffness and LV hypertrophy. Together, our findings indicate that tobacco smoke/aerosol and marijuana smoke exposure adversely affect the cardiovascular system at multiple levels. The transient reductions in cardiac function observed immediately after smoke or aerosol exposure likely reflect an acute physiological response, whereas the increased infarct size following repeated exposure suggests a persistent reduction in myocardial tolerance to I/R injury. Although the present study was not designed to establish a direct mechanistic link between these endpoints, the results collectively support the concept that repeated exposure creates a cardiovascular milieu that increases susceptibility to ischemic myocardial injury. These cumulative cardiovascular alterations may impair myocardial resilience to ischemic stress and help explain why cigarette smoking remains one of the strongest modifiable risk factors for cardiovascular morbidity and mortality.

Extensive evidence demonstrates that smoking increases cardiovascular morbidity and mortality through autonomic dysregulation (19, 37, 44, 46, 51–55, 64), oxidative stress (50, 62, 65), endothelial dysfunction (4–8, 13, 34, 35), systemic and cardiac inflammation (20, 62, 65–68), platelet activation (3, 9, 12, 24, 50, 52, 69–71), and pathological remodeling of cardiovascular tissues (48, 62, 65, 66). Longer-term exposure has been associated with LV hypertrophy, atrial enlargement, myocardial interstitial fibrosis, and heightened thrombotic risk, contributing to arrhythmias (21, 72, 73), myocardial infarction (40, 74), heart failure (75), stroke, and sudden cardiac death (69, 76–79), particularly in individuals with pre-existing CVD. Epidemiologic studies report that smoking confers equal or greater cardiovascular risk in women than in men (74, 80, 81), and smoking-related cardiovascular morbidity in women has increased substantially over recent decades (1). Consistent with prior epidemiologic evidence, our findings further indicate that tobacco and marijuana smoking potentiate platelet aggregation. Exploratory sex-stratified analyses suggested that these effects may be more pronounced in females, which, if confirmed, could have implications for thrombotic risk. Moreover, prior smoke and aerosol exposure similarly increased infarct size in both sexes, indicating that the exploratory differences in platelet aggregation did not translate into detectable sex differences in myocardial injury in our I/R model. These findings highlight the need for adequately powered studies to determine whether sex-specific differences in cardiovascular responses to tobacco and marijuana exposures truly exist.

Limitations

The study was powered for combined-sex analyses and small sample sizes; therefore, subgroup analyses by sex are underpowered and may have missed additional sex-specific effects, and the observed differences were preliminary. Some cardiac functional endpoints demonstrated variability and only borderline statistical significance, which may reflect an acute physiological response and inherent biological variability. Echocardiography and pressure-volume analyses provide complementary assessments of cardiac function but were performed under distinct experimental conditions and at different time points after exposure, which may account for differences in sensitivity across functional endpoints. In addition, echocardiographic indices of diastolic function, including the ratio of the early (E) to late (A) transmitral inflow velocity (E/A) and LV global strain, were not obtained, limiting a more comprehensive assessment of cardiac function and the interpretation of the observed changes. Platelet aggregation was assessed by a single agonist (collagen). Although collagen is a physiologically relevant agonist that reflects platelet responses to vascular injury, inclusion of additional agonists such as ADP would strengthen mechanistic conclusions. The estrous cycle was not monitored in females, which may have contributed to variability, because fluctuations in sex hormones can influence cardiovascular physiology, future studies controlling for estrous cycle stage will help further define potential sex-specific responses to smoke or aerosol exposure. While this study focused on the cardiovascular effects of individual tobacco smoke/aerosol and marijuana smoke exposure, concurrent (dual use) exposure was not evaluated despite the increasing prevalence of combined product use. Future studies are warranted to determine whether dual-use exposure induces additive, synergistic, or distinct cardiovascular effects compared with exposure to each product alone.

Conclusions

Smoking and vaping of tobacco and marijuana products acutely impair cardiac function, promote platelet hyperreactivity, and increase myocardial tissue death following MI. These findings underscore clinically relevant cardiovascular risks associated with both combustible and non-combustible smoking products and raise concern that even short-term exposure may prime the cardiovascular system toward dysfunction and thrombosis.

Supplementary Material

Supplemental Tables S1-S2: https://doi.org/10.5061/dryad.vt4b8gv7r

Supplemental Figs. S1-S6: https://doi.org/10.5061/dryad.vt4b8gv7r

DATA AVAILABILITY

Data in this study are available upon reasonable request.