Adverse Cardiovascular Effects of Nicotine Delivered by Chronic Electronic Cigarettes or Standard Cigarettes Captured by Cardiovascular Intrinsic Frequencies
Journal of the American Heart Association
Abstract
Background
Electronic cigarettes have gained popularity as a nicotine delivery system, which has been recommended by some as an aid to help people quit traditional smoking. The potential long‐term effects of vaping on the cardiovascular system, as well as how their effects compare with those from standard cigarettes, are not well understood. The intrinsic frequency (IF) method is a systems approach for analysis of left ventricle and arterial function. Recent clinical studies have demonstrated the diagnostic and prognostic value of IF. Here, we aim to determine whether the novel IF metrics derived from carotid pressure waveforms can detect effects of nicotine (delivered by chronic exposure to electronic cigarette vapor or traditional cigarette smoke) on the cardiovascular system.
Methods and Results
One hundred seventeen healthy adult male and female rats were exposed to purified air (control), electronic cigarette vapor without nicotine, electronic cigarette vapor with nicotine, and traditional nicotine‐rich cigarette smoke, after which hemodynamics were comprehensively evaluated. IF metrics were computed from invasive carotid pressure waveforms. Standard cigarettes significantly increased the first IF (indicating left ventricle contractile dysfunction). Electronic cigarettes with nicotine significantly reduced the second IF (indicating adverse effects on vascular function). No significant difference was seen in the IF metrics between controls and electronic cigarettes without nicotine. Exposure to electronic cigarettes with nicotine significantly increased the total IF variation (suggesting adverse effects on left ventricle‐arterial coupling and its optimal state), when compared with electronic cigarettes without nicotine.
Conclusions
Our IF results suggest that nicotine‐containing electronic cigarettes adversely affect vascular function and left ventricle‐arterial coupling, whereas standard cigarettes have an adverse effect on left ventricle function.
Nonstandard Abbreviations and Acronyms
- |Δω|
- absolute value of total intrinsic frequency variation
- ω 1
- first intrinsic frequency
- ω 2
- second intrinsic frequency
- EC
- electronic cigarette
- EC NIC(−)
- electronic cigarette vapor without nicotine
- EC NIC(+)
- electronic cigarette vapor plus nicotine
- IF
- intrinsic frequency
Electronic cigarettes (ECs) have gained popularity as a nicotine delivery system, which has been recommended by some as an aid to help people quit traditional smoking. However, their use has greatly expanded, especially among adolescents and teenagers.1 After becoming commercially available in 2004, the use of ECs (ie, known as vaping) has grown significantly.2 ECs are often released commercially without clinical safety. However, the possibility of developing long‐term adverse effects on the cardiovascular system after vaping, and comparing such effects with standard cigarettes, is poorly understood, mainly due to the short presence of ECs in the market.3 Several studies demonstrated that nicotine vaping results in significant acute increases in both blood pressure and heart rate.4, 5, 6, 7, 8, 9, 10 A study on nicotine‐free vaping also found significant increases during nicotine‐free vaping.5 Both nicotine and nicotine‐free vaping have been shown to induce vascular impairment.11, 12, 13 Acute EC use significantly increased pulse wave velocity, a clinically relevant index of arterial stiffness, in both nicotine‐ and nicotine‐free vaping4, 5, 10, 14; however, some studies showed that acute vaping might have less significant effects on vascular function when compared with traditional cigarette smoking.6, 10, 12 It is also shown that the use of traditional cigarettes increases the risk of cardiovascular diseases (eg, hypertension, heart failure, atherosclerosis).15, 16 Additionally, it was demonstrated by survey data17 (n=69 395) that daily EC use is independently associated with increased odds of having had a myocardial infarction (MI). The odds of MI were about 1.7 for daily EC users, compared with subjects who had never used ECs (prospective studies and clinical trials have not yet been performed). In a preclinical study by Dai et al,18 chronic exposure to ECs before inducing an acute MI did not alter MI size or no‐reflow but did alter left ventricle (LV) hypertrophy and vascular function. The aforementioned studies collectively suggest that vaping can pose long‐term cardiovascular health risks for chronic users.
The intrinsic frequency (IF) method was introduced as a systems‐based approach for quantitative understanding of cardiovascular physiology and hemodynamic analysis of heart and vascular function.19, 20, 21, 22, 23, 24 This method can be applied to any single uncalibrated arterial waveform measured either invasively or noninvasively and computes the 2 dominant frequencies during systole (ie, ω1) and diastole (ie, ω2). ω1 is mainly dominated by the LV contractile function, whereas ω2 is mainly related to the arterial dynamics (eg, arterial stiffness, wave reflections, and peripheral vascular tone).19, 20, 21, 22, 23, 24 It also demonstrated that lower total frequency variation (Δω=ω1−ω2) is associated with more optimal states of the ventricular‐vascular coupling and vascular health.19, 20, 23, 25 Previous clinical and preclinical studies have demonstrated that IF metrics provide clinically valuable information about the dynamic state and underlying pathologies of the LV, the arterial system, and the LV‐arterial interactions.19, 20, 21, 22, 26, 27, 28, 29, 30 In a clinical study, Cooper et al19 assessed the prognostic value of IFs using the longitudinally followed large cohort of the Framingham Heart Study. They found that higher ω1 and lower ω2 are associated with higher risks for heart failure incidents.19
Some studies have shown clear pulmonary consequences of EC use; however, effects of chronic vaping on the cardiovascular system remain speculative and controversial. Here, we aimed to evaluate effects of nicotine delivered by chronic EC exposure or standard cigarettes on the cardiovascular system using the IF metrics computed from carotid pressure waveforms. In this study, adult male and female rats were exposed to purified air (control), EC vapor without nicotine, EC vapor with nicotine, and standard nicotine‐containing cigarette smoke, after which the IF metrics were extracted.
METHODS
The data that support the findings of this study are available from the corresponding author upon reasonable request. We used the Animal Research Reporting of In Vivo Experiments checklist when writing our report.31
Preclinical Data
In this study, we used a total of 117 healthy adult male and female Sprague‐Dawley rats (49% [n=57] female, 6 weeks old, body weight ≈260 to 370 g). All of the procedures were conducted according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication number 85–23, National Academy Press, Washington, DC, revised 2011). The procedures used for the exposures and the invasive hemodynamic measurements (see Exposure effects on the first intrinsic frequency and Exposure effects on the second intrinsic frequency for details) are approved by the Institutional Animal Care and Use Committee at the University of California, Irvine, and Huntington Medical Research Institutes. These rats are part of our larger study (ie, the smoking‐acute MI study18 based on acute MI protocols), but this study only focuses on smoking because we used carotid pressure waveforms after EC exposure and before inducing MI (before coronary artery occlusion). We specifically focused on the new IF metrics computed from carotid pressure waveforms that have not been previously reported.
Vaping and Smoking Procedures
A total of 117 rats were randomized and exposed to the following exposure groups: (1) purified air group (control), n=32; (2) EC vapor without nicotine group (EC NIC[−]), n=26; (3) EC vapor plus nicotine group (EC NIC[+]), n=27; and (4) standard cigarette smoke group from reference combustion cigarettes (1R6F), n=32. Test atmospheres were generated using a custom‐built smoking system (Figure 1A). Rats were exposed to smoke or vapor in a nose‐only inhalation exposure system (In‐Tox Products, Clinton, MS) with puff parameters and dilution factors set to mimic human smoking behavior. All exposures were conducted at the Air Pollution Health Effects Laboratory of the University of California, Irvine, following procedures approved by the Institutional Animal Care and Use Committee. During exposure, animals were restrained in individual tubes and connected to an exposure manifold with only the snout exposed to the exposure atmosphere, which served to minimize dermal exposure and ingestion. The test atmosphere (1) enters the manifold, (2) is delivered to each rat's breathing zone, and (3) the excess is removed through an exhaust port. Any exhaled air from the rats is entrained and removed with the exhaust air, thereby minimizing the chance for rebreathing and CO2 buildup. All exposures took place for a duration of 5 hours per day, 4 days per week, for a total of 8 weeks. A commercially available, third‐generation, mod‐type vaporizer (VaporFi VEX 150 TC mod with Volt Tank and 0.5 Ohm stainless steel atomizer; 80 W power) was used to generate all EC vapor. The e‐liquid was tobacco‐flavored with a 50/50 propylene glycol/vegetable glycerin volume ratio (www.VaporFi.Com). For the EC NIC(+) group, pure nicotine (LNicotine; Acros Organics; lot A0382410) with 15 mg/mL concentration was added to the propylene glycol/vegetable glycerin mixture to match specifications based on Standardized Research Electronic Cigarette (www.nida.nih.gov). For the combustion cigarette smoke group, the 1R6F standard research cigarettes were used (Center for Tobacco Reference Products, University of Kentucky). Combustion cigarette concentrations were diluted 1:50 to reduce CO levels down to ≈150 ppm (average±SD 134.77±50.16 ppm) to limit any CO‐derived health effects. For all the exposure groups, we used the International Organization for Standardization smoking conditions of puff volume=35 mL, puff frequency=1 puff/min, and puff duration=2 seconds (at a flow rate of 1.67 L/min; our system uses a square puff profile). These standardized conditions simulate the average human smoking behavior for the purpose of laboratory testing and allow for the consistent measurement of various cigarette components, providing a basis for comparison across different products and brands.
Invasive Hemodynamic Measurements
After 8 weeks of exposure, the rats were transferred to the Huntington Medical Research Institutes and allowed to acclimate for 2 to 4 days (according to our Institutional Animal Care and Use Committee guidelines for transferring animals in chronic studies) before measuring hemodynamic markers such as carotid pressure waveform, invasively. Intraperitoneal ketamine (75 mg/kg) and xylazine (5 mg/kg) were used as the anesthesia drug. Depth of anesthesia was evaluated using different indicators (ie, respiration rate, heart rate, response to toe pinch reflexes, and response to saline dripped into the eyes). Anesthesia was also correspondingly increased immediately after observation of any body movement or discomfort. After achieving the anesthesia, rats were shaved and cleaned in the neck area, and the rats were intubated and mechanically ventilated with room air (60 cycles/min with tidal volume 1 mL/100 g of body weight). The major steps of our rat experimental surgeries are summarized as (1) full anesthesia (described earlier in this section), (2) intubation and mechanical ventilation, (3) neck incisions to expose and isolate the right carotid artery and jugular vein (the rat's body was positioned appropriately in the lateral recumbency, and corresponding cutdowns were performed), (4) insertion of a 2F high‐fidelity piezo‐tipped micromanometer (model SPR‐869; Millar Mikro‐Cath Pressure Catheter) into the carotid artery and subsequently into the LV, and (5) recording of the carotid/LV pressure waveforms (see Figure 1B). ECG signal and body temperature were also recorded and monitored over each surgical operation. A rectal thermocouple probe attached to a continuous reading system was used for body temperature measurements. A water‐circulating heating pad and covering towels were used to maintain the body temperature at ~37 °C. Clean‐room techniques were followed, and we ensured that all instruments were sterilized. Vital signs were carefully monitored throughout the surgical operations.
Hemodynamic Analysis
Different hemodynamic parameters and indices were used to analyze the measured waveforms. These parameters included the IF metrics19, 20, 23, 24 (eg, ω1, ω2, ) computed from carotid waveforms, minimum and maximum pressure values (of the same carotid waveforms selected for the IF analysis), heart rate, cardiac output, arterial resistance, compliance, and left ventricular fractional shortening. Two common measures of ventricular contractility, including contractile element velocity of shortening () and left ventricular wall stress at end‐systole (), were also used in this study. and are defined as:where and refer to left ventricular fractional shortening and left ventricular ejection time (ie, systolic time), respectively. In Equation (1b), , , and are left ventricular end‐systolic pressure (millimeters of mercury), left ventricular end‐systolic internal dimension (centimeters), and left ventricular posterior wall thickness at end‐systole (centimeters), respectively. Such parameters were measured using echocardiography (via Philips Sonos 5500).
(1a)
(1b)
Cardiovascular Intrinsic Frequency
The IF metrics are extracted from a single arterial waveform (uncalibrated waveform) by minimizing the following objective function:subjected to 2 nonlinear constraints defined for ensuring waveform continuity at decoupling time (ie, time of the dicrotic notch), and waveform periodicity (for the subsequently reconstructed waveform), as expressed as:
(2)
(3)
is the pressure waveform (any arterial waveform), and refers to an indicator function such that for , and otherwise. The above minimization problem is solved for obtaining the following unknown parameters (optimization variables): (1) intrinsic frequencies (ω1, ω2), (2) fitting parameters (, , ), and (3) a translation constant (c). The total frequency variation (Δω) is calculated as the difference between and .19, 20, 21, 22, 23, 24 Secondary IF‐derived parameters (defined with the help of the trigonometric circle concept) are the intrinsic envelopes (either systolic [Rs] or diastolic envelope [Rd]) and the intrinsic envelope ratio (ER)19, 20, 21, 22, 23, 24:where (i=1,2) are unknowns of the IF method optimization problem as described above. Using the secondary IF metrics, along with the main intrinsic frequencies (ω1, ω2), the IF methodology can be visualized for each of the systolic and diastolic phases separately (see Figure 1C). This method results in a new representative space called the intrinsic frequency space (see Figure 1C).
(4)
Statistical Analysis
Different statistical methods were used in this study to evaluate the significance of the differences among the 4 groups of the study. The Levene test, which evaluates the homogeneity of the variances, was conducted to assess whether the variances for all groups were significantly different from each other or not.32 The Shapiro‐Wilk test was used to test the normality of the data.33 If homogeneity and normality of the data were satisfied, the ANOVA and the Tukey‐Kramer tests were used, respectively, to test the overall difference and pairwise comparison of variables with normal distribution.34, 35 The null hypothesis for ANOVA analysis indicates that all means for different treatment groups are equal. A small P value (corresponding to the chosen significant level) rejects the null. Furthermore, the Kruskal‐Wallis rank sum test and Dunn test with Bonferroni adjustment were also used, respectively, in our analyses for overall and 2‐by‐2 comparisons of the parameters with a nonnormal distribution.36, 37 The software used for statistical analyses was R version 4.1.2. The significance level for the tests was 95% (α=0.05); however, higher levels of statistically significant differences were also highlighted at P<0.0001, 0.001, and 0.01. The flowchart of the statistical analysis methods is presented in Figure S1.
RESULTS
The total data consist of 117 rats from 4 groups in which different rats were exposed to purified air (Group 1, control), EC vapor without nicotine (Group 2), EC vapor plus nicotine (Group 3), and standard cigarette smoke (Group 4). The IF metrics were computed from carotid pressure waveforms of each rat. Figure 2 shows samples of raw carotid waveforms (dashed black) overlaid on top of their IF‐reconstructed waveforms (blue) for each exposure group. The obtained IF values as well as other hemodynamic parameters were analyzed to study effects of different vaping and smoking conditions. The distribution summary for such parameters is presented in Table 1 and Table S1.
Variable | Air (control) | EC NIC(−) | EC NIC(+) | Standard cigarette |
---|---|---|---|---|
n=32 (50% female) | n=26 (38% female) | n=27 (56% female) | n=32 (50% female) | |
452.6±43.3 | 454.3±37.2 | 450.8±48.8 | 495.1±48 | |
285.9±108.8 | 285.6±99.2 | 217±71.5 | 269.2±79.4 | |
|| | 166.7±128.1 | 168.8±94 | 233.7±63.7 | 225.9±77.9 |
Pmin, mm Hg | 55.8±13 | 60.1±16 | 56.3±13 | 55±13.3 |
Pmax, mm Hg | 78.6±17 | 83.6±18.9 | 77.1±14 | 77.7±15.7 |
Cardiac output, mL/min | 61.9±20 | 57.2±17.3 | 50.8±18.2 | 58.2±15.5 |
Arterial resistance, mm Hg.min/mL | 1.35±0.461 | 1.35±0.505 | 1.45±0.477 | 1.37±0.442 |
LVFS, % | 41.4±7.3 | 45.4±10.7 | 43.0±7.65 | 41.1±6.63 |
Vce, | 5.93±1.08 | 6.59±1.48 | 6.4±1.53 | 6.55±1.13 |
Stresses, | 20.7±8.63 | 14.7±7.34 | 18.1±10.1 | 16.1±6.35 |
Heart rate, bpm | 251.6±25.2 | 251.7±35.5 | 244.7±28.5 | 258.5±30.5 |
Compliance*, mL/mm Hg | 0.0105±0.003 | 0.0106±0.003 | 0.0105±0.003 | 0.0105±0.004 |
Data are presented as mean±SD. Pmin and Pmax are minimum and maximum pressure values, respectively, of the same carotid waveforms selected for the intrinsic frequency analysis. |Δ𝜔| indicates absolute value of total intrinsic frequency variation; 𝜔1, first intrinsic frequency; 𝜔2, second intrinsic frequency. EC, electronic cigarette; EC NIC(−), electronic cigarette vapor without nicotine; EC NIC(+), electronic cigarette vapor plus nicotine; LVFS, left ventricular fractional shortening; Stresses, left ventricular wall stress at end‐systole; and 𝑉ce, contractile element velocity of shortening.
*
Numbers of available rat data for compliance measurements are 32, 25, 25, and 31 for purified air (Group 1), EC vapor without nicotine (Group 2), EC vapor plus nicotine (Group 3), and standard cigarette smoke (Group 4), respectively.
In the next step, multiple statistical tests, including the Levene test, Shapiro‐Wilk test, ANOVA, or Kruskal‐Wallis rank sum test, were applied to the parameters of interest (eg, IF metrics) using the total data from all the exposure groups. The P value results corresponding to the statistical tests are presented in Table 2 and Table S2. Because the data for , (Table 2) and (see Data S1 and Table S2, as well as Figure S2 for ER distribution) passed the normality test by the Shapiro‐Wilk test, we conducted the ANOVA to see if there was an overall difference between the exposure groups. ANOVA for ω1 and ω2 results in P values of 0.000224 and 0.0177, respectively, showing a significant difference over the exposure groups for such parameters. Then, the Tukey‐Kramer test was used for pairwise comparisons of the groups for these parameters (see Tables S3 through S5). The normality assumption for the remaining parameters of Table 2 (eg, Pmin, Pmax, and heart rate) was rejected after conducting the Shapiro‐Wilk test. Consequently, the Kruskal‐Wallis test was used to assess potential overall differences among these parameters (see Table 2 for P value results; |∆ω| was not normal by the definition of the normal distribution). Except for |∆ω|, the Kruskal‐Wallis test demonstrated no overall significant difference over the exposure groups for the remaining parameters. Results of the Dunn test that was used for pairwise comparisons of the groups for |∆ω| are presented in Table S6.
Variable | Levene test | Shapiro‐Wilk test | ANOVA | Kruskal‐Wallis test |
---|---|---|---|---|
0.78 | 0.17 | 0.0002* | ||
0.18 | 0.203 | 0.02† | ||
|| | 0.25 | N/A | 0.01† | |
P min | 0.4 | 0.01† | 0.45 | |
P max | 0.5 | 0.00§ | 0.41 | |
Cardiac output | 0.45 | 0.00§ | 0.08 | |
Arterial resistance | 0.99 | 0.02† | 0.73 | |
LVFS | 0.28 | 0.00§ | 0.38 | |
V ce | 0.75 | 0.00§ | 0.09 | |
Stresses | 0.46 | 0.004‡ | 0.06 | |
Heart rate | 0.71 | 0.0007* | 0.24 | |
Compliance | 0.88 | 0.002‡ | 0.98 |
Data are presented as P values. Pmin and Pmax are minimum and maximum pressure values, respectively, of the same carotid waveforms selected for the intrinsic frequency analysis. |Δ𝜔| indicates absolute value of total intrinsic frequency variation; 𝜔1, first intrinsic frequency; 𝜔2, second intrinsic frequency; LVFS, left ventricular fractional shortening; N/A, not applicable; Stresses, left ventricular wall stress at end‐systole; and 𝑉ce, contractile element velocity of shortening;
*
P < 0.001.
†
P < 0.05.
‡
P < 0.01.
§
P < 0.0001.
Exposure Effects on the First IF
Figure 3 shows distribution of the first IF (ω1) for different exposure groups including purified air (Group 1), EC vapor without nicotine (Group 2), EC vapor plus nicotine (Group 3), and standard cigarette smoke (Group 4). The results of the Tukey‐Kramer test for ω1 are presented in Table S4, where significant differences were found between Group 1 and Group 4 (P=0.0013538), Group 2 and Group 4 (P=0.0043204), and Group 3 and Group 4 (P=0.0013665).
Exposure Effects on the Second IF
The distribution of the second IF (ω2) for different exposure groups (ie, Group 1: purified air, Group 2: EC vapor without nicotine, Group 3: EC vapor plus nicotine, and Group 4: standard cigarette smoke) is demonstrated in Figure 4. The Tukey‐Kramer test results for ω2 are presented in Table S5. Significant differences were found between Group 1 and Group 3 (P=0.0236134) and between Group 2 and Group 3 (P=0.0361873).
Exposure Effects on the Total IF Variation
Figure 5 shows distribution of the absolute value of total IF variation () for different exposure groups of purified air (Group 1), EC vapor without nicotine (Group 2), EC vapor plus nicotine (Group 3), and standard cigarette smoke (Group 4). Because the data for |∆ω| were not normal by the definition of the normal distribution, the Kruskal‐Wallis test was used to examine the overall difference between groups, and the overall difference between groups was established by P=0.01389 (Table 2). Then, the Dunn test was performed for pairwise comparison of the groups (see Table S6). The results showed a significant difference only between Group 2 and Group 3 in |∆ω| (P=0.02788259).
DISCUSSION
The results suggested that cardiovascular IF can capture adverse cardiovascular effects of nicotine delivered by chronic EC exposure or standard cigarette smoke. We investigated trends of novel IFs (, , and ) derived from carotid waveforms for different smoking conditions. We studied the difference in the value of , , and among the 4 exposure groups of rats and found significant differences for such parameters. ANOVA for and yielded differences in their mean value. We further used the Tukey‐Kramer test and found that was significantly different in 2‐by‐2 comparisons of all the groups with Group 4 (ie, standard cigarette). was also significantly different between Group 1 (ie, purified air, control) and Group 3 (nicotine EC), and also between Group 2 and Group 3 (nicotine‐free and nicotine ECs). The 2‐by‐2 comparisons of among the exposure groups showed a significant difference only between Group 2 and Group 3 (ie, nicotine‐free and nicotine ECs) based on the Dunn test with Bonferroni adjustment.
We found that standard cigarettes significantly increased when compared with all of the other exposure groups (ie, EC with nicotine, EC without nicotine, or purified air). is the IF of the coupled heart and vascular system during systole, and it is mainly dominated by the LV contractile function.19, 20, 21, 23, 38, 39 Previous studies showed that was higher among heart failure patients with systolic dysfunction.19, 23 Therefore, standard cigarettes, which significantly affected according to our results, may lead to LV contractile dysfunction. We also found that ECs with nicotine significantly reduced compared with ECs without nicotine or purified air exposure groups. represents the IF of the decoupled vasculature during diastole, and it is mainly dominated by the dynamics of the aorta and the rest of the arterial system (related to arterial stiffness, wave reflections, and peripheral vascular tone).19, 20, 22, 23, 40 was previously found significantly lower among patients with peripheral artery disease and hypertension.23 Based on our findings, the use of ECs with nicotine, which significantly reduced , seems to adversely affect the vasculature, mainly leading to arterial stiffening. We found no significant difference in any of the IF metrics between purified air (control) and ECs without nicotine, so the effects between these 2 exposure groups were not significant enough to be captured through the IF metrics. Moreover, looking at the results of total frequency variation ( for EC, the addition of nicotine to an EC significantly increased . Previous studies showed that lower is associated with more optimal states of the ventricular‐vascular coupling and vascular health.19, 20, 23, 25 was previously found to be ≈0 among young healthy individuals (who have ideal LV‐arterial coupling), but it was found significantly higher among older people and also among patients with prevalent heart failure. Therefore, the use of ECs with nicotine seems to adversely affect the LV‐arterial coupling and its optimal state when compared with ECs without nicotine.
When compared with other studies, our approach for detecting adverse cardiovascular effects of nicotine only requires measurement of carotid pressure waveforms, and neither ECG nor flow/velocity is needed. The IF method requires only a single arterial waveform, because it operates based on waveform morphology alone; therefore, there is no need for calibration of the measured waveform data, which facilitates the measurement process by allowing noninvasive, instantaneous, and inexpensive waveform measurement techniques (eg, measurements can be acquired by arterial applanation tonometry,19 a smartphone,20 or Vivio41). Overall, our findings indicate that novel IF metrics, derived from carotid pressure waveforms, can capture the detrimental effects of standard cigarettes on LV function, as well as the negative impacts of ECs on vascular function and LV‐arterial coupling, which suggests that vaping can pose long‐term cardiovascular health risks for chronic users.
Strengths and Limitations
The proposed approach for detecting adverse effects of nicotine only requires measurement of carotid pressure waveforms without any need for ECG or flow/velocity measurements. Our IF‐based approach is instantaneous42, 43, 44 and uses carotid pressure waveforms that can be measured noninvasively (eg, using tonometry or a smartphone).20, 41, 45, 46, 47, 48, 49 Previous studies have validated noninvasive measurements against invasively measured arterial pressure waveforms.50, 51, 52, 53 The scalability of IF metrics between different mammals (rats and rabbits) and humans has also been shown.24 Therefore, although this study is an invasive preclinical validation on rats, our proposed approach can be translated to humans for noninvasive and instantaneous applications in clinics or at home. Moreover, our results may suggest that IF parameters are more sensitive markers for capturing the adverse effects of different exposures compared with some non‐IF parameters in this study that also changed after 8 weeks of exposure (eg, cardiac output), but their corresponding P values were borderline on or marginally significant (see Table 2). We believe adding more rats in future studies can change these borderline/marginally significant P values to significant ones. However, in our recent study,18 we did capture significant changes of other non‐IF parameters, such as negative (dP/dt)Max, left ventricular end‐diastolic pressure, and systolic blood pressure.
The proposed approach was developed using only healthy rats before the smoke exposure. Using healthy rats was necessary as the first step to identify and confirm the true changes of the novel IFs during different smoking conditions. However, it is acknowledged that further studies should confirm such approach on rats with various cardiovascular complications/diseases (eg, spontaneously hypertensive rats). We also acknowledge that our results are not specific to only 1 sex by performing additional sex‐specific analysis (see Data S2, Tables S7 and S8 results). However, adding more rats (both male and female) to each exposure group in future studies can enhance the statistical power and change the borderline/marginally significant P values to the significant ones (similar to our non‐sex specific results). Looking into our sex‐specific results (see Data S2, Tables S7 and S8) with the current male and female rat numbers, relatively higher P values corresponding to female rats could suggest that female rats show some degree of cardiovascular protection against nicotine‐induced effects when compared with male rats (in agreement with recent findings by Neczypor et al2). Because we used relatively young rats, our results suggest that estrogen could be playing this protective role in female rats.54, 55 However, we did not measure the estrogen level in this study. Future studies are needed to investigate the relation between estrogen and cardiovascular intrinsic frequency metrics. Future studies are also needed to investigate different formulations of propylene glycol/vegetable glycerin volume ratio (eg, 70:30) besides 50:50, as well as future studies to analyze the changes in histological structure of cardiovascular tissues and therefore understand the underlying mechanisms in more detail. This study was designed as a proof of concept for investigating changes of cardiovascular intrinsic frequencies across different exposure groups; however, we acknowledge that further studies should include measurements of nicotine's metabolites56, 57 (eg, cotinine levels), and also evaluate if there is any relation between the level of the metabolites and cardiovascular intrinsic frequencies.
CONCLUSIONS
In this study, we showed that novel IF metrics can capture adverse effects of nicotine (delivered by either chronic EC exposure or standard cigarette smoke) on the cardiovascular system by using only carotid pressure waveforms. We used adult male and female rats exposed to different smoking conditions including purified air (control), EC vapor without nicotine, EC vapor with nicotine, and standard nicotine‐containing cigarette smoke. The outcomes implied that standard cigarettes significantly increased the first IF () and might result in LV contractile dysfunction. Also, ECs with nicotine significantly reduced the second IF () and seemed to adversely affect the vasculature and lead to arterial stiffening. The evidence suggested no significant difference in any of the IF metrics between control and ECs without nicotine. Moreover, exposure to ECs with nicotine significantly increased the total frequency variation () and seemed to adversely affect the LV‐arterial coupling as well as its optimal state, when compared with ECs without nicotine.
Sources of Funding
R.A. holds the James G. Boswell Postdoctoral Fellowship at the California Institute of Technology and the Huntington Medical Research Institutes. The rats used in this study were part of our larger study (smoking‐acute MI study) funded by the University of California Office of the President Special Research Programs: Tobacco‐Related Disease Research Program study (grant number: 28IR‐0057). This study was made possible, in part, by generous support from the James G. Boswell Foundation.
Disclosures
N.M. Pahlevan holds equity in Ventric Health and has a consulting agreement with Ventric Health. The remaining authors have no disclosures to report.
Footnotes
This article was sent to Julie K. Freed, MD, PhD, Associate Editor, for review by expert referees, editorial decision, and final disposition.
Supplemental Material is available at Supplemental Material
For Sources of Funding and Disclosures, see page 10.
Supplemental Material
Data S1
Tables S1–S8
Figures S1–S2
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© 2024 The Author(s). Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
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Received: 14 March 2024
Accepted: 31 July 2024
Published online: 11 September 2024
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University of California Office of the President Special Research Programs: Tobacco‐Related Disease Research Program study: 28IR‐0057
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