An Examination of the Impact of Habitual Cannabis Use on Vascular Health, Cardiac Function, and Cardiac Structure in Healthy Adults

An Examination of the Impact of Habitual Cannabis Use on Vascular Health, Cardiac Function, and Cardiac Structure in Healthy Adults.

Christian Plinio Cheung

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Human Health and Nutritional Sciences

Guelph, Ontario, Canada

AN EXAMINATION OF THE IMPACT OF HABITUAL CANNABIS USE ON VASCULAR HEALTH, CARDIAC FUNCTION, AND CARDIAC STRUCTURE IN HEALTHY ADULTS

Christian Plinio Cheung Advisor: University of Guelph, 2020 Professor J.F. Burr

Cigarette smoking is one of the most detrimental behaviours to cardiovascular health.

Its use leads to arterial stiffening, endothelial dysfunction, and structural/functional alterations to the myocardium. Apart from alcohol, cannabis is the most commonly used recreational substance in the world and is often smoked, similar to cigarettes. This thesis explores whether cannabis users manifest altered baseline levels of arterial stiffness, endothelial function, or cardiac structure and function. Using a cross-sectional design measures of pulse wave velocity (PWV), flow-mediated dilation (FMD) and echocardiography were performed in young healthy cannabis smokers (CU) and non- users (NU). CU had higher PWV compared to non-users. FMD was similar between CU and NU. There were no differences in cardiac morphology, or measures of cardiac function between CU and NU. However, CU demonstrated reduced peak apical rotation.

This suggests young apparently healthy cannabis users demonstrate arterial stiffening and altered cardiac mechanics compared to young apparently healthy non-users.

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DEDICATION

This thesis is dedicated to my Yaya. He is the best teacher I have ever had and was my first ever science teacher. I am incredibly thankful that he gets to see the grandson he hosted “Science Tuesdays” for, become a scientist.

ACKNOWLEDGEMENTS

The work encompassed in this thesis would not exist without the unwavering support of a number of people. I hope that each and every one of you mentioned in this section understand that the words here express just a fraction of the thanks you deserve.

First and foremost, I’d like to thank my advisor Jamie Burr. In the past three years you’ve overseen my transition from a third-year student in your class, to a fourth-year research student, and finally to an MSc student. In that time you’ve not only pushed me to become a better scientist, but also a better person. You’ve also provided me opportunities I never could’ve imagined and have provided an incredible amount of support. You’ve been a better mentor than I could’ve asked for. For all of these things I express my utmost thanks. I am excited for what we can achieve together in the future.

I also received a great deal of support from Phil Millar. It has become clear to me that the opportunity to collaborate with your group is invaluable, and that your perspective vastly improves my own work. I want to give special thanks for your willingness to have a conversation or give feedback. Despite not being under your direct supervision I have always felt that my requests for support or help have been given great care and attention. If you ever need tips on your jumper, I would be happy to return the favour.

Mom, Dad, and Nicole, you guys have also been incredibly important these past two years. Your love for me and support for what I do is so special, and I can’t thank you guys enough. You guys make it easy to understand why family is important. Taylor, you deserve a special thank you. You’re always understanding of long hours of work, and the life-related sacrifices that come with me working in an area that I have a passion for. You’ve helped me through my most stressful of times and have been there to celebrate my successes as they come. I hope you know that my successes are also yours, and that I truly believe I wouldn’t be where I am without you.

The participants who volunteered their time to this study also deserve special recognition. Recreational cannabis use is still stigmatized. I believe that this is a large part of why cannabis is under-studied. The participants who volunteered went against the grain to make this study possible and for that I am thankful.

I need to thank my lab mates for the role they’ve played in my life the past three years. This includes Alex, Brett, Chris, Heather, Jeremy, Josh, Kyle, Megan, Shane, Trevor, and all the others I’ve worked with. Not only have you been great friends, but you’ve been a part of a lab culture where everyone helps each other, no questions asked. More importantly, you’ve helped create a lab where everyone knows they are cared for, and where individual success is synonymous with group success. I wouldn’t trade this group for any other lab group out there. Alex and Trevor you guys deserve special thanks, as you two have mentored me in the techniques necessary to complete my work. Thank you for being patient with me and for being amazing teachers. I hope one day I am as fit and strong as all of you guys. Just know, I’m not going to stop chasing.

TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iii

Acknowledgements ...... iv

Table of Contents ...... v

List of Symbols, Abbreviations and Nomenclature ...... vii

1 Introduction and Review of the Literature ...... 1

1.1 Cardiovascular Disease ...... 1

1.2 Prediction of CVD and Assessment of CVD Risk ...... 2

1.2.1 Arterial Stiffness ...... 2

1.2.2 Endothelial Dysfunction ...... 4

1.2.3 Assessment of Cardiac Structure and Function via Echocardiography ...... 7

1.3 Cigarette Smoking and CVD ...... 8

1.3.1 Cigarette Smoking and Arterial Stiffness ...... 9

1.3.2 Cigarette Smoking and Endothelial Dysfunction ...... 10

1.3.3 Cigarette Smoking, Cardiac Morphology, and Cardiac Function ...... 12

1.4 Cannabis and the Cardiovascular System ...... 14

1.4.1 Similarities Between Cannabis Use and Cigarette Smoking ...... 15

1.4.2 Acute Effects of Cannabis Use on Cardiovascular Physiology ...... 17

1.4.3 Chronic Effects of Cannabis Use on the Heart and Vasculature ...... 30

2 Statement of Problem ...... 36

2.1 Rationale ...... 36

2.2 Purpose ...... 36

2.3 Hypotheses ...... 36

3 Original Investigation ...... 37

3.1 Candidate’s Contribution ...... 37

3.2 Manuscript ...... 38

4 Interpretation of Findings, Summary, and Conclusions ...... 72

4.1 Arterial Stiffness ...... 72

4.2 Endothelial Function ...... 76

4.3 Cardiac Morphology and Function ...... 80

4.4 Limitations and Future Directions ...... 84

4.5 Conclusions...... 86

References ...... 87

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LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE

Body Mass Index: BMI

Body Surface Area: BSA

Cannabinoid Receptor 1: CB1

Cannabinoid Receptor 2: CB2

Cannabis User: CU

Carbon Dioxide: CO2

Carbon Monoxide: CO

Cardiovascular Disease: CVD

Delta-9-Tetrahydrocannabinol: THC

Diastolic Blood Pressure: DBP

Early to Late Transmitral Filling Velocity Ratio: E:A

Electrocardiogram: ECG

Flow-Mediated Dilation: FMD

Interventricular Septal Wall Thickness during Diastole: IVSd

Left Ventricle: LV

Maximal Voluntary Contraction: MVC

Mean Arterial Pressure: MAP

Mean Mitral Annular Peak Early Velocity: E’

Mean Mitral Annular Peak Late Velocity: A’

Mean Mitral Annular Peak Systolic Velocity: S’

Nitric Oxide: NO

Nitric Oxide Synthase: eNOS

Non-Cannabis User: NU

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Peak Early Transmitral Filling Velocity: E

Peak Late Transmitral Filling Velocity: A

Polycyclic Aromatic Hydrocarbons: PAHs

Posterior Wall Thickness during Diastole: LVPWd

Pulse Wave Velocity: PWV

Reactive Oxygen Species: ROS

Right Ventricle: RV

Shear Rate Area Under the Curve: SR AUC

Systolic Blood Pressure: SBP

Tetrahydrocannabivarin: THCV

Total Peripheral Resistance: TPR

Tricuspid Annular Plane Systolic Excursion: TAPSE

1 1 Introduction and Review of the Literature

2 1.1 Cardiovascular Disease

3 Cardiovascular disease (CVD) can be broadly defined as any pathology of the

4 heart and/or the vasculature. Most recent data suggests that annually, between 2007

5 and 2017, CVD represented the greatest cause of death in the world,1 and was

6 responsible for 330 million years of life lost in 2017 alone.2 The etiology of CVD is

7 therefore of great interest, and has inspired large clinical trials such as the Framingham

8 Heart Study and the CARDIA study. Results from these trials and others, have

9 established the relationship between modifiable lifestyle factors and the risk of

10 development of CVD.3–5 Use of tobacco is among the most detrimental lifestyle factors

11 to cardiovascular health. For example, cigarette smoking increases the risk of stroke,6

12 myocardial infarction,7 coronary artery disease,8 and accelerates the progression of

13 atherosclerosis.9 Although the mechanisms underlying the contribution of cigarette

14 smoking to cardiovascular disease are not completely understood, cigarette smoking is

15 associated with arterial stiffening,10 dysfunction of the vascular endothelium,11 and

16 alterations in cardiac function and morphology12; all of which are risk factors for the

17 development of various CVDs. Around the world cannabis is widely used, currently

18 making it the most commonly used recreational drug apart from alcohol.13 Plants of the

19 Cannabis genus are often smoked, similar to cigarette products. Despite the

20 commonality of its use, and shared methods of consumption with cigarette smoking, the

21 link between cannabis smoking and CVD risk in humans is not known. Furthermore,

22 there is limited knowledge regarding how cannabis smoking chronically alters

23 cardiovascular function, and how it may contribute to CVD. The aim of this thesis is to

24 examine whether young apparently healthy habitual cannabis users demonstrate

25 impairments in cardiovascular function, suggestive of greater CVD risk, compared to

26 young apparently healthy non-users.

27 1.2 Prediction of CVD and Assessment of CVD Risk

28 Epidemiological data has identified clinical measures that provide value for

29 assessing general CVD disease risk, such as blood pressure and blood lipid

30 profiles.14,15 While these measures have substantial prognostic value, they are non-

31 specific indicators of cardiovascular function, in that they do not assess the function of

32 the heart or vasculature individually. Specific functional and/or structural alterations to

33 the heart and vasculature often represent key events in the development of CVD, and

34 manifest before traditional clinical measures distinguish compromised, or sub-optimal,

35 cardiovascular health. Thus, tests that directly assess the structure and function of the

36 heart and vasculature can identify subclinical differences in cardiovascular function.

37 These tests may bare particular importance in evaluating the early effects of a given

38 lifestyle factor on cardiovascular health in young, seemingly healthy individuals.

39 Previous work has identified the importance of arterial stiffness, vascular endothelial

40 function, and cardiac function in assessing CVD disease risk. In this thesis each of

41 these areas, and the methods used to assess them, will be highlighted.

42 1.2.1 Arterial Stiffness

43 The aorta is one of the most important arteries in the body because all of the

44 blood from the heart must pass through this large conduit vessel before reaching the 2

45 rest of the peripheral systemic circulation. One of the key features of this artery is its

46 elastic nature, resulting in high compliance compared to other arteries. The high

47 compliance and distensibility associated with the elasticity of the aorta enables pressure

48 dampening during systole and consistent flow to more peripheral muscular arteries

49 within each cardiac cycle.16 This elastic characteristic also reduces central systolic

50 pressure and thereby minimizes the afterload of the left ventricle (LV).16 Stiffening of the

51 aorta inherently reduces the vessel’s elastic characteristic and therefore subjects the LV

52 to greater central aortic pressures, which can lead to development of adverse

53 concentric LV remodeling and reduced systolic function.17,18 Therefore, the elastic

54 nature of the aorta is an important structural feature in the vessels role in optimal

55 cardiovascular function.

56 A highly consistent finding across studies is increased aortic stiffening and

57 reduced compliance with age. This stiffening process is largely attributed to collagen

58 deposition and loss of elastin in the arterial wall, arterial wall thickening, and alterations

59 in tonic vascular tone.19 Certain lifestyle factors have a significant impact on the natural

60 stiffening of the aorta that occurs with aging. For example, age-associated arterial

61 stiffening is attenuated with greater levels of physical activity20 and is accelerated in

62 obese individuals, possibly secondary to increases in the baseline inflammatory

63 profile.21,22 Due to the clear functional significance of the elasticity of the aorta and the

64 relationship between age associated stiffening and different lifestyle factors related to

65 CVD risk, arterial stiffening has garnered interest as a predictor for CVD development.

66 The measurement of arterial stiffness has become largely accessible in research

67 and clinical settings due to the development of multiple non-invasive techniques. A

68 simple, non-invasive measure known as pulse wave velocity (PWV) can be used to

69 estimate stiffness of a specific vascular segment of interest. PWV involves the

70 calculation of the propagation velocity of a pressure wave across the vessel of interest.

71 This is done by measuring the distance between two measurement sites and inferring

72 the transit time of the pulse between sites. While these techniques can be applied using

73 many different capacitance vessels throughout the central and peripheral circulation,

74 carotid-femoral PWV (i.e. aortic stiffness) has emerged as the gold-standard measure,

75 largely because it is the best predictor of CVD risk and all-cause mortality.23 Using large

76 scale data sets in humans, it has been demonstrated that an increase in carotid-femoral

77 PWV of 1m/s is associated with a 15% greater risk of cardiovascular mortality and all-

78 cause mortality.24 Therefore, carotid-femoral PWV is a valuable tool in assessing aortic

79 stiffness and assessing CVD risk.

80 1.2.2 Endothelial Dysfunction

81 The innermost layer of cells of a human blood vessel is known as the vascular

82 endothelium.25 The endothelium has an integral role in regulation of the cardiovascular

83 system and is directly involved in the regulation of vessel diameter in response to

84 changes in blood flow via the release of vasoactive factors. The structural integrity and

85 ability of the endothelium to regulate diameter in response to hemodynamic stimuli is an

86 essential defense against the development of atherosclerosis.26 Furthermore,

87 endothelial dysfunction precedes key steps in the progression of atherosclerosis, such

88 as lesion and plaque development.27 Endothelial damage and subsequent endothelial

89 dysfunction is also heavily implicated in inflammatory processes that are critical to the

90 pathogenesis of atherosclerosis.28 Thus, assessment of the essential function of the

91 endothelium has garnered interest as a clinical measure of CVD risk.

92 A functional in vivo assessment of endothelial function in the brachial artery was

93 first put forth by Celermajer and colleagues29 in 1992 and has since become a widely

94 used technique. This test, known as flow-mediated dilation (FMD), assesses the ability

95 of the endothelium to induce vasodilation in response to an induced reactive hyperemia.

96 The most common reactive hyperemia FMD protocol involves a 1-minute baseline

97 measurement period, a 5-minute occlusion period, and a 3-minute post-occlusion

98 recovery period.30 Vessel diameter and blood velocity are measured throughout an

99 FMD test, with calculated post-occlusion shear rate considered as the stimulus, and

100 magnitude of vessel dilation relative to baseline diameter considered as the functional

101 outcome.

102 During reactive hyperemia, mechano-sensitive receptors on endothelial cell

103 membranes are stimulated by the change in the frictional shear force of blood that

104 accompanies increases in blood flow.31 This begins a signaling cascade resulting in the

105 generation of prostaglandins, endothelial-derived hyperpolarizing factor, and most

106 importantly nitric oxide (NO) by cyclooxygenase, cytochrome P450, and endothelial

107 nitric oxide synthase (eNOS) enzymes, respectively.32 These molecules then diffuse to

108 the interstitium, and subsequently into vascular smooth muscle cells, thereby reducing

109 intracellular calcium concentration leading to smooth muscle relaxation and vessel 5

110 dilation.33 As the endothelium releases multiple vasoactive factors after experiencing

111 shear force, test conditions such as occlusion duration and tourniquet location appear to

112 partially dictate which vasoactive factor drives vasodilation.34,35 The traditional FMD

113 protocol involves a 5 minute occlusion distal to the site of measurement, and NO is

114 considered the primary driver of vasodilation in these testing conditions. This is

115 supported by work that demonstrates that the FMD response to these conditions is

116 nearly completely abolished with competitive inhibition of eNOS.36 Furthermore, FMD is

117 reduced in situations where inactivation of endothelial NO by reactive oxygen species

118 (ROS) occurs.37 NO bioavailability is therefore tightly coupled to FMD in response to

119 these traditional testing conditions.

120 The clinical value of FMD as an assessment of endothelial function has become

121 of substantial interest for its potential predictive value of the development of CVD. FMD

122 is an independent predictor of cardiovascular events, with greater FMD responses being

123 inversely correlated with CVD risk.38 Furthermore, FMD has been correlated with

124 structural and functional outcomes strongly involved in the development of various

125 CVD’s in healthy non-diseased individuals, such as carotid-intima media thickness,

126 coronary microvascular function, coronary endothelial function, and PWV.39–41

127 Therefore, impairments in FMD consequent to subclinical endothelial dysfunction

128 represent an early indication of suboptimal cardiovascular health in non-clinical

129 populations.

130 1.2.3 Assessment of Cardiac Structure and Function via Echocardiography

131 The use of ultrasound imaging to assess myocardial structural and functional

132 parameters has become widespread in clinical and research settings since it was first

133 reported in the 1950s.42 Innovations in echocardiographic techniques have resulted in a

134 vast array of outcomes that can be captured during an exam. Structural elements of the

135 heart that can be assessed using echocardiography include quantification of LV, right

136 ventricle (RV), and atrial chamber sizes, and thickness of myocardial wall segments.

137 Outcomes such as atrial areas, LV mass and RV areas, allow for identification of

138 cardiac chamber abnormalities.43,44 Structural changes to the myocardium can be

139 associated with augmentation or impairment of systolic and/or diastolic function. Both

140 systolic and diastolic function can also be well characterized using B-Mode ultrasound

141 and Doppler ultrasound. B-Mode ultrasound allows visualization of the LV throughout

142 the cardiac cycle, and in combination with geometric transformations allows for

143 quantification of key measures of systolic function, such as ejection fraction. Doppler

144 ultrasound is commonly used for the measurement of the ratio between early and late

145 transmitral filling velocities (E:A), which describes diastolic function. These measures

146 are just two of numerous outcomes that describe systolic and diastolic function

147 obtainable from the combination of B-Mode and Doppler echocardiography.45

148 Recent innovations in echocardiographic techniques have expanded the range

149 of measures used by clinicians and researchers for the assessment of cardiac function.

150 Relevant innovations include techniques such as tissue Doppler imaging, which

151 involves direct measurement of tissue velocities throughout the cardiac cycle. Measures

152 derived from this technique, such as mean mitral annular peak early velocity (E’) and its

153 ratio to peak early transmitral filling velocity (E), have proven particularly useful in

154 expanding the range of measures assessing diastolic function. Two-dimensional

155 speckle tracking, another big step forward in ultrasonography capability, allows for

156 automated motion tracking to map tissue translocation in time and space, thus giving

157 information about its deformation throughout the cardiac cycle, often referred to as

158 cardiac mechanics. This provides detailed information about myocardial strain and

159 twisting patterns; aspects of which are related to either systolic or diastolic function.

160 Given its more recent introduction, it is not completely understood how sub-clinical

161 alterations in cardiac mechanics are implicated in the development of CVD.

162 Nonetheless, speckle-tracking technology provides a means of identifying subtle

163 differences in cardiac mechanics between different populations.

164 1.3 Cigarette Smoking and CVD

165 It is well established that tobacco use and cigarette smoking is a strong

166 independent risk factor for a range of CVD, including stroke,6 coronary artery disease,8

167 and myocardial infarction.7 Furthermore, smoking has been demonstrated to interact

168 with other CVD risk factors such as hypertension and serum lipid profiles, such that

169 CVD increases multiplicatively with each factor.46 The global burden of cigarette and

170 tobacco use is readily apparent, as annual population attributable deaths are predicted

171 to reach 7 million by 2030.47 Prior to the development of overt CVD, the negative

172 systemic effects of cigarette smoking are apparent in both the vasculature and the

173 heart, in otherwise healthy individuals.

174 1.3.1 Cigarette Smoking and Arterial Stiffness

175 Prior to the development of CVD in smokers, cigarette use results in structural

176 changes to the peripheral and central vasculature. Cigarette smoking results in transient

177 arterial stiffening48 and cigarette smokers have altered brachial artery pulse pressure

178 waveforms.49 Cigarette smokers consistently demonstrate greater augmentation index

179 (AIx), a measure of systolic wave reflection and LV afterload which is partially

180 contributed to by arterial stiffeness.50 This finding holds even when controlling for

51 181 confounders for AIx, such as heart rate. A causal relationship between cigarette

182 smoking and increased AIx is supported by a linear relationship between AIx and

51 183 cigarette consumption and a reduction in AIx with smoking cessation. Cigarette use

184 also appears to have an effect on arterial stiffness measured by peripheral PWV, as the

185 rate of age-associated arterial stiffening measured by brachial-ankle PWV is increased

186 in cigarette smokers.52 The relationship between arterial stiffening and smoking may be

187 partially explained by observations of increased intimal-media thickening within arterial

188 walls of smokers, similar to what is observed in hypertension.53 Cigarette smoking has

189 also been associated with altered autonomic function,54 and chronic sympathetic

190 activation in hypertensives,55 suggesting that increased vascular tone partially explains

191 increased arterial stiffness in cigarette smokers. It is important to note, however, that

192 although AIx is consistently elevated in smokers, PWV is not, with some investigations

193 demonstrating no differences between smokers and non-smokers in middle-aged or

194 younger populations.56–58

195 Although some adverse vascular outcomes, such as elevated AIx, are

196 consistently shown to occur in cigarette smokers, other more traditional risk factors for

197 CVD, such as brachial blood pressure, are not different between young healthy smokers

198 and non-smokers.59 This may be explained in part by elevated aortic systolic pressures,

199 independent of brachial pressures, in smokers secondary to increased arterial

200 stiffness.48 Thus, arterial stiffness may represent an important early step in the

201 pathogenesis of CVD brought upon by cigarette smoking. Given this, and the fact that

202 aortic stiffness is also an established independent predictor of CVD in asymptomatic

203 individuals,24 measurements of arterial stiffness provide valuable insight into the degree

204 of smoking related decrements in cardiovascular function in healthy individuals prior to

205 the manifestation of CVD.

206 1.3.2 Cigarette Smoking and Endothelial Dysfunction

207 Cigarette use increases the risk of atherosclerosis.9 Prior to the development of

208 the FMD protocol, it was understood that endothelial dysfunction was an early-stage

209 occurrence involved in the development of atherosclerosis, as in vitro evidence

210 suggested that endothelial dysfunction presented before plaque development in the

211 arterial wall.27 Thus, the effects of smoking on FMD have been of interest since the

212 inception of the test. The original investigation revealed that FMD was reduced in

213 smokers compared to healthy individuals.29

214 Subsequent investigations into the effects of cigarette smoking on FMD provide

215 further evidence for a causal, dose-response relationship between cigarette smoking

216 and impairments in FMD. Volume of cigarette use, measured by pack years, has been 10

217 inversely related to FMD using multivariate linear regression60 and heavy smokers

218 demonstrate greater impairments in FMD than moderate smokers.61 The effects of

219 smoking on FMD appears to occur regardless of whether traditional or “light” cigarettes

220 are most often consumed,62 suggesting that the volume of smoking is a more important

221 variable than are the more subtle differences in cigarette composition. Importantly,

222 smokers do not demonstrate differences in endothelial-independent vasodilation,29,63 or

223 concentrations of vasoconstrictor endothelin-164; directly implicating the endothelium

224 and NO bioavailability as the points of compromised physiological function.

225 There likely exist multiple mechanisms explaining impaired FMD in chronic

226 smokers. An acute bout of cigarette smoking increases circulating endothelial cell

227 concentration, suggesting damage to the endothelium.65 ROS are also strongly

228 implicated in impairments of FMD in smokers. Smokers have been shown to have

229 greater circulating products of lipid peroxidation66,67 and a greater oxidized to reduced

230 glutathione ratio.62 This oxidative stress likely occurs due to acute increases in

231 superoxide anions generated by cigarette smoke.68 Chronic increases in ROS result in

232 reduced NO bioavailability in smokers, and consequently a compromised FMD

233 response to reactive hyperemia. Reduced NO bioavailability due to chronic elevations of

234 ROS is supported as a mechanism by evidence of reconciled NO bioavailability in

235 former smokers.69 Improvements in NO bioavailability with smoking cessation are

236 functionally important as former smokers demonstrate lesser impairments in FMD

237 compared to current smokers.60,70

238 Although many studies demonstrate that FMD is impaired in smokers who have

239 presented in clinical settings, comparisons of young healthy smokers do not always

240 reveal differences in FMD compared to healthy controls.71–73 Given the dose-response

241 relationship that has been demonstrated with smoking and impairments in FMD it could

242 be that young smokers may have not yet accumulated sufficient cigarette usage to

243 develop endothelial dysfunction. Indeed, it appears that cumulative cigarette exposure

244 is an important factor in determining the presence of smoking-induced endothelial

245 dysfunction.

246 1.3.3 Cigarette Smoking, Cardiac Morphology, and Cardiac Function

247 The relationship between smoking and CVDs affecting the heart are well

248 established.7,8 However, studies have largely focused on prevalence of CVD in smokers

249 with less attention given to the sub-clinical alterations in cardiac function that precede a

250 CVD diagnosis. A number of cross-sectional studies have addressed this deficit by

251 performing echocardiography in young and middle-aged healthy smokers and non-

252 smokers. Although early studies demonstrated no differences between smokers and

253 non-smokers,74,75 later studies have revealed a number of morphological and functional

254 differences between these populations, suggesting that smoking exerts detrimental

255 effects on cardiac function. Of these studies, there is limited data to suggest that

256 smoking is associated with reductions in systolic function. Data from a middle-aged

257 population in the ECHO-SOL trial is the only evidence that suggests reductions in

258 systolic function, as current smokers had reduced ejection fraction compared to never

259 smokers, while former smokers had a lesser but still significant reduction compared to

260 never smokers.12 This trial also demonstrated a dose-response relationships between

261 reduced RV systolic function and cigarette smoking intensity and duration. In contrast to

262 measures of systolic function, studies have frequently demonstrated differences in

263 diastolic function between healthy smokers and non-smokers. Decrements in diastolic

264 function in smokers are supported by findings of reduced E,76–78 mitral inflow

265 deceleration time,79 greater peak late transmitral filling velocity (A),80 and a reduction in

266 the E:A ratio.76–78

267 Analysis of cardiac mechanics by tissue Doppler- and speckle tracking-derived

268 measures have yielded more data supporting the existence of subclinical cardiac

269 differences between healthy smokers and non-smokers. Studies employing tissue-

270 doppler imaging demonstrate reductions in E’,76,78,80 greater E:E’,12 reduced systolic

271 tissue velocity and reduced RV systolic and diastolic tissue velocities.80 Additionally,

272 altered cardiac mechanics observed in smokers include reduced diastolic longitudinal

273 strain rate,76,81 reduced systolic longitudinal strain rate,80 and both LV and RV global

274 longitudinal strain.78

275 Collectively, these studies demonstrate that there are few differences in LV

276 systolic function between healthy smokers and non-smokers prior to the development of

277 CVD. In contrast, healthy smokers appear to demonstrate reduced systolic and diastolic

278 RV function, LV diastolic function, and altered cardiac mechanics compared to healthy

279 age-matched non-smokers.

280 1.4 Cannabis and the Cardiovascular System

281 Recreational and medicinal cannabis products are derived from plants of the

282 Cannabis genus, and typically contain over 500 different chemical compounds,

283 including over 100 different phytocannabinoids.82 Phytocannabinoids are molecules

284 unique to the Cannabis genus. Typically, the most abundant phytocannabinoids within

285 recreational cannabis products are delta-9-tetrahydrocannabinol (THC) and cannabidiol

286 (CBD), the former of which is the main psychoactive component of cannabis.83 The

287 primary cannabinoid receptors in the human body are cannabinoid receptor 1 (CB1) and

288 cannabinoid receptor 2 (CB2). CB1 receptors are present in the brain, peripheral

289 nervous system, heart, and vasculature, while CB2 receptors are primarily expressed by

290 immune tissues.84–87 THC is a weak partial agonist of CB1 and CB2, while CBD does

291 not act at either receptor.82 The effects of chronic exposure to these cannabinoids, and

292 the role of these receptors in cardiovascular physiology has been studied in animal and

293 in-vitro models, however their role in human cardiovascular health is less studied.88

294 The epidemiology of CVD with cannabis use is also incompletely understood, and

295 our characterization of the alterations in CV physiology are far less developed than that

296 of cigarette smoking. Studies examining the relationship between CVD and cannabis

297 use have been equivocal.89–94 Cannabis use has been linked to increased risk of severe

298 cardiovascular events. The earliest reports linking cannabis use to increased

299 cardiovascular events suggested that acute cannabis use was associated with ~4.5

300 times increased risk of myocardial infarction in the hour following use.89 Cannabis use

301 has also been associated with increased risk of cerebrovascular events and heart

302 failure.90,91 The risk of developing certain cardiac arrhythmias has also been reported to

303 be greater in cannabis users.92

304 These associations between cannabis use and serious cardiovascular events

305 generally mirror the risks associated with cigarette use. Unlike cigarette use, however, a

306 recent investigation has identified lower prevalence of coronary heart disease in

307 cannabis users compared to non-users presenting with angina.93 Additionally the

308 CARDIA study found no associations between cannabis use and CVD.94 Notably,

309 controlled laboratory studies investigating how cannabis could alter measures of

310 subclinical cardiovascular function that may be altered prior to traditional clinical risk

311 factors for CVD are lacking. These studies are necessary to understand whether

312 cannabis negatively impacts the cardiovascular system and could help reconcile

313 equivocal findings from larger trials. Currently, a thorough understanding of how chronic

314 cannabis use impacts cardiovascular function in seemingly healthy individuals and what

315 mechanisms could underly a relationship with CVD disease risk remains elusive.

316 1.4.1 Similarities Between Cannabis Use and Cigarette Smoking

317 Both tobacco and cannabis are most commonly consumed via smoking; with

318 users inhaling the smoke produced from the combusted material. Compared to other

319 methods of cannabis consumption this common method of use yields high bioavailability

320 of THC. The efficiency of this consumption method and desire for psychoactive effects

321 associated with recreational use likely explains the popularity of smoking.95

322 Although cigarettes contain a number of compounds known to be detrimental to

323 human health, a substantial degree of the adverse cardiovascular effects of cigarette

324 smoking are attributable to products associated with the combustion requisite to

325 smoking. Both cigarette smoke and cannabis smoke contain polycyclic aromatic

326 hydrocarbons (PAHs), carbon monoxide (CO), and tar.96 Each of these compounds

327 presents a hazard to cardiovascular health. PAHs have been demonstrated to be

328 cardiotoxic in animal models,97 and to interfere with autonomic function,98 while chronic

329 exposure to PAHs has been associated with greater prevalence of CVD independent of

330 smoking status.99 CO exerts negative effects on both the heart and the vasculature. CO

331 exerts negative inotropic effects on the heart100 and reduces myocardial oxygen delivery

332 due to the strong affinity of CO for hemoglobin. Both theoretical and animal models

333 have also implicated CO for a role in atherogenesis. Arterial wall hypoxia likely occurs

334 with elevated blood CO,101 as does damage and monocyte recruitment to the

335 endothelium102; all of which contribute to the atherosclerotic process. Cigarette smokers

336 who inhale smoke into their lungs also experience greater rates of coronary heart

337 disease with increasing cigarette tar content.103

338 Traditional characteristics of cannabis and cigarette smoking behaviours may

339 also play an important role in determining the relationship between cannabis smoking

340 and cardiovascular health. Although cannabis is smoked less frequently in comparison

341 to cigarettes amongst users,104 cannabis is smoked with longer breath hold times and

342 greater puff volumes, both of which have been demonstrated to lead to greater tar

343 consumption and CO absorption.105,106 Collectively, these factors suggest that based on

344 smoking behaviours alone, cannabis smoking could be more harmful to cardiovascular

345 health compared to cigarettes. Consideration must be given to the comparison of

346 cigarette contents and the chemical composition of cannabis. While numerous

347 hazardous chemicals have been clearly identified in cigarettes,107 a comprehensive

348 understanding of the effects of compounds within cannabis, and thus potential dangers

349 of such compounds, is still lacking.

350 Cannabis and cigarette smoking share important similarities in consumption

351 method which may implicate cannabis as being similarly hazardous to cardiovascular

352 health. Indeed, certain traditional smoking behaviours associated with cannabis use

353 such as breath hold times and puff volume suggest harm may actually be amplified in

354 cannabis users compared to cigarette users with equivalent exposure.104–106 A more

355 complete understanding of the mechanistic actions of the many compounds within

356 cannabis is needed to fully compare the health risks of cannabis to cigarette products.

357 1.4.2 Acute Effects of Cannabis Use on Cardiovascular Physiology

358 The acute effects of cannabis use on human cardiovascular physiology have

359 been examined in studies involving smoked cannabis and isolated THC. THC is

360 typically the most abundant cannabinoid within dried cannabis flower, and has thus

361 been researched extensively.108 Therefore, the current understanding of the acute

362 effects of cannabis use on cardiovascular physiology rely largely on studies of the

363 effects of isolated THC. The existing body of literature examining the acute effects of

364 cannabis and isolated THC on cardiovascular physiology are summarized in Table 1.

365 The effects of THC on the cardiovascular system were first studied in 1967 in a

366 work examining the psychological and physiological effects of both orally administered

367 and smoked THC.109 This was the first study to report that THC acutely induced sinus

368 tachycardia following consumption. Tachycardia has since become a near universal

369 finding in studies administering isolated THC or cannabis to healthy users, and this

370 effect has been determined to occur in a dose-response manner.110 This effect also is

371 not dependent on sex, and occurs regardless of menstrual cycle phase.111 Other early

372 investigations consistently reported supine systolic and/or diastolic pressure blood

373 pressure to be moderately increased by oral THC.112,113 In contrast, reductions in

374 upright blood pressure and orthostatic hypotension occur with acute doses, suggesting

375 that the effects of cannabis and THC on blood pressure are posture dependent.114 Early

376 studies also reported that smoked cannabis increased calf and forearm blood flow.115

377 Following early reports of cannabis and THC on heart rate and blood pressure,

378 subsequent investigations sought to understand the underlying mechanisms of these

379 effects. It had been reported that epinephrine concentration was increased following

380 THC consumption116 and that reflex responses to a Valsalva maneuver were impaired

381 following cannabis smoking.110 This raised the question of whether the cardiovascular

382 effects of cannabis and THC were mediated by autonomic nervous mechanisms.

383 Multiple studies115,117–120 performed sympathetic and parasympathetic blockades using

384 propranolol and atropine before cannabis or THC consumption to assess autonomic

385 contributions to the previously reported effects. These studies generally reported that

386 the THC and cannabis induced tachycardia could be reduced by pre-treatment with

387 propranolol while pre-treatment with atropine augmented both tachycardia and

388 hypertensive effects. Future work would demonstrate increases in circulating

389 norepinephrine following cannabis smoking, further implicating autonomic nervous

390 mechanisms.121 It should also be noted that these effects are also dependent upon

391 cannabinoid receptors, as blockade of CB1 also attenuates the cardiovascular effects of

392 cannabis and THC blockade.122–127

393 Further disruption of homeostatic cardiovascular function was evidenced by

394 development of electrocardiographic abnormalities with acute doses of THC, such as

395 premature ventricular contractions (PVC).112,128 These initial findings prompted

396 investigation into the direct effects of THC and cannabis on cardiac function. Cannabis

397 smoking was shown to alter systolic time intervals, specifically by increasing the length

398 of the pre-ejection period and left ventricular ejection time.118 Echocardiographic studies

399 have demonstrated somewhat equivocal findings regarding the effects of THC and

400 cannabis on cardiac function. Studies reported both reduced systolic function,129 and

401 augmented left ventricular tissue velocity.121,130

402 Only a small number of studies have assessed the functional impact of the acute

403 cardiovascular effects of cannabis. Two investigations demonstrated that patients with

404 coronary artery disease had earlier onset of angina during exercise after cannabis

405 smoking in comparison to placebo and cigarette smoking.131,132 In healthy participants,

406 cannabis use has been shown to reduce maximal work capacity on an incremental

407 exercise test.133,134 Acute cannabis use prior to exercise has also been shown to

408 increase heart rate at submaximal exercise intensities.135 Reduced myocardial oxygen 19

409 supply due to inhaled CO, in combination with greater rate-pressure product at a given

410 intensity, may explain the earlier onset to angina in clinical populations and reduced

411 work capacity in healthy individuals.

412 The acute effects of cannabis and THC on rudimentary hemodynamic measures

413 have been well investigated. However, it is less understood how cannabis and THC

414 interact with more specific aspects of cardiovascular function. It is, perhaps worth noting

415 that more than 75% of the work in this area was performed 40-50 years ago, most prior

416 to the 1980s. Considerable technological innovation has occurred since publication of

417 many of these seminal studies, leaving substantial knowledge gaps. There are currently

418 no studies which have examined how cannabis acutely affects direct measurements of

419 sympathetic or parasympathetic activity, arterial stiffness, or endothelial function.

420 Furthermore, there are no studies examining cardiac function following cannabis use

421 that have maximized the range of measurements that can be obtained with modern

422 echocardiographic techniques. Future studies should work to advance our

423 understanding of interactions between cannabis, THC, and the cardiovascular system.

424 Table 1. Summary of studies examining the acute effects of cannabis or isolated THC on cardiovascular physiology in

425 human participants.

Study Methodology Dosage Primary Outcomes General Findings Conclusion

Oral: THC elicited no changes in blood pressure, but Measured brachial blood pressure and 120/480mcg/kg THC induces tachycardia Isbell et al. Heart Rate increased heart rate in heart rate prior to and following THC when smoked and 1967109 both conditions. Heart smoked and oral THC administration. Brachial Blood Pressure consumed orally. Smoked: rate increases were 50/200mcg/kg THC greatest after cannabis.

Epinephrine was Semi-acute Measured plasma cortisol and Hollister et al. significantly elevated two cardiovascular response catecholamine concentrations Oral: 15-70mg THC Plasma Epinephrine 1970116 hours post-THC to THC may involve following oral THC administration. consumption. epinephrine.

Heart rate increased in a Smoked cannabis Measured heart rate in experienced Smoked: 62.5- dose-response manner. induces tachycardia in a Renault et al. and non-experienced smokers during 435mg Cannabis Heart Rate Heart rate slowing during dose-response manner 1971110 Valsalva maneuver after smoking (1.5% THC) Valsalva was attenuated and interacts with cannabis. after cannabis smoking. Valsalva reflex.

Heart rate increased in a Smoked cannabis Performed 3-lead ECG and measured Heart Rate dose-response manner Smoked: 300mg induces tachycardia in a Johnson and blood pressure and heart rate with THC. Slight increase Cannabis (2.9% Blood Pressure dose-response manner Domino 1971112 following smoking cannabis of varying (<10mmHg) in supine THC) and increases supine THC content. SBP and DBP. Observed ECG blood pressure. PVCs.

Both cannabis and Smoked: 500mg placebo increased heart THC induced tachycardia Martz et al. Pre-treated participants with Cannabis (25μg/kg Heart Rate rate. Propranolol is at least partially beta- 1972117 propranolol prior to smoking cannabis. THC) prevented tachycardia in adrenergic mediated. both cases.

Heart rate, forearm blood flow, and calf blood flow increased following smoking. Propranolol Pre-treated participants with either Limb Blood Flow Both parasympathetic Smoked: 1g prevented rises in blood Beaconsfield et propranolol, atropine, epinephrine, or and sympathetic nervous Cannabis (10mg Heart Rate flow and heart rate. al. 1972115 norepinephrine and assessed reflex systems are implicated in THC) Atropine augmented heart responses after smoking cannabis. THC-induced tachycardia. Blood Pressure rate response. Cold pressor and mental stress reflexes were suppressed.

Supine blood pressure Heart Rate and heart rate increased Blood Pressure after THC administration. THC reduced upright THC increases heart rate Measured forearm blood flow, venous Forearm Blood Flow blood pressure and and blood pressure and Weiss et al. tone, urinary catecholamines, and induced orthostatic Oral: 0.3mg/kg THC Forearm Conductance induces peripheral 1972114 systolic time intervals following hypotension. Forearm vasodilation. THC also administering oral THC or placebo. blood flow and Venous Tone interferes with reflexes. epinephrine concentration Systolic Time Intervals increased after THC. Reflex venoconstriction Urinary Catecholamines was reduced after THC.

THC induces tachycardia Used a parallel group design to Intravenous: 10mg Tachycardia was Perez-Reyes et whether delivered as examine cardiovascular responses to THC or THC Heart Rate observed in both al. 1972136 active metabolite or THC or an active THC metabolite. metabolite conditions. precursor.

Measured ECG following infusions of Kochar and Intravenous: 200- Heart Rate Tachycardia observed in Low and high doses of high and low doses of THC in regular Hosko 1973128 400μg/kg both conditions. THC induce tachycardia. cannabis smokers. ECG

Tachycardia occurred Smoked: 1g Cannabis smoking does Roth et al. Measured ECG following cannabis Heart Rate following smoking. No Cannabis (20mg not cause ECG 1973137 smoking. ECG changes following THC) ECG abnormalities. smoking.

Heart Rate Heart rate and venous Performed brachial artery pulse wave pressure increased post analysis to determine dp/dt and Smoked: 600mg Cannabis smoking has Clark et al. Peripheral dp/dt smoking. Increases were measured blood flow during Valsalva Cannabis (1.5% sympatho-excitatory 1974113 observed in dp/dt, DBP, and cold pressor test after smoking THC) Intravascular Pressure effects. blood flow, and arterial cannabis or placebo. Limb Blood Flow resistance.

Exercise time to angina Heart Rate was reduced in both Performed exercise testing following conditions, but more so Cannabis reduces 10 “Puffs” of Aronow and cannabis or placebo smoking in Blood Pressure after cannabis smoking. exercise capacity in cannabis cigarette Cassidy 1974138 patients with coronary artery disease Rate pressure product patients with coronary (19.8mg THC) Venous Carboxyhemoglobin and stable exertional angina. increased in THC artery disease. Exercise Time to Angina condition due to tachycardia.

Exercise time to angina reduced in both Performed exercise testing following Smoked: 10 “Puffs” Heart Rate conditions, but more so Cannabis reduces Aronow and cannabis or cigarette smoking with of cannabis after cannabis smoking. exercise capacity in Blood Pressure Cassidy 1975132 patients with coronary artery disease cigarette (19.8mg Rate pressure product patients with coronary and stable exertional angina. THC) Exercise Time to Angina increased in THC artery disease. condition due to tachycardia.

ECG Measured ECG, blood pressure, and THC increased heart rate THC induces tachycardia Karniol et al. heart rate after oral THC and/or Oral: 25mg THC Heart Rate but had no effect on blood but does not affect blood 1975139 cannabinol administration. pressure. pressure. Blood Pressure

Ventilation Tachycardia and Heart Rate increased cardiac output Administered THC to individuals who Cannabis increases Johnstone et al. Intravenous: 27- were observed following had been anaesthetized prior to oral Blood Pressure cardiac output through 1975140 134μg/kg THC THC administration. A surgery. tachycardia. Cardiac Output reduction in TPR was observed following THC. Total Peripheral Resistance

Sensitivity of ventilatory Measured ventilatory responses to Ventilation Cannabis alters Malit et al. Intravenous: 135- drive to CO2 was reduced CO2 and hemodynamics following regulatory mechanisms of 1975141 201μ/kg THC and heart rate was intravenous THC administration. Heart Rate ventilation. increased following THC.

Heart Rate Cannabis smoking Performed maximal exercise testing in Smoked: 1.4g induced tachycardia, Steadward and Cannabis reduces healthy individuals after smoking either Cannabis (18.2mg Blood Pressure increased blood pressure, Singh 1975133 exercise capacity. cannabis or placebo. THC) and reduced physical Physical Work Capacity work capacity.

Heart rate, SBP, and DBP increased after smoking Smoked: 10 “Puffs” Heart Rate Cannabis reduces Performed echocardiography in a cannabis. End diastolic Prakash et al. of cannabis diastolic and systolic double-blind fashion after individuals Blood Pressure volume, ejection fraction, 1975129 cigarette (18mg function, but effects are smoked cannabis or placebo. and stroke index reduced THC) likely related to smoking. Echocardiographic Measures after smoking placebo and cannabis.

Tachycardia occurred post THC before chronic Measured heart rate, blood pressure, dosing. Cold pressor test and orthostatic tolerance after Oral: 0-30mg THC responses were impaired. Cannabis induces Benowitz and Heart Rate 142 administering THC in increasing doses Smoked: Cannabis Supine blood pressure tachycardia and interferes Jones 1975 Blood Pressure every 4 hours to inpatients for up to 21 (20mg THC) decreased regardless of with reflexes. days. dosing phase. Orthostatic hypotension was reported following THC.

Tachycardia occurred after smoking cannabis Measured ECG, heart rate, and blood ECG Cannabis induces and blood pressure was Bernstein et al. pressure daily during a 21 day period tachycardia and has Not Reported Heart Rate variable but largely 1976143 where participants could smoke variable effects on blood unchanged. No ECG cannabis ad libitum. pressure. Blood Pressure abnormalities were reported.

Effects of THC on blood Administered THC to individuals who Hypotension and Gregg et al. Intravenous 0.022- Heart Rate pressure and heart rate had been anaesthetized prior to oral tachycardia occurred 1976144 0.044mg/kg THC are not affected by surgery. Blood Pressure following THC. anesthetic.

Heart Rate Pre-ejection period and Measured systolic time intervals LV ejection time were Cannabis effects the Kanakis et al. Intravenous: 25μ/kg following THC administration with and Blood Pressure increased with THC. heart through beta- 1976118 THC without pre-treatment of propranolol. Propranolol attenuated adrenergic mechanisms. Systolic Time Intervals these efects.

Stroke volume decreased, heart rate increased, and cardiac output was unchanged Measured cardiac function and cardiac Cardiac Output Smoked: 900mg after smoking. Pre- Tashkin et al. output using echocardiography and Cannabis smoking alters Cannabis (2.2% Echocardiographic Measures ejection period and LV 1977130 dye-dilution following cannabis cardiac tissue velocities. THC) ejection time were smoking. Systolic Time Intervals unchanged, but circumferential fiber shortening velocity was increased.

Neither response to beta- or alpha adrenergic Pre-treated participants with Parasympathetic activity stimulation was altered by Benowitz and isoproterenol, phenylephrine, atropine, Heart Rate likely counters pressor Oral: 10mg THC THC. Blood pressure Jones 1977119 or propranolol prior to THC responses to oral THC Blood Pressure increases after THC were administration. consumption. exaggerated with prior atropine treatment.

Sinus Length Recorded electrograms of the bundle THC alters the Miller et al. of his and right atrium, and performed Intravenous: Sinus Recovery Time THC reduced conduction electrophysiology of the 1977145 atrial pacing before and after THC 25μg/kg THC and refractory times. Sinoatrial Conduction Time heart. administration. A-H Interval

Cannabis increases heart Smoked: Cannabis Propranolol prevented Pre-treated participants with rate and blood pressure Sulkowski and (No total dose Heart Rate THC induced increases in propranolol prior to THC or placebo at least partially through Vachon 1977146 amount reported, heart rate and blood treatment. Blood Pressure beta-adrenergic 10mg THC) pressure. mechanisms.

Velocity of circumferential Performed echocardiography and Smoked: Cannabis fiber shortening was Cannabis smoking likely Gash et al. measured plasma norepinephrine (No total dose Plasma Norepinephrine increased with cannabis increases sympathetic 1978121 concentrations after smoking amount reported, smoking. Norepinephrine Echocardiographic Measures activity. cannabis. 2.1% THC) was elevated 30 and 120 minutes after smoking.

Measured heart rate and blood Smoked: 217- Neither systolic nor Cannabis smoking has no Pihl et al. 1978147 pressure before and after smoking low 400mg Cannabis Blood Pressure diastolic blood pressure effect on blood pressure. dose, high dose, or placebo cannabis. (1.5% THC) was affected by smoking.

Heart rate response to Heart Rate exercise was Performed submaximal exercise Smoked: 500mg Cannabis accelerates Avakian et al. exaggerated following testing following smoking cannabis or Cannabis (1.54% Blood Pressure heart rate during 1979135 smoking. Blood pressure placebo THC) submaximal exercise. Submaximal Exercise response to was not altered.

Blood pressure, heart rate, and forearm blood flow increased following Pre-treated participants with Heart Rate THC. Individual Cannabis acts through Benowitz et al. propranolol, atropine, or both atropine Intravenous: blockades did not parasympathetic and Blood Pressure 1979120 and propranolol prior to THC 30μg/kg THC attenuate heart rate or sympathetic nervous administration. Forearm Blood Flow blood pressure effects. All mechanisms. blockades prevented increases in forearm blood flow.

Combined blockade Pre-treated participants with Heart Rate Cannabis acts through completely ablated all Kanakis et al. propranolol and atropine prior to THC Intravenous: parasympathetic and Blood Pressure effects of THC on heart 1979148 administration and measuring systolic 25μg/kg sympathetic nervous rate, blood pressure, and time intervals. mechanisms. Systolic Time Intervals systolic time intervals.

Smoking cannabis and Measured heart rate in never, Oral: 17.5mg THC Cannabis and THC oral THC induced Mendelson and intermediate, or experienced cannabis induce tachycardia in Smoked: 1g Heart Rate tachycardia in Mello 1984149 smokers following cannabis, oral THC, familiar individuals but not Cannabis (1.83% intermediate users and or placebo administration. naïve individuals. THC) experienced users.

Smoking cannabis Cannabis induced Measured heart rate after smoking Smoked: Cannabis induced tachycardia tachycardia occurs cannabis or placebo in women in the (No total dose Lex et al. 1984111 Heart Rate regardless of menstrual independently of luteal, follicular, and ovulatory phases amount reported, phase and previous menstrual cycle phase of the menstrual cycle. 1.8% THC) smoking experience. and smoking experience.

Submaximal intensities elicited greater heart Cannabis smoking Time to Exhaustion rates after cannabis Smoked: 7mg/kg reduces maximal exercise Renaud and Performed maximal exercise testing smoking. Exercise time Cannabis (1.7% Heart Rate performance through Cormier 1986150 after administration of THC or placebo. was reduced following THC) accelerated heart rates at cannabis smoking. No Oxygen Consumption submaximal intensities. difference was observed in oxygen consumption.

Heart rate increased with Smoked: Cannabis Heart rate was measured after non-placebo cannabis Kelly et al. (No total dose Smoking cannabis smoking cannabis of varying THC Heart Rate and following smoking 1993151 amount reported, 2- induces tachycardia. concentrations. both 2.0 and 3.5% THC 3.5% THC) cannabis.

Administered oral treatments of Smoked: Cannabis Tachycardia induced by Tachycardia induced by Huestis et al. varying doses of selective cannabinoid (No total dose THC was 60% attenuated Heart Rate cannabis smoking is 2001122 receptor antagonist SR141716 prior to amount reported, by higher doses of partially CB1 mediated. smoking cannabis 2.64% THC) SR141716.

Cannabis did not consistently induce hypotension. In those Administered oral treatments of Smoked: 764mg CB1 is implicated in Gorelick et al. who experienced rimonabant, a CB1 receptor antagonist Cannabis (2.64% Blood Pressure hypotensive response 2006123 hypotension with prior to smoking cannabis. THC) only in some individuals. smoking, hypotension was attenuated with rimonabant.

Administered rimonabant over a 15- Cannabis induced Smoked: 764mg Tachycardia induced by Huestis et al. day dosing schedule, and tachycardia was Cannabis (2.78% Heart Rate cannabis smoking is 2007124 subsequently assessed tachycardic attenuated following THC) partially CB1 mediated. responses to THC. rimonabant treatment.

Measured heart rate in experienced THC induced tachycardia THC induces tachycardia D’Souza et al. Intravenous: 0-5mg cannabis users and non-users after Heart Rate in a dose-dependent in a dose-dependent 2008152 THC administration of intravenous THC. manner. manner.

Smoking cannabis Measured heart rate after smoking Smoked: 800mg All preparations of Cooper and induces tachycardia cannabis in different cigarette Cannabis (1.8-3.6% Heart Rate cannabis induced Haney 2009153 regardless of cigarette preparations. THC) tachycardia. preparation.

Tachycardia occurred in a Administered oral treatments of dose-response manner Tachycardia induced by Zuurman et al. AVE1625, a CB1 receptor antagonist Inhaled: THC 2-6mg Heart Rate with THC. AVE1625 cannabis smoking is 2010125 prior to administering varying doses of completely attenuated partially CB1 mediated. THC. THC induced tachycardia.

THC induced tachycardia was inhibited in Administered oral treatments of Tachycardia induced by Klumpers et al. Heart Rate proportion to surinabant surinabant, a CB1 receptor antagonist Inhaled: THC 2-6mg cannabis smoking is 2013126 dose. Blood pressure prior to varying doses of THC. Blood Pressure partially CB1 mediated. remained unchanged by THC.

THC induced tachycardia Administered THCV for 5 days, a was inhibited with chronic Tachycardia induced by Englund et al. phytocannabinoid CB1 receptor Intravenous; THC Heart Rate THCV treatment. cannabis smoking is 2016127 antagonist prior to administration of 1mg Hypotensive effects of Blood Pressure partially CB1 mediated. THC or placebo. THC persisted regardless of condition.

426 Abbreviations: Carbon Dioxide (CO2), Delta-9-Tetrahydrocannabinol, Diastolic Blood Pressure (DBP), Electrocardiogram

427 (ECG), Left Ventricle (LV), Premature Ventricular Contraction (PVC), Systolic Blood Pressure (SBP),

428 Tetrahydrocannabivarin (THCV), Total Peripheral Resistance (TPR)

429 1.4.3 Chronic Effects of Cannabis Use on the Heart and Vasculature

430 In comparison to transient physiological changes, the effects of chronic use of

431 cannabis on cardiovascular function are much less studied. There has only been limited

432 study on chronic effects of cannabis use on rudimentary cardiovascular outcomes such

433 as blood pressure, and more specific outcomes such as cardiac function, morphology,

434 and arterial stiffness. Despite a large body of literature revealing detrimental effects of

435 cannabinoids and related mechanisms on endothelial cells in animal models and in

436 vitro,88 there have been no studies that assess the effects of chronic cannabis use on

437 endothelial function in human participants. The existing body of literature examining the

438 chronic effects of THC and cannabis use on human cardiovascular physiology is

439 summarized in Table 2.

440 Controlled laboratory studies have attempted to understand short-term adaptive

441 cardiovascular responses to chronic cannabis smoking or chronic THC consumption.

442 These studies have utilized dosing periods ranging from as short as 6 days154 to as long

443 as 94 days.134 The time period selected for these studies appears to be an important

444 indicator of whether or not tolerance to the acute effects of THC are observed. Studies

445 employing dosing periods less than 12 days tend to reveal no change in tolerance,154,155

446 while longer studies consistently demonstrate attenuations in THC induced tachycardia

447 and orthostatic hypotension with prolonged exposure.119,134,142,156 In addition to the

448 development of tolerance to the acute effects of THC and cannabis, resting

449 hemodynamics have also been reported to be altered with chronic THC dosing.

450 Benowitz and Jones142 reported reductions in resting systolic blood pressure, diastolic

451 blood pressure, and heart rate with chronic oral THC. These effects likely represent

452 residual drug effects rather than permanent cardiovascular adaptations, as these

453 measures return to baseline levels upon cessation of THC use. In contrast to these

454 short-term effects, a retrospective analysis of national survey data in the United States

455 revealed that current cannabis users had greater prevalence of hypertension compared

456 to non-users, but the influence of other lifestyle factors cannot be ruled out, and the fact

457 that cannabis remains an illicit substance must be considered.157 While pseudo-

458 longitudinal laboratory-controlled studies provide proof-of-concept that cannabis and

459 THC can exert chronic effects on the cardiovascular system, further studies are

460 necessary to disentangle residual drug effects and physiological adaptations to

461 cannabis use.

462 There are currently no controlled laboratory studies that have examined the

463 chronic effects of cannabis use on cardiovascular function. The best assessments of

464 potential cardiovascular effects to date include a retrospective analysis of a large

465 cardiac MRI database158 and an ecological study which performed radial artery

466 tonometry to indirectly examine aortic pressure wave reflection properties and vascular

467 age in patients admitting to an addictions treatment clinic.159 The former investigation

468 revealed that cannabis users present with different morphological and functional cardiac

469 traits than non-users. Cannabis users demonstrated greater end diastolic volume, end

470 systolic volume, and stroke volume of both the LV and RV, greater LV mass, and

471 reduced global circumferential strain. After multivariable adjustment and adjustment for

472 age and sex alone only greater end diastolic volume, greater end systolic volume, and

473 reduced global circumferential strain remained significant. This investigation was cross-

474 sectional and therefore cannot identify a causal relationship between cannabis use and

475 these outcomes, however it does provide preliminary evidence in support of altered

476 cardiac morphology and function associated with chronic cannabis use. Reece and

477 colleagues demonstrated a number of differences in the vasculature of cannabis users

478 compared to tobacco users, opioid users, and concomitant tobacco and cannabis users.

479 Cannabis users demonstrated greater AIx and advanced vascular aging compared to

480 each of these groups. While these findings implicate cannabis use as particularly

481 detrimental to vascular health, it should be noted that this study has a number of key

482 limitations. Due to the observational nature of the study the acute behaviours of

483 participants were not controlled. Given that measures of arterial stiffness may be

484 significantly affected by a range of acute behaviours such as food and caffeine

485 consumption, and smoking,160 this design may have introduced significant confounds to

486 these measurements. Furthermore, this study included only 11 users of cannabis only.

487 Currently, there is a paucity of science examining the effects of chronic cannabis

488 use on human cardiovascular physiology. Preliminary evidence suggests that cannabis

489 use may indeed have chronic cardiovascular effects. Cross-sectional controlled

490 laboratory studies examining these effects and prospective longitudinal studies are

491 needed to further the understanding of cardiovascular adaptations associated with

492 cannabis use.

493 Table 2. Summary of studies examining the chronic effects of cannabis or isolated THC on cardiovascular physiology in

494 human participants.

Study Methodology Dosage Primary Outcomes General Findings Conclusion

Tachycardia and Administered cannabis smoke twice Smoked: 435mg No tolerance developed to Heart Rate orthostatic Renault et al. daily to participants to assess Cannabis tachycardia or orthostatic hypotension persist 1974155 tolerance to the effects of THC on (2.8%THC) twice hypotension induced by with chronic cannabis heart rate and orthostatic hypotension. daily for 10 days Blood Pressure cannabis smoking. use.

Resting blood pressure THC alters resting and heart rate were heart rate, blood reduced with prolonged pressure, and Administered oral THC every 4 hours THC use. Tolerance increases plasma Oral: Up to 210mg Heart Rate Benowitz and for up to 21 days. Assessed developed to THC induced volume chronically. THC Daily for 21 Jones 1975142 cardiovascular adjustments to standing tachycardia and orthostatic Tolerance develops to days and heart rate. Blood Pressure hypotension. A reduction in acute tachycardia and hemoglobin concentration orthostatic and increase in body hypotension with weight occurred. longer dosing periods.

Development of tolerance Smoked: Cannabis Tolerance to to THC induced Participants smoked cannabis ad- (Minimum 20mg Heart Rate tachycardia induced by tachycardia occurred. Cohen 1976134 libitum for 94 days with a minimum THC daily) Average cannabis smoking Physical work capacity was smoking requirement. daily THC exposure occurs with long Physical Work Capacity reduced with prolonged was 100mg. dosing periods. exposure.

Pre-treated participants with Propranolol slowing of Prolonged THC isoproterenol, phenylephrine, atropine, Oral: Up to 210mg Heart Rate Benowitz and heart rate was attenuated administration has and propranolol after 14 days of THC Daily for up to Jones 1977119 after prolonged THC residual sympatho- chronic THC dosing, prior to THC 12 days Blood Pressure administration. excitatory effects. administration.

Smoked: Cannabis Tolerance to Participants smoked ad-libitum for 94 (Minimum 20mg Development of tolerance tachycardia induced by Nowlan and days with a minimum smoking THC daily) Average Heart Rate to acute THC induced cannabis smoking Cohen 1977156 requirement. daily THC exposure tachycardia occurred. occurs with long was 100mg. dosing periods.

Ventilation Cannabis use is not No differences in any associated with Performed maximal exercise testing in Heart Rate physiological or Maksud and chronic effects on cannabis users, cigarette smokers, N/A performance outcome was Baron 1980161 exercise capacity or concomitant users, and controls. found between cannabis Oxygen Consumption whole-body responses smokers and non-smokers. to exercise. Work Capacity

THC induced tachycardic and hypotensive effects for Tolerance to Administered oral THC every 3.5-6 all 6 days of study. No Heart Rate cardiovascular effects Gorelick et al. hours for 6 days to prior to THC and tolerance was developed. Oral: 20mg THC of oral THC is not 2013154 assessed acute cardiovascular Baseline blood pressure developed in short responses to THC. Blood Pressure decreased by day 6 and dosing periods. baseline heart rate increased by day 6.

Cannabis users Cannabis users have experienced advanced accelerated arterial Performed radial artery tonometry in Vascular Age vascular aging and greater stiffening and greater Reece et al. cannabis users admitting to an N/A AIx compared to tobacco wave reflection 2016159 addiction clinic. Central Augmentation Index users, opioid users and augmentation users of tobacco and compared to users of cannabis. other substances.

Ventricular volumes, stroke Regular cannabis use Retrospectively assessed cardiac volume, and LV mass were is associated with Khanji et al. MRIs of cannabis users to examine greater in cannabis users. alterations to cardiac N/A Cardiac MRI Measures 2019158 chronic effects of cannabis on the Cannabis users also had chamber volumes, heart. lower global circumferential cardiac function, and strain. cardiac mechanics.

Cannabis users had a Retrospectively examined national higher prevalence of Cannabis use is survey data from the USA comparing Vidot et al. 2019157 N/A Blood Pressure elevated blood pressure, associated with higher blood pressure in cannabis users and stage one, and stage two blood pressure. non-users. hypertension.

No baseline differences in Regular cannabis use Assessed cardiovascular responses to Heart Rate blood pressure or is not associated with DeAngelis and mental stress and cold pressor test in N/A cardiovascular responses alterations to Al’Absi 2020162 cannabis users and non-users. Blood Pressure to stress were observed cardiovascular stress between groups. responses.

495 Abbreviations: Delta-9-Tetrahydrocannabinol (THC), Left Ventricle (LV), Right Ventricle (RV),

496 2 Statement of Problem

497 2.1 Rationale

498 Existing evidence suggests that cannabis has pronounced acute effects on the

499 cardiovascular system. Preliminary evidence suggests that there are also chronic

500 cardiovascular effects of habitual cannabis use. At present, it is not clear how chronic

501 cannabis use impacts arterial stiffness, endothelial function, or cardiac morphology and

502 function. Cannabis use shares similarities to cigarette use, an established detrimental

503 behaviour to each of these aspects of cardiovascular physiology. Understanding the

504 effects of cannabis on these outcomes is important given the widespread use of the

505 drug, and the importance of these outcomes in cardiovascular health.

506 2.2 Purpose

507 The purpose of this study was to quantify baseline arterial stiffness, endothelial

508 function, and both cardiac morphology and function in habitual cannabis users, while

509 comparing these outcomes to healthy age-matched controls.

510 2.3 Hypotheses

511 1) Arterial stiffness measured by PWV will be greater in habitual cannabis users

512 compared to age-matched controls.

513 2) Endothelial function measured using reactive-hyperemia FMD will be impaired in

514 habitual cannabis users compared to age-matched controls.

515 3) Cardiac morphology and function will be altered in habitual cannabis users

516 compared to age-matched controls.

517 3 Original Investigation

518 3.1 Candidate’s Contribution

519 The manuscript included in the present chapter of this thesis is formatted for

520 publication to an academic journal. The candidate was involved in study design, data

521 collection, data analysis, and manuscript writing. The contributions of the co-authors are

522 detailed at the end of the manuscript.

523 3.2 Manuscript

524 Title: Habitual Cannabis Use is Associated with Altered Cardiac Mechanics and

525 Arterial Stiffening, but not Endothelial Function in Young Healthy Smokers

526 Christian P. Cheung, Alexandra M. Coates, Philip J. Millar, Jamie F. Burr

527 Department of Human Health and Nutritional Sciences, University of Guelph, Guelph,

528 Ontario, Canada

529

530 Running Title: Cannabis Use, Arterial Stiffness, Endothelial Function and Cardiac

531 Function

532 Keywords: Flow-Mediated Dilation, Pulse Wave Velocity, Smoking, Marijuana,

533 Echocardiography

534

535 Corresponding Author:

536 Jamie F. Burr

537 University of Guelph

538 Department of Human Health and Nutritional Sciences

539 Guelph, ON, Canada, N1G 2W1

540 Email: [email protected]

541 Abstract

542 Purpose: Cigarette smoking is amongst the most detrimental behaviours to

543 cardiovascular health, and results in arterial stiffening, endothelial dysfunction, and

544 structural/functional alterations to the myocardium. Cannabis is the most commonly

545 used recreational substance in the world exclusive of alcohol and, similar to cigarettes,

546 is often smoked. Little is known about the long-term cardiovascular effects of cannabis.

547 This study explored the associations of cardiovascular form and function with cannabis

548 use. Methods: We cross-sectionally performed echocardiography and measured

549 carotid-femoral pulse wave velocity (PWV) and brachial flow-mediated dilation (FMD) in

550 18 young healthy cannabis users (CU) and 17 young healthy non-users of cannabis

551 (NU) Results: CU demonstrated reduced peak apical rotation compared to NU (CU:

552 5.5±3.8, NU: 9.6±1.5; p = 0.02). There were no differences in cardiac morphology or

553 traditional resting measures of systolic or diastolic function between CU and NU (All

554 p>0.05). CU also had higher PWV compared to NU (CU: 5.8±0.6m/s, NU: 5.3±0.7m/s; p

555 = 0.05). FMD was similar between CU and NU (8.3±3.3% vs. 6.8±3.6%; p=0.7).

556 Conclusions: Young, apparently healthy, cannabis users demonstrate altered cardiac

557 mechanics and arterial stiffening. Further studies should explore causal links between

558 cannabis smoking and altered cardiovascular function.

559 Introduction

560 Cardiovascular disease (CVD) remains the world’s leading cause of global

561 mortality1 despite global public health efforts to combat lifestyle factors which increase

562 CVD risk. Tobacco use and cigarette smoking are amongst the most detrimental

563 behaviours to cardiovascular health, as regular use is associated with increases in CVD

564 risk.163 Prior to the development of CVD, cigarette smokers experience detrimental

565 structural and functional adaptations in both the heart and vasculature. These include

566 increases in arterial stiffness,10,48 reductions in vascular endothelial function,60,62,164,165

567 and alterations to cardiac morphology and function.12,77,81,166

568 Despite long-standing global prohibition that has limited targeted scientific

569 inquiry, cannabis is the most commonly used recreational substance in the world, next

570 to alcohol.13 Cigarette smoking and recreational cannabis use share a number of

571 important similarities, particularly in their methods of consumption, which require the

572 inhalation of combusted material for chemical absorption through the lung membrane

573 into the blood stream. Although the chemical profile of cannabis differs from that of

574 cigarettes, substantial overlap exists in the chemicals contained in the smoke of each

575 product,96 including polycyclic aromatic hydrocarbons, carbon monoxide, and tar; each

576 of which has been identified as a hazard to cardiovascular health.103,167,168 Despite

577 these similarities, there are also some notable differences in the characteristic smoking

578 behaviours associated with cannabis use. Cannabis smoking typically involves greater

579 inhalation volumes and prolonged breath holds, both of which augment the deposition of

580 tar and absorption of carbon monoxide.105,106,169 These factors alone could suggest that 40

581 cannabis could present a greater threat to cardiovascular health than cigarettes.

582 However, it is notable that cannabis is typically smoked less frequently,105 and certain

583 cannabinoids possess notable anti-inflammatory properties.170,171 Thus, how each of

584 these factors associated with cannabis smoking interact to impact cardiovascular

585 function warrants investigation. At present, the degree to which habitual cannabis

586 smoking impacts the cardiovascular system remains unclear.

587 There are limited investigations that examine the effects of cannabis use on

588 human cardiovascular function, or cardiovascular risk factors that precede the

589 development of CVD. Recent work has identified altered cardiac dimensions and

590 mechanics in cannabis users.158 A single ecological study performed in a clinical setting

591 has demonstrated that cannabis users may experience an accelerated loss of arterial

592 compliance compared to concomitant users of tobacco and cannabis, and non-users.159

593 In an animal model, acute exposure to second-hand cannabis smoke has also been

594 demonstrated to reduce endothelial function.172 There are currently no studies that

595 directly compare measures of arterial or cardiac structure and function in cannabis

596 users in a controlled laboratory setting.

597 The aim of the present study was to examine the effects of chronic cannabis use

598 on cardiac morphology and function, arterial stiffness, and endothelial function in

599 healthy young cannabis smokers. We hypothesize that cannabis users will demonstrate

600 altered cardiac function, greater arterial stiffness, and impairments in endothelial

601 function compared to young healthy non-users of cannabis.

602 Methods

603 Subjects

604 The present study employed a cross-sectional design, comparing cardiovascular

605 variables of interest in cannabis users and non-users. Participants were recruited by

606 print advertisement and word of mouth in and around the university campus. Inclusion

607 criteria captured those between 19-45 year of age and required participants to have

608 either smoked cannabis < 5 times in their lifetime or have smoked cannabis for a

609 minimum of three “cannabis years”. One cannabis year was defined as a year where

610 cannabis was smoked once per week, or an equivalent usage (i.e. 6 months where

611 cannabis was smoked twice per week). This definition was adapted from “pack years”

612 traditionally used in cigarette literature, with consideration given to the aforementioned

613 differences between smoking behaviors of cigarettes and cannabis. Exclusion criteria

614 comprised stage ≥1 hypertension,173 diagnoses of chronic cardiovascular or metabolic

615 disease, engagement in ≤150 minutes of physical activity per week in the preceding

616 year, active use of prescription medication exclusive of oral contraceptives, previous

617 use of recreational drugs other than cannabis, cigarette smoking, and a BMI>30kg•m-2.

618 Testing of female participants occurred during the early follicular phase, or during the

619 placebo phase for participants taking oral contraceptives.

620 General Procedure

621 Participants visited the laboratory on two separate occasions. In visit 1,

622 participants provided written informed consent and completed an investigator generated

623 questionnaire to confirm eligibility. Information was also collected regarding physical

624 activity, frequency of cannabis use, duration of cannabis use, and total lifetime cannabis

625 use. Participants were then classified as either cannabis users (CU) or non-users (NU)

626 using stated criteria.

627 Prior to the second laboratory visit, participants were asked to abstain from:

628 cannabis use (48 hours), alcohol, vitamins, caffeine, and exercise (24 hours), and any

629 food or calorie containing beverages (minimum 4 hours). Upon arrival, participant height

630 and weight were measured using a standard stadiometer (SECA; Hamburg, Germany)

631 and a digital scale (Tanita; Tokyo, Japan), calibrated prior to each measurement.

632 Brachial blood pressure was measured in triplicate, with subjects in the seated position

633 using an automated sphygmomanometer (BpTRU; Coquitlam, Canada) according to the

634 guidelines of the American Heart Association.174 Participants were then transitioned to

635 an examination table where they rested awake in the supine position for a 10-minute

636 period, after which brachial blood pressure was again measured in triplicate. Arterial

637 stiffness and vascular endothelial function were then assessed using non-invasive

638 measures of carotid-femoral pulse wave velocity (PWV) and brachial artery reactive

639 hyperemia flow-mediated dilation (FMD). Assessments of PWV and FMD were

640 performed prior to a resting echocardiographic scan with and without the added stress

641 of isometric handgrip exercise. All testing was performed in a private thermoneutral

642 humidity-controlled lab environment.

643

644 Echocardiography

645 Resting transthoracic echocardiographic scans were performed with the

646 participant in the left lateral decubitus position. Hearts were imaged from the apical 4-

647 chamber view, parasternal long axis view, and the parasternal short axis view at the

648 basal, mid-papillary, and apical planes in accordance with current recommendations

649 from the American Society of Echocardiography.175 All images were collected using an

650 ultrasound device (Vivid-Q, General Electric; Boston, USA), and a 1.5-3.6MHz phased

651 array transducer. Two-dimensional B-mode imaging and tissue Doppler imaging was

652 performed at a minimum frame rate of 50 and 100 frames per second, respectively.

653 After completion of resting echocardiographic scans, participants performed two

654 maximal voluntary contractions (MVC) using a handgrip dynamometer (Baseline; White

655 Plains, USA). Each MVC was separated by 5 minutes. In an identical setting to the

656 resting echocardiographic scans, subjects were required to perform an isometric

657 contraction targeting 30% of MVC with the left hand for a period of three minutes while

658 images were collected from the apical 4-chamber, and the parasternal short axis view at

659 the basal and apical planes. Brachial blood pressures were measured using an

660 automated sphygmomanometer placed around the right arm. The sphygmomanometer

661 recorded brachial blood pressures and heart rate at each minute during the stress test

662 with the first measurement occurring at the start of the test, and the final measurement

663 occurring at the end of the 3-minute test. All images collected during rest and during the

664 isometric handgrip stress test were saved and stored for offline analysis.

665 Pulse Wave Velocity

666 PWV measures the propagation velocity of a systolic pressure wave through the

667 aorta by measuring the time delay between systolic ejection and pulse arrival times at

668 peripheral sites of known distances from the heart. PWV is a non-invasive surrogate of

669 central artery stiffness given the relationship between stiffness and propagation

670 velocity.176 Assessments were completed using a commercially-available tonometry

671 system (Sphygmocor CPVH, Atcor Medical; Sydney, Australia) with subjects in the

672 supine position. Subjects were instrumented with a single-lead (Lead II)

673 electrocardiogram, and the carotid and femoral (at the inguinal ligament) arteries were

674 palpated to find the strongest pulse, which was marked with an indelible marker for

675 repeat measures. A high-fidelity tonometer (Millar Instruments; Houston, USA) was

676 placed on each pulse site until 10 high-quality arterial pulse waves were recorded. The

677 time of arrival of the pulse at the carotid site and the femoral site from each cardiac

678 cycle was used to determine a foot-to-foot pulse transit time for each pulse pressure

679 waveform via the intersecting tangent method. If heart rate differed by greater than 5

680 beats per minute between measurement sites the measurement was discarded, in

681 accordance with expert consensus.177 For velocity calculations, distance was measured

682 with a standard anthropometric tape using a straight line above the body from the

683 carotid to femoral sites, with an adjustment for the distance from the carotid site to the

684 supra-sternal notch. Pulse transit distance was calculated by subtracting the distance

685 from the carotid site to the suprasternal notch from the distance between the carotid site

686 and the femoral site.176 PWV was determined as the average quotient of pulse transit

687 distance and pulse transit time of all measured cycles. Three measures of PWV were

688 collected and the mean PWV was taken as the value for each participant.

689 Reactive Hyperemia Flow-Mediated Dilation

690 The FMD test assesses endothelial function by measuring the vasodilatory

691 response to hyperemia. The right arm was supinated and abducted 90º at the shoulder

692 while supported in a custom-made foam block at the level of the heart. A manual

693 sphygmomanometer was placed around the subjects’ forearm immediately distal to the

694 antecubital fossa. An ultrasound device (Vivid-Q, General Electric; Boston, USA) with a

695 6-13MHz linear array transducer was used to image the brachial artery ~5-10cm

696 proximal to the edge of the tourniquet. Duplex ultrasound with an insonation angle of

697 60º and a sample volume spanning arterial diameter was used to simultaneously

698 measure blood velocity and arterial diameter throughout the reactive hyperemia FMD

699 test using continuous edge detection software (Quipu Medical; Pisa, Italy). After a

700 satisfactory image was obtained, wherein the blood velocity signal was optimized and

701 the arterial luminal border could be clearly defined, reactive hyperemia was assessed,

702 starting with a 1-minute baseline imaging period, after which the sphygmomanometer

703 was inflated to a pressure of 250mmHg for 5 minutes. Upon tourniquet deflation imaging

704 was continued for a 3-minute recovery period. The selected durations of each phase of

705 the reactive hyperemia FMD test were selected to match most frequently used

706 protocols, which adequately capture baseline diameter, occlude blood flow, and

707 adequately capture FMD.30

708 Data Analysis

709 Cardiac Structure and Function: All cardiac analyses were performed by one

710 investigator blinded to participant sex and group using EchoPac software (General

711 Electric Healthcare; Boston, USA). Each cardiac measurement was determined as the

712 average of measurements derived from three consecutive cardiac cycles. Left

713 ventricular (LV) volumes were determined using the modified Simpson’s Biplane

714 analysis and LV mass was calculated using the area-length method.178 Fractional

715 shortening, LV internal diameter and both interventricular septal (IVSd) and posterior

716 wall thickness (LVPWd) during diastole and systole were measured in the parasternal

717 long-axis view. Relative wall thickness was determined as 2 • (LVPWd • IVSd-1). LV

718 length was determined as the distance between the mitral plane and the most distal

719 endocardial border observable in the 4-chamber view. Peak early transmitral filling

720 velocity (E) and peak late transmitral filling velocity (A) were measured with Doppler

721 sampling distal to the mitral valve at the point where the mitral valve leaflets no longer

722 entered the sample volume. Mean mitral annular peak early velocity (E’), mean mitral

723 annular peak late velocity (A’), mean mitral annular peak systolic velocity (S’), and

724 isovolumetric contraction and relaxation times were obtained via tissue Doppler imaging

725 and were determined as averages recorded from the interventricular septum and LV

726 lateral wall at the mitral annular plane. Right ventricular (RV) areas were measured in

727 the 4-chamber view at end systole and end diastole, and right ventricular fractional area

728 change was calculated as ((RV end diastolic area – RV end systolic area) • RV end

729 diastolic area-1 • 100). Arterial elastance was calculated by dividing end systolic

730 pressure (brachial systolic blood pressure • 0.9) by stroke volume. Ventricular elastance

731 was calculated by dividing end systolic pressure by end systolic volume. Vascular

732 ventricular coupling was determined as the ratio of arterial elastance to ventricular

733 elastance.

734 LV Mechanics: Parasternal short-axis views at the basal and apical levels were

735 used to capture 2D images for speckle tracking analysis of LV twist. Rotation and

736 rotation rate were determined by the average values of six individual myocardial wall

737 segments. Rotation in the counterclockwise direction was defined as positive, while

738 rotation in the clockwise direction was defined as negative. LV twist was defined as the

739 difference between the peak rotation values at the basal and apical levels. LV peak

740 twisting rate and untwisting rate were defined as the peak rotational velocity during

741 systole and diastole, respectively.

742 LV strain was assessed via speckle tracking analysis of apical 4-chamber and

743 parasternal short-axis views. Specifically, radial and circumferential strain and strain

744 rates were assessed from parasternal short-axis views at the mid-papillary level, and

745 longitudinal strain was assessed from the apical 4-chamber view. Strain in each plane

746 was determined as the average of strain of six individual myocardial wall segments.

747 All speckle tracking analysis was performed for three cardiac cycles for a given

748 measure. If an individual myocardial wall segment was not imaged with significant

749 quality for speckle tracking analysis or could not be accurately tracked as determined by

750 the automated software, the segment was not included in the calculation of the average

751 value for that particular cardiac cycle.

752 FMD: Each reactive hyperemia FMD test was analyzed using continuous edge

753 detection software by one investigator blinded to participant sex and group. Arterial

754 diameter was averaged into 3-second time bins throughout the test and missing data

755 from tracking errors was interpolated from surrounding time bins. Antegrade and

756 retrograde blood velocity was averaged to determine mean blood velocity, which was

757 also averaged into 3-second time bins. Shear rate was calculated as the quotient of

758 mean blood velocity and arterial diameter. Shear rate area under the curve (SR AUC)

759 was calculated as the sum of shear rate 3 second time bins in the first 60 seconds of

760 recovery and up to the time of peak diameter change for each individual. Peak FMD%

761 was determined as the percentage increase in diameter from baseline during the

762 recovery period of the reactive hyperemia FMD test. Peak FMD% normalized to SR

763 AUC for 60s, and SR AUC up to the time of peak diameter change was also determined

764 by dividing peak FMD% by each respective SR AUC. To further account for potential

765 confounds of between group differences by baseline diameter, allometric scaling was

766 applied according to previously published methods.179 Peak FMD%, peak FMD%

767 normalized to SR AUCs, and allometrically scaled peak FMD% are all presented

768 separately.

769 Statistical Analysis

770 A Fisher Exact Test was used to compare sex distribution in each group. For

771 non-repeated measures, independent samples t-tests were used to compare group

772 means of a given outcome between CU and NU. This applied to all measures of

773 anthropometrics, resting hemodynamics, resting measures of cardiac structure and 49

774 function, PWV, and measures collected during the reactive hyperemia FMD test.

775 Allometric scaling of each FMD test was performed by first calculating the natural

776 logarithm of the absolute diameter change and baseline diameter. An analysis of co-

777 variance was performed with group as a fixed factor, log-transformed absolute diameter

778 change as the outcome variable, and log-transformed baseline diameter as a co-variate.

779 Estimated marginal means were calculated for each group and were exponentiated and

780 converted from a ratio statistic to a percentage change. In order to evaluate group

781 differences during the isometric hand grip stress test, a 2(group) x 2(exercise condition)

782 x 2(sex) mixed-model repeated measures analysis of variance with Bonferroni corrected

783 post-hoc comparisons was performed with each cardiac measure as the outcome

784 variable. For all tests, a two-sided p ≤ 0.05 was defined as significant a priori.

785 Results

786 Subjects

787 A total of 35 participants we recruited for this study, 18 CU (12M, 6F) and 17 NU

788 (10M, 7F). Descriptive participant statistics for each group are presented in Table 3.

789 There were no differences in anthropometric measurements between groups and

790 neither blood pressure nor heart rate differed at rest. Echocardiographic data at rest and

791 during isometric handgrip is presented for a subset of 28 (14 CU, 14 NU) subjects

792 matched for height, weight, age, sex, body surface area (BSA) and ethnicity. Full LV

793 twist and LV strain data is presented for 16 matched subjects (8 CU, 8 NU). None of

794 age, height, weight, sex, and BSA differed between groups in the speckle tracking

795 subset, nor were there differences in brachial blood pressure or heart rate between

796 groups.

797 Table 3. Participant characteristics and absolute values of brachial artery diameter and

798 shear rate during a reactive hyperemia flow-mediated dilation (FMD) test in cannabis

799 users (CU) and non-cannabis using controls (NU). Heart rate, blood pressure, and

800 FMD were measured in the supine position.

NU CU p

Sex (M/F) 10/7 12/6 0.9 Age (years) 21±3 22±2 0.8 Height (cm) 174±8 174±6 0.9 Weight (kg) 71±10 72±12 0.8 BMI (kg/m2) 23.4±2.3 24.7±3.8 0.8 BSA (m2) 1.9±0.2 1.9±0.2 0.9 Heart Rate (bpm) 68±8 64±8 0.2 SBP (mmHg) 111±8 110±8 0.6 DBP (mmHg) 69±8 66±5 0.3 MAP (mmHg) 83±7 81±5 0.3 Baseline Diameter (mm) 3.77±0.64 3.96±0.70 0.4 Occlusion Diameter (mm) 3.77±0.60 3.97±0.65 0.4 Peak Diameter (mm) 4.00±0.60 4.24±0.70 0.3 Baseline Shear Rate (s-1) 61.5±31.8 62.1±37.5 0.9 Peak Shear Rate (s-1) 477.8±163.3 417.8±165.9 0.3

801 Abbreviations: Body Mass Index (BMI), Body Surface Area (BSA), Systolic Blood

802 Pressure (SBP), Diastolic Blood Pressure (DBP), Mean Arterial Pressure (MAP). Shear

803 rate is calculated as 4 • mean blood velocity • artery diameter-1. Data is presented as

804 mean ± standard deviation.

805 Echocardiography

806 Measures of cardiac structure and function collected at rest and their change in

807 response to sustained isometric handgrip exercise are displayed in Table 4. Traditional

808 2D imaging, Doppler, and tissue Doppler imaging did not reveal any differences

809 between CU and NU at rest. Each group demonstrated similar LV and RV structural

810 characteristics. Traditional measures of systolic function were similar between groups at

811 rest and during handgrip exercise. Diastolic function also appeared similar between

812 groups at rest and during handgrip exercise. There were no differences in atrial

813 measurements at rest but right atrial area at end systole was decreased during handgrip

814 exercise irrespective of group (p = 0.01). Handgrip exercise also resulted in increases in

815 A, A’ and E:E’, and reductions in E:A irrespective of group (all p < 0.05).

816 Measures of LV mechanics at rest and their change in response to sustained

817 isometric handgrip exercise are displayed in Table 5. Peak global longitudinal,

818 circumferential, and radial strain were similar between groups at rest. Peak systolic and

819 diastolic longitudinal, circumferential and radial strain rates were also similar between

820 groups, with the exception of peak longitudinal diastolic strain rate, which was greater in

821 CU at rest (p = 0.02). At rest peak global LV twist and peak basal rotation were similar

822 between groups. Peak systolic twisting and diastolic untwisting velocities were similar

823 between groups, as were basal systolic and diastolic rotation velocities. Peak LV twist

824 and peak systolic twist velocity were increased during handgrip exercise irrespective of

825 group (both p = 0.04). Despite no observed difference in peak global LV twist at rest,

826 peak apical rotation was lower in CU compared to NU (p = 0.02). Peak apical rotation

827 velocities also differed between groups, as CU demonstrated lower peak systolic

828 rotation velocity (p = 0.01) and lower peak diastolic rotation velocity than NU (p = 0.01).

829 Table 4. Indices of cardiac structure and function at rest and changes with sustained isometric handgrip exercise in CU and NU.

NU CU Significance Time x Group x Rest Δ HGEX Rest Δ HGEX Baseline Time Time x Group Sex LV Structure LV Internal Diameter (cm) 4.6±0.3 - 4.7±0.4 - 0.7 - - - LV End Diastolic Volume (mL) 98±14 8±14 98±33 -0.4±11 0.9 0.1 0.7 0.9 LV Length (cm) 7.0±0.8 - 7.0±0.5 - 0.8 - - - LV Mass (g) 105±29 - 98±22 - 0.5 - - - LV Mass Index (g • m-2) 57±12 - 53±8 - 0.8 - - - Relative Wall Thickness 0.41±0.04 - 0.43±0.07 - 0.6 - - - LV Systolic Function S’ (cm • s-1) 0.09±0.02 -0.02±0.03 0.08±0.01 -0.01±0.01 0.3 0.1 0.1 0.7 Isovolumetric Contraction Time (ms) 72±8 4±17 67±11 0.05±10 0.2 0.4 0.9 0.8 Ejection Fraction (%) 61±6 -1±5 61±5 0.1±7 0.8 0.7 0.6 0.7 Fractional Shortening (%) 33±3 - 34±4 - 0.7 - - - Stroke Volume (mL) 59±7 4±11 63±12 -0.4±11 0.4 0.4 0.4 0.6 Cardiac Output (L • min-1) 2.5±0.3 2.2±1.0 2.6±0.4 2.0±0.9 0.6 <0.001 0.5 0.6 Arterial Elastance (mmHg • mL-1) 2.4±0.4 - 2.4±0.5 - 0.9 - - - Ventricular Elastance (mmHg • mL-1) 2.9±0.8 - 2.6±0.5 - 0.4 - - - Vascular Ventricular Coupling 0.91±0.2 - 0.97±0.2 - 0.4 - - - LV Diastolic Function E’ (cm • s-1) 0.14±0.02 -0.02±0.03 0.13±0.02 -0.01±0.02 0.6 0.1 0.2 0.9 A’ (cm • s-1) 0.06±0.01 0.02±0.02 0.06±0.01 0.02±0.03 0.2 <0.001 0.9 0.4 E (cm • s-1) 0.84±0.14 -0.002±0.1 0.82±0.17 0.01±0.1 0.7 0.5 0.9 0.9 A (cm • s-1) 0.41±0.09 0.20±0.16 0.39±0.08 0.11±0.13 0.5 <0.001 0.1 0.3 E:A 2.2±0.6 -0.70±0.55 2.2±0.4 -0.45±0.53 0.9 <0.001 0.2 0.4 E:E’ 6.1±1.0 1.6±1.9 6.3±1.7 0.35±1.7 0.7 0.01 0.1 0.8 Isovolumetric Relaxation Time (ms) 77±15 4±21 74±8 -2±12 0.5 0.7 0.7 0.6 RV Structure End Diastolic Area (cm2) 16.6±2.9 -0.71±1.9 17.5±3.9 1.0±5.4 0.5 0.4 0.9 0.9 End Systolic Area (cm2) 8.9±1.9 0.3±1.6 9.5±2.6 -1.0±1.7 0.5 0.6 0.3 0.7 RV Fractional Area Change (%) 46.8±5.5 -3.8±7.3 45.8±5.4 2.8±10.2 0.7 0.9 0.3 0.8 TAPSE (cm) 2.5±0.4 - 2.4±0.1 - 0.8 - - - Atria Left Atrial Area (cm2) 15.5±3.4 -1.1±1.8 16.1±3.7 -0.8±2.5 0.5 0.3 0.6 0.9 Right Atrial Area (cm2) 13.4±3.1 -0.9±1.6 14.0±3.3 -1.5±1.2 0.7 0.01 0.5 0.8

830 Abbreviations: Left Ventricle (LV), Hand-Grip Exercise (HGEX), Mean Mitral Annular Peak Systolic Velocity (S’), Mean

831 Mitral Annular Peak Early Velocity (E’), Mean Mitral Annular Peak Late Velocity (A’), Peak Early Transmitral Filling

832 Velocity (E), Peak Late Transmitral Filling Velocity (A), Right Ventricle (RV), Tricuspid Annular Plane Systolic Excursion

833 (TAPSE). Data are presented as mean ± standard deviation.

834 Table 5. LV twist and LV strain at rest and changes with sustained isometric handgrip exercise in CU and NU.

NU CU Significance

Rest ΔHGEX Rest ΔHGEX Baseline Time Time x Group Time x Group x Sex

LV Twist Global Peak Twist (°) 10.0±4.4 4.8±5.3 7.1±5.7 6.0±5.5 0.3 0.04 0.8 0.9 Peak Systolic Twist Velocity (° • s-1) 73.0±21.1 33.5±38.0 59.3±31.9 34.7±30.6 0.3 0.04 0.9 0.8 Peak Untwisting Velocity (° • s-1) -77.6±24.7 -15.7±30.6 -61.9±25.3 -22.8±29.6 0.2 0.2 0.8 0.6

Basal Peak Rotation (°) -3.1±2.3 -3.5±5.9 -3.0±1.7 -2.9±3.1 0.9 0.1 0.9 0.9 Peak Systolic Rotation Velocity (° • s-1) -49.0±14.8 -2.5±63.7 -50.0±17.3 -19.9±26.6 0.9 0.8 0.6 0.8 Peak Diastolic Rotation Velocity (° • s-1) 30.5±12.3 16.1±35.4 33.3±17.9 14.6±19.2 0.7 0.3 0.9 0.5

Apical Peak Rotation (°) 9.6±1.5 -0.4±2.0 5.5±3.8 2.3±5.1 0.02 0.5 0.3 0.9 Peak Systolic Rotation Velocity (° • s-1) 71.8±15.5 10.7±38.0 45.2±20.7 31.1±28.0 0.01 0.07 0.4 0.7 Peak Diastolic Rotation Velocity (° • s-1) -68.5±19.0 2.8±25.6 -47.9±13.8 -7.4±12.1 0.01 0.8 0.3 0.2

LV Strain Radial Peak Strain (%) 32.5±16.7 - 35.7±24.1 - 0.8 - - - Peak Systolic Strain Rate (% • s-1) 1.6±0.4 - 1.8±1.0 - 0.6 - - - Peak Diastolic Strain Rate (% • s-1) -1.9±0.6 - -1.8±0.8 - 0.9 - - -

Circumferential Peak Strain (%) -15.2±3.9 - -16.4±4.5 - 0.6 - - - Peak Systolic Strain Rate (% • s-1) -0.9±0.2 - -1.0±0.2 - 0.2 - - - Peak Diastolic Strain Rate (% • s-1) 1.1±0.3 - 1.2±0.4 - 0.4 - - -

Longitudinal Peak Strain (%) -17.4±3.2 -0.2±3.0 -18.9±3.8 1.2±4.1 0.4 0.4 0.6 0.4 Peak Systolic Strain Rate (% • s-1) -0.9±0.1 -0.04±0.3 -1.0±0.1 -0.05±0.2 0.7 0.4 0.9 0.9 Peak Diastolic Strain Rate (% • s-1) 1.3±0.4 0.06±0.3 1.7±0.3 -0.2±0.6 0.02 0.5 0.3 0.6

835

836 Abbreviations: Left Ventricle (LV). Data are presented as mean ± standard deviation.

837 Pulse Wave Velocity

838 PWV was recorded in all but one male CU, due to an immeasurably low femoral

839 pulse. PWV was found to be significantly greater in CU compared to NU (CU:

840 5.8±0.6m/s, NU: 5.3±0.7m/s; p = 0.05; Figure 1).

841

842 Figure 1. Carotid-femoral PWV measured in CU and NU. Bars and error bars represent

843 Mean and SD, respectively. Circles represent individual values.

844 Reactive Hyperemia Flow-Mediated Dilation

845 Absolute artery diameters and both baseline and peak shear rate during the

846 reactive hyperemia FMD test are displayed in Table 3. Brachial artery diameter and

847 shear rate during the baseline period of the test were not significantly different between

848 groups. Artery diameter during the last minute of the 5-minute occlusion period was also

849 similar between groups, and there was no difference in peak artery diameter or peak

850 shear rate during the recovery portion of the test. Peak FMD% (CU: 8.3±3.3%, NU:

851 6.8±3.6%; p = 0.7, Figure 2) did not differ between groups. SR AUC in the first 60

852 seconds of the recovery period and SR AUC prior to peak diameter change are

853 displayed in Figures 3A and 3B, respectively. SR AUC in the first 60 seconds of the

854 recovery period (CU: 4432±1630 a.u., NU: 4900±2222 a.u.; p = 0.7), and SR AUC to the

855 time of peak diameter change (CU: 4177±1808, NU: 4179±1752; p = 0.8) were similar

856 between groups. Peak FMD% normalized to both 60-second SR AUC and SR AUC to

857 peak vasodilation are displayed in Figures 3C and 3D, respectively. Normalization to

858 shear rate did not reveal any between group differences, as peak FMD% normalized to

859 60-second SR AUC (CU: 6.7±3.5%, NU: 5.3±2.8%; p = 0.2) and peak FMD%

860 normalized to SR AUC to peak diameter change (CU: 7.4±3.6%, NU: 6.0±2.7%; p = 0.2)

861 were similar between groups. When an allometric scaling approach was applied, peak

862 FMD% still remained similar between groups (CU: 7.5±0.6%, NU: 6.4±0.6%; p = 0.2).

863

864 Figure 2. Non-normalized peak FMD expressed as a percentage of baseline diameter

865 in CU and NU. Bars and error bars represent mean and SD, respectively. Circles

866 represent individual values.

867

868 Figure 3. (A) SR AUC in the first 60 seconds of the recovery period of the reactive

869 hyperemia FMD test in CU and NU. (B) Total SR AUC to the time of peak diameter

870 change during the recovery period of the reactive hyperemia FMD test in CU and NU.

871 (C) Peak FMD expressed as a percentage of baseline diameter normalized to SR AUC

872 in the first 60 seconds of recovery period of the reactive hyperemia FMD test in CU and

873 NU. (D) Peak FMD expressed as a percentage of baseline diameter normalized to SR

874 AUC prior to peak diameter change in CU and NU. Bars and error bars represent mean

875 and SD, respectively. Circles represent individual values.

876 Discussion

877 The major novel finding of this investigation is that young, otherwise healthy,

878 cannabis users demonstrate alterations in cardiac mechanics and greater central

879 arterial stiffness compared to healthy age-matched controls. In contrast, endothelial

880 function and cardiac responses to isometric handgrip exercise did not differ between

881 cannabis users and non-users. Therefore, cannabis users experience altered cardiac

882 mechanics and vascular structure but not function compared to non-users of similar

883 age. These differences suggest that this group may be at greater risk for the

884 development of CVD.

885 The present study identified altered cardiac mechanics at rest in cannabis users,

886 in spite of similar resting heart rates and blood pressures. Cannabis users presented

887 with reductions in peak apical rotation, peak apical systolic rotation velocity, and peak

888 apical diastolic rotation velocity, and while the precise functional significance of these

889 alterations of cardiac mechanics amongst cannabis users remains unclear, peak apical

890 rotation is a strong indicator of LV contractility.180 Studies on the effects of cannabis use

891 have revealed that acute cannabis consumption alters systolic time intervals and tissue

892 velocity,118,130 and reduces both stroke index and ejection fraction.129 Experimental data

893 also supports the potential negative effects of cannabis use on systolic function, as

894 reduced contractility in human tissue has been demonstrated in vitro with cannabinoid

895 receptor 1 (CB1) activity.84 The most abundant cannabinoid in recreational cannabis

896 products is typically delta-9-tetrahydrocannabinol (THC), a partial CB1 agonist. Thus,

897 acute reductions in contractility, and consequently systolic function associated with

898 cannabis use, may be CB1-mediated. Whether these acute effects translate to chronic

899 effects and explain the reduced peak apical rotation and rotation velocities in cannabis

900 users in our study requires further investigation.

901 Similar to our study, a previous investigation of the chronic effects of cannabis

902 use also found an association between altered cardiac mechanics with normal systolic

903 function and cannabis use. Khanji and colleagues used cardiac MRI to examine cardiac

904 function in cannabis users, and reported no differences in ejection fraction between

905 regular cannabis users and abstinent controls.158 They did, however, identify reduced

906 global circumferential strain in cannabis users. While our ejection fraction data agrees

907 with these findings, here we did not identify impaired peak global circumferential strain

908 in cannabis users. However, our finding of reduced peak apical rotation aligns with the

909 notion that traditional measures of systolic function are not sensitive to the effects of

910 cannabis on the myocardium, which may primarily exist as subtle alterations in cardiac

911 mechanics. Whether these subtle alterations manifest in clinically significant reductions

912 in cardiac function is currently unknown. An important null finding in ours and past work

913 is the lack of difference in LV mass between cannabis users and non-users, as LV mass

914 is an independent predictor of increased CVD.181 It is also important to note that we did

915 not observe any impairment in the cardiac response to isometric exercise in CU.

916 Although sustained isometric handgrip exercise presents an afterload challenge to the

917 heart, whereas dynamic exercise presents a volume challenge, this finding may be

918 considered preliminary evidence supporting preserved cardiac function during exercise

919 in young healthy cannabis users. This is important given recent reports that suggest

920 cannabis use with exercise is popular within certain populations.182 Further research

921 should seek to understand how acute cannabis consumption affects cardiac function

922 both at rest and during dynamic exercise.

923 While novel, the finding that cannabis users demonstrated higher PWV

924 compared to non-users is perhaps unsurprising when considering the effects of

925 cigarette smoking on arterial stiffness. Although not a universal finding, cigarette users

926 have been reported to have advanced arterial stiffening compared to age-matched non-

927 smokers, independent of other risk factors such as hypertension.53,183 Acute cigarette

928 smoking has far more consistently been shown to transiently increase PWV; however,

929 no study has examined whether the effects of cannabis use parallel the effects of

930 cigarette use in this manner. One large study of patients in an addiction care clinic

931 demonstrated that vascular aging (an age-adjusted metric of arterial stiffness) and

932 central augmentation index measured by radial artery tonometry was greater in

933 cannabis-only users compared to opioid users, tobacco users, and users of both

934 tobacco and cannabis.159 The present study differs from the previous work in that we

935 demonstrate a much lower degree of stiffening in cannabis users, but it is important to

936 highlight some important differences between the two studies that may explain the

937 difference in magnitudes of stiffening. The present study was performed in a controlled

938 laboratory setting which provided the opportunity to regulate participant behaviours that

939 may have confounded outcomes, whereas measurement in the previous study occurred

940 upon patient intake. It seems likely that stress levels could also have differed greatly

941 between these settings which could have acutely altered measures of arterial

942 stiffness.184 Additionally, the relatively low degree of additional stiffening in this study

943 compared to previous findings may be explained by participant characteristics.

944 Volunteers in our study were young (22±2yr), as opposed to the middle aged (40 ±2yr)

945 adults studied in previous work. The younger population may not yet manifest changes

946 in stiffness that are hypothesized to occur with cannabis use and likely had less

947 cannabis exposure. Cannabis users in the previous study had used cannabis for an

948 average of 37.67 gram-years, where one gram-year is the product of self-reported

949 grams of cannabis consumed daily and years of use.185 Although we quantified

950 “cannabis years” and not gram-years, it is highly unlikely our population had similar

951 exposure owing to their young age. Either or both of these key differences may explain

952 the degree of arterial stiffening we observed in our population of cannabis users. It is

953 also important to note the absolute PWV measured in each of our groups, and the

954 relative difference in cannabis users. Both groups demonstrated PWV values (CU:

955 5.8±0.6m/s, NU: 5.3±0.7m/s) that were within the expected range for individuals <30

956 years of age with normal blood pressure.186 This and the difference of 0.5m/s between

957 groups suggests that the arterial stiffening in young healthy cannabis users is relatively

958 small. The only slightly greater arterial stiffness in cannabis users could represent an

959 early maladaptation that worsens with more exposure; this observation could represent

960 a maladaptation of a magnitude that is not yet fully realized. Examining PWV in

961 cannabis users with more cannabis exposure or who are of older age could reveal more

962 dramatic arterial stiffening. Despite the relatively small difference in arterial stiffness in

963 cannabis users that we report, even minor increases in PWV indicate increased risk for

964 CVD and should be considered an adverse vascular characteristic.24

965 Contrary to our hypotheses, we did not observe impairments in endothelial

966 function measured by reactive-hyperemia FMD in cannabis users. Given the similarities

967 between cannabis and cigarettes, the effects of cigarette use on FMD, and evidence

968 that implicates cannabinoid receptor-mediated pathways in impaired endothelial

969 function we had expected that such changes would occur, but ultimately must reject this

970 hypothesis. Studies of cigarette smokers have revealed impairments in reactive

971 hyperemia FMD compared to age-matched controls.60,62,164,165 Carbon monoxide and

972 hydrocarbons, which are present in both cigarette and cannabis smoke, have also been

973 shown to induce damage to the endothelium.101 The negative effects of cigarette smoke

974 on FMD are strongly related to generation of ROS and consequent reductions in nitric

975 oxide availability68; cannabis smoke has also been shown to produce ROS.187

976 Endothelial cells also express CB1, the cannabinoid receptor which THC acts upon.188

977 CB1 activation may negatively affect endothelial function as CB1 activation has been

978 demonstrated to increase ROS generation by both macrophages189 and endothelial

979 cells.190 Thus, reduced nitric oxide availability due to generation of intravascular or

980 endothelial ROS provides a potential mechanism for CB1 mediated impairments in

981 FMD. Additionally, second-hand cannabis smoke has been demonstrated to reduce

982 FMD in an animal model, independent of THC content,102 making similar FMD between

983 groups in our study a notable null finding.

984 Although FMD was not different between groups, it is notable that non-users had

985 numerically greater SR and smaller baseline artery diameter, which would not be

986 explained by healthier lifestyle choices (such as exercise), as these are in the opposite

987 direction. Further, by conventional knowledge, these conditions would predispose the

988 control group to greater FMD than cannabis users in the current study. Despite these

989 conditions, FMD remained similar between groups. The similarity in FMD between

990 cannabis users and non-users may also be explained by the relatively young age of our

991 participants, and insufficient lifetime exposure to cannabis smoking to meaningfully

992 impair endothelial function. We believe this is the most likely explanation, as unlike their

993 middle aged and older counterparts, young healthy cigarette smokers do not always

994 present with measurably impaired reactive hyperemia FMD compared to non-

995 smokers.71–73

996 An additional explanation for the discrepancy in endothelial function between

997 cannabis users in our study who had preserved FMD and cigarette smokers typically

998 demonstrate reduced FMD could be related to the anti-inflammatory activity of

999 cannabinoids acting at the endogenous cannabinoid receptor 2 (CB2). Previous work85

1000 has revealed that ApoE-/- mice fed a high cholesterol diet for 11 weeks have attenuation

1001 of atherosclerotic lesion development, leukocyte adhesion, and macrophage infiltration

1002 compared to control mice when exposed to 1.0mg/kg of THC daily. These effects are

1003 completely abolished when a CB2 antagonist is administered alongside THC treatment.

1004 Additionally, CB1-mediated ROS generation by macrophages can be attenuated with

1005 CB2 activation189 and CB2 activation interferes with inflammatory pathways in

1006 endothelial cells.191 Another intriguing possibility is the notion that compounds within

1007 cannabis such as cannabidiol (CBD) mitigate the effects of smoke exposure on the

1008 endothelium. In vitro evidence suggests that CBD may also mitigate ROS generation

1009 and inflammatory responses in endothelial cells exposed to a damaging hyperglycemic

1010 preparation.192 Thus, it is possible that endothelial damage from cannabis smoke may

1011 be partially reconciled by cannabinoid and/or CB2-mediated interference of oxidative

1012 stress and inflammatory pathways which detrimentally affect endothelial function; an

1013 effect not present with cigarette smoke. Mitigation of the eventual inflammatory

1014 consequences of smoking-induced endothelial damage may explain why cannabis

1015 users demonstrated no impairment in FMD in the present study. Future studies are

1016 needed to confirm if cannabis smoking is indeed minimally detrimental to endothelial

1017 function and should attempt to elucidate the mechanisms underlying any discrepancies

1018 between the effects of cigarette use and the effects of cannabis use on the endothelium.

1019 As with any study, the current work is not without limitations. Owing to the use of

1020 a cross-sectional design, we cannot establish causality between cannabis smoking and

1021 the observed differences in cardiovascular indices. Interventional studies and/or studies

1022 employing longitudinal designs are needed to assess cause and effect relationships.

1023 Additionally, we employed self-reported cannabis use and did not implement objective

1024 screening tests confirming recent cannabis use in our population of users, although

1025 there is no apparent reason one group of participants in particular would have

1026 disproportionally attempted to intentionally misrepresent their pattern of use. This study

1027 did not sample blood to assess the inflammatory profile of participants. Hypothetically,

1028 greater levels of inflammatory markers in cannabis users compared to non-users could

1029 help to mechanistically explain the observed differences in arterial stiffness, as a

1030 relationship between chronic inflammation and both atherosclerosis and PWV has been

1031 reported previously.193 Indeed, a recent study identified that cannabis users are at

1032 greater risk of CVD compared to non-users based on circulating levels of c-reactive

1033 protein.194 This hypothesis should, however, be approached cautiously, as other studies

1034 have found contradictory results,195 and the relationship between cannabinoid receptor

1035 signaling and inflammation is multifactorial.88 Finally, the current study did not assess

1036 central blood pressures or pulse wave reflection characteristics. The existence of

1037 discrepancies in central pressure and/or pulse wave augmentation in cannabis users

1038 could provide valuable mechanistic insight on the increased arterial stiffness and altered

1039 cardiac function that we observed and is an area for future investigation.

1040 The present study identified differences in both cardiac mechanics and vascular

1041 wall properties between cannabis users and non-users. Specifically, cannabis users

1042 demonstrated sub-optimal apical cardiac function and higher aortic stiffness, which are

1043 hypothesized to represent cardiovascular maladaptations to cannabis smoking. These

1044 differences in cardiac function and vascular structure suggest that cannabis users may

1045 be at greater risk for the development of CVD. Future work should prospectively explore

1046 causal links between cannabis smoking and altered cardiovascular function, with a goal

1047 of characterizing the relationship between cannabis use and the development of CVD.

1048 Conflict of Interest:

1049 None of the authors have conflicts of interest to report.

1050 Author Contributions: 1051 CPC: Study Design, Data Collection, Data Analysis, Manuscript Preparation, Manuscript

1052 Revision - AMC: Data Collection, Manuscript Revision – PJM: Manuscript Revision -

1053 JFB: Study Design, Manuscript Revision.

1054 Funding:

1055 This work was supported by a Discovery Grant from the Natural Sciences and 1002

1056 Engineering Research Council of Canada (JFB 03974), the Canadian Foundation for 1003

1057 Innovation (JFB 35460), and the Ontario Ministry of Research, Innovation, and Science

1058 1004 (JFB 460597).

1059 Acknowledgements:

1060 We would like to thank all the participants who volunteered their time to be a part of this

1061 study.

1062 4 Interpretation of Findings, Summary, and Conclusions

1063 This thesis work consisted of a novel cross-sectional examination of arterial

1064 stiffness, endothelial function, and cardiac morphology and function in young healthy

1065 cannabis users and non-users. In comparison to non-users, cannabis users exhibited

1066 greater central PWV and reduced resting apical rotation, suggesting that cannabis use

1067 is associated with greater central arterial stiffness and altered cardiac mechanics.

1068 Brachial artery endothelial function, and cardiac function during isometric exercise, were

1069 not different between these groups. These novel findings provide preliminary evidence

1070 that habitual cannabis use may be associated with early alterations to cardiac

1071 mechanics and vasculature structure but not function.

1072 4.1 Arterial Stiffness

1073 The present work agrees with previous work that cannabis users demonstrate

1074 altered vascular structure compared to non-users of cannabis. We are the first to report

1075 that arterial stiffness is greater in cannabis users compared to non-users, as past work

1076 by Reece and colleagues159 did not measure PWV, the gold standard measure of

1077 arterial stiffness.160 This distinction is important for a number of reasons. First,

1078 commonly reported measures, such as AIx, are not solely measurements of arterial

1079 stiffness, as AIx is also influenced by a number of other factors including vasomotor

1080 tone, heart rate, and blood pressure.196 Secondly, the prognostic value peripheral

1081 measures of PWV such as brachial-ankle PWV have not been demonstrated in non-

1082 Asian populations197 Our study also is the only study to examine arterial stiffness in

1083 cannabis users in a controlled laboratory study, which provides the advantage of 72

1084 restricting acute behaviours such as exercise, smoking, caffeine, alcohol and food

1085 consumption, which may confound measures of PWV. Therefore, this study provides

1086 the current best evidence associating cannabis use with greater CVD-risk attributable to

1087 arterial stiffening.

1088 The findings of our study confirmed our hypothesis that cannabis users would

1089 present with greater PWV compared to non-users. This was hypothesized for two

1090 reasons. First, the only existing literature examining this hypothesis reported that

1091 cannabis users demonstrated greater peripheral arterial stiffness compared to users of

1092 cigarettes and non-cannabis users.159 Second, cigarettes users generally demonstrate

1093 greater central arterial stiffness than non-users, and important similarities exist between

1094 cigarette smoke and cannabis smoke.96

1095 The present and past findings are notable given the common observation that

1096 cigarette smokers have advanced peripheral and central arterial stiffening compared to

1097 non-users.10 These studies may be considered preliminary evidence that rationalizes

1098 future studies to examine whether cannabis smoking is indeed more detrimental to

1099 arterial stiffening than is cigarette smoking. It is important to note that our study did not

1100 have a cigarette comparator group and was not equipped to address this research

1101 question. However, our finding of greater central arterial stiffening in cannabis users

1102 contrasts some studies comparing cigarette smokers to non-users; which include

1103 individuals who were older than those included in the present study, and show no

1104 differences in PWV between groups.56–58 Indeed, advanced arterial stiffening in the

1105 present cohort of cannabis users is ominous in light of the young age of the participants 73

1106 and relatively lower exposure to cannabis use in the present study. However, the

1107 relative magnitude of difference was 0.5m/s, which may be considered relatively modest

1108 given that a 1.0m/s greater PWV corresponds to an approximately ~15% increase in

1109 CVD risk.24 The absolute values of PWV in our study should also be taken in context.

1110 Both groups in the present study demonstrated PWV in the expected range for

1111 individuals younger than 30 years of age with normal blood pressure.186 Nonetheless,

1112 any advancement of arterial stiffening is associated with greater risk of CVD; greater

1113 arterial stiffness in cannabis users should thus be viewed as an adverse vascular

1114 characteristic.

1115 The differences in exposure to cannabis between our study and past parallel

1116 differences in arterial stiffening in the two studies. This may indicate that cannabis use

1117 contributes to arterial stiffening in a dose-response manner. The present study recruited

1118 cannabis users with similar amounts cannabis use owing to their young age, and thus

1119 was not well equipped to examine the existence of a dose-response relationship

1120 between cannabis and arterial stiffness. Additionally, the cohorts of our study and that of

1121 Reece and colleagues differed in age and their use of other substances, so future work

1122 should more systematically and rigorously examine whether cannabis use contributes to

1123 arterial stiffening in a dose-response manner. If, however, cannabis contributes to

1124 arterial stiffening similarly to cigarettes, a dose-response relationship may not exist, as

1125 numerous studies have failed to demonstrate significant correlations between cigarette

1126 use and PWV within smokers.58,198,199 Other variables, such as inflammatory status and

1127 chronic health conditions, could also interact with cannabis use and arterial stiffness;

1128 these variables should be explored in future work as they were outside the scope of the

1129 present study. Understanding the mechanisms by which cannabis may contribute to

1130 arterial stiffening is imperative to fully understanding the relationship between cannabis

1131 use and PWV.

1132 It is currently unknown what mechanism underlies the potential cannabis-induced

1133 arterial stiffening. It is also unknown whether these effects occur due to the chemical

1134 constituents of cannabis, or due to the smoke inhalation associated with consumption.

1135 Studies of the underlying mechanisms of arterial stiffening in cigarette smokers suggest

1136 that nicotine and not CO is the primary driver of stiffening.200,201 Given this, and the fact

1137 that nicotine is only present in cigarettes, factors other than CO in cannabis smoke are

1138 likely to be the primary drivers of the arterial stiffening observed in our cohort of

1139 cannabis users. A more likely explanation for the greater arterial stiffness in cannabis

1140 smokers could be related to greater levels of chronic inflammatory markers. Previous

1141 reports have demonstrated associations between c-reactive protein and PWV,193 and

1142 recent evidence suggests that circulating c-reactive protein is greater in cannabis

1143 smokers compared to non-smoking controls.194 This area warrants further study, as

1144 there does exist contradictory data regarding c-reactive protein in cannabis smokers. 195

1145 Moreover, the relationship between cannabinoid receptor signaling and inflammation

1146 depends on multiple factors, including THC dose,85 and content of anti-inflammatory

1147 cannabinoids such as CBD.88 It is also worth investigating whether greater baseline

1148 vascular tone explains the greater PWV in cannabis users. Administration of propranolol

1149 abolishes the tachycardic effects of THC,115,117–120 strongly implicating the autonomic

1150 nervous system in the cardiovascular effects of cannabis. Direct measures of

1151 sympathetic outflow in cannabis users and appropriate controls should be used in future

1152 studies to evaluate this hypothesis.

1153 Future studies should be designed to critically assess our conclusion of increased

1154 PWV in cannabis users. Future work should also seek to understand whether stiffness

1155 is increased similar to that observed in cigarette smokers, if stiffening occurs in a dose-

1156 response manner with cannabis exposure, and what mechanisms underly cannabis-

1157 associated arterial stiffening.

1158 4.2 Endothelial Function

1159 We did not identify any differences in brachial artery reactive hyperemia FMD

1160 between cannabis users and non-users. This was contrary to our hypothesis which was

1161 based on a number of previous findings. Cannabis smoking exposes individuals to

1162 similar compounds as cigarette smoke96 and it is well-established that cigarette smoking

1163 is detrimental to FMD.29,60,62,71 Additionally, smoking associated increases in ROS are

1164 thought to be primarily responsible for impairments in FMD due to reduced NO

1165 bioavailability68 and cannabis smoke similarly induces increases in ROS.202 CB1 is

1166 widely expressed by vascular endothelial cells203 and its signaling also activates

1167 apoptotic pathways190 and increases ROS production in macrophages and endothelial

1168 cells.189,190 Each of these factors could, hypothetically, contribute to impairments in

1169 FMD. Finally, existing evidence suggests that cannabis impairs FMD in rats, regardless

1170 of whether it is inhaled as smoke or aerosolized vapor.172,204 These findings are difficult

1171 to reconcile, however, we propose numerous possible explanations for this null finding. 76

1172 It is possible that our cohort of cannabis users did not experience impairments in

1173 FMD due to an insufficient exposure to cannabis smoking. Some evidence exists to

1174 suggest that cigarette smoking has a negative dose-response effect on FMD.60,61 This

1175 may explain why certain studies examining FMD in young cigarette smokers do not

1176 reveal impairments in FMD.71–73 If such an effect existed with cannabis use and FMD,

1177 inclusion of cannabis smokers who had more cumulative cannabis exposure or used

1178 cannabis containing higher cannabinoid concentrations, may have revealed between

1179 group differences. This phenomenon may be particularly exaggerated in young

1180 cannabis users, as cannabis is typically smoked less frequently than cigarettes by

1181 users.104,105 The choice of FMD test used in the present study may also be of particular

1182 importance in explaining our null finding. One study of FMD in young cigarette users

1183 identified no impairments of reactive-hyperemia FMD in smokers, but did report

1184 impairments when FMD was assessed with a sustained shear stimulus instead.71

1185 Sustained shear FMD tests have also shown to be more sensitive in detecting

1186 endothelial dysfunction in other populations,205 suggesting that it may be a better test for

1187 identifying subclinical endothelial dysfunction. We did not perform sustained shear FMD,

1188 however, future studies may use it as a tool to further investigate the effects of chronic

1189 cannabis use on endothelial function.

1190 It must also be acknowledged that, at present, no study has administered

1191 cannabis to healthy participants and measured the acute effects on FMD. The only

1192 existing in vivo evidence to suggest that FMD is acutely impaired by cannabis comes

1193 from measurements of FMD in rats after being exposed to aerosolized cannabis and

1194 cannabis smoke.172,204 While this evidence may be viewed as preliminary evidence that

1195 FMD is impaired by cannabis, previous studies of the acute cardiovascular effects of

1196 cannabis have revealed distinct species differences in the cardiovascular responses of

1197 humans and animal models.112,206 Species differences in the FMD response to acute

1198 cannabis exposure cannot be discounted until it is examined in a controlled setting.

1199 Another important factor to consider in the determination of the effects of cannabis

1200 on endothelial function is the complex interplay of ROS, inflammation, cannabinoid

1201 receptor signaling, and FMD. Although CB1 receptor signaling has been shown to

1202 increase ROS,189,190 and ROS is increased by cannabis smoke itself,187 certain

1203 cannabinoids that do not act at CB1 also possess antioxidant actions. One such

1204 cannabinoid is CBD,207 which is often the second most abundant cannabinoid within

1205 cannabis. The potential for CBD contained in cannabis to suppress increases in ROS

1206 and thus prevent impairments in FMD is supported by evidence that shows acute

1207 decrements in FMD associated with cigarette smoking can be rescued by antioxidant

1208 supplementation.208 CBD has also been shown to exert anti-inflammatory effects in

1209 models of tissue damage,209 and mitigate both mitochondrial superoxide generation and

1210 inflammatory pathways in endothelial cells in vitro.192 Thus, CBD may be of particular

1211 importance in combatting cannabis smoking-associated mechanisms which could impair

1212 endothelial function and consequently FMD.

1213 Despite being a partial agonist of CB1, the actions of THC at a second

1214 cannabinoid receptor may also contribute to the lack of chronic impairment observed in

1215 FMD in cannabis users. THC is also a partial agonist of CB2, which is expressed in 78

1216 immune tissues.210 Despite not having any direct cardiovascular effects, CB2 agonists

1217 have been shown to be cardioprotective in situations of myocardial infarction and stroke

1218 due to anti-inflammatory actions.211,212 CB2 activation also appears to mitigate CB1-

1219 mediated increases in ROS in macrophages189 and interfere with inflammatory

1220 pathways in endothelial cells.191 Theoretically, mitigation of excess tissue damage,

1221 inflammation, and ROS by CB2 activation could combat the consequent decrements in

1222 endothelial function and FMD associated with smoking-induced endothelial injury. The

1223 most compelling evidence in favour of this notion comes from previous work that shows

1224 ApoE-/- mice fed a high cholesterol diet have attenuations in atherosclerotic lesion

1225 development, leukocyte adhesion, and macrophage infiltration when exposed to chronic

1226 doses of THC, but not when administered a CB2 antagonist.85

1227 More recently discovered novel cannabinoid receptors may also be involved in

1228 atherogenic processes and, thus, interact with endothelial function. For example,

1229 antagonism of the novel cannabinoid receptor GPR55213 has recently been shown to

1230 have anti-atherogenic effects.214 Given the vast array of cannabinoids and other

1231 molecules present in cannabis smoke, continuing work to understand the entirety of

1232 cellular mechanisms associated with cannabis use will be necessary to fully understand

1233 its effect on the endothelium.

1234 The relationship between cannabis smoking and endothelial function is complex,

1235 and likely depends on a wide range of variables, which may include consumption

1236 methods, cumulative cannabis exposure, species-dependent effects, and the chemical

1237 composition of cannabis. While we have speculated on numerous mechanisms which 79

1238 may be critical to elucidating the effects of cannabis on endothelial function, much work

1239 must be done to address our hypotheses. Each of these hypotheses largely pertain to

1240 the acute effects of cannabis on FMD. Although the acute effects of cannabis on FMD

1241 are outside of the scope of this thesis, knowledge of the acute effects of cannabis on

1242 FMD are pertinent to further understanding how FMD is affected chronically by habitual

1243 cannabis use. Understanding if, and why, cannabis use does not chronically impair

1244 FMD should be the goal of future work and should consider all these variables which we

1245 have discussed.

1246 4.3 Cardiac Morphology and Function

1247 This study is the first to examine cardiac morphology, cardiac function, and cardiac

1248 mechanics in cannabis users in a controlled laboratory setting. Cannabis users

1249 demonstrated lower apical rotation, peak systolic rotation velocity, and peak diastolic

1250 rotation velocity at rest. However, LV twist was not different between groups, nor were

1251 there any differences in structural or Doppler derived measures of cardiac structure and

1252 function.

1253 The only other examination of the hearts of cannabis users was performed by

1254 Khanji and Colleagues.158 Cannabis users had greater end diastolic volume, end

1255 systolic volume, and stroke volume of both the LV and RV. As well, they reported that

1256 cannabis users had greater LV mass and reduced global circumferential strain. When

1257 performing multivariable regression and accounting for age and sex, this group also

1258 observed reductions in global circumferential strain, and greater LV end diastolic

1259 volume and LV end systolic volume. Our findings share some similarity with this work, 80

1260 but also some key differences. We did not identify any differences in LV volumes or LV

1261 mass between cannabis users and non-users. A lack of difference in LV mass is an

1262 important null finding, as LV mass can be a key indicator of LV remodeling, and

1263 increased LV mass is an independent predictor of CVD.181 Despite the lack of

1264 differences in LV mass in the current study, it is interesting to compare our finding of

1265 greater PWV with the greater LV mass in the work of Khanji and colleagues. Arterial

1266 stiffness, indicated by greater PWV, could serve as a mechanism to increase LV

1267 afterload and potentially induce LV remodeling.50 Although these findings were not

1268 observed concurrently, it is possible that increased arterial stiffness in cannabis users

1269 precedes increases in LV mass. At present, this suggestion is purely speculative, and

1270 the work of Khanji and colleagues cannot verify this hypothesis as they did not measure

1271 PWV. Future studies should further investigate central arterial pressures, and whether

1272 accelerated arterial stiffening occurs in conjunction with cannabis use, and if this

1273 contributes to increases in LV mass. Similar to Khanji and colleagues, we demonstrated

1274 altered cardiac mechanics assessed by cardiac motion analysis. However, while they

1275 previously identified reductions in global circumferential strain, we identified reduced

1276 apical rotation in cannabis users. Although these two findings represent differences in

1277 function of two different myocardial regions, each have value in the assessment of left-

1278 ventricular function. Apical rotation has been shown to be an indicator of LV

1279 contractility180 and global circumferential strain has been shown to be well correlated

1280 with cardiac magnetic resonance measured ejection fraction.215 It is important to

1281 acknowledge that despite this difference in tissue mechanics, we did not identify

1282 impairments in systolic function. It is possible that cardiac function is preserved in light

1283 of this finding due to non-statistically significant compensations in other measures of

1284 contractility (e.g. radial strain), or that altered cardiac mechanics precede functional

1285 impairments in cardiac physiology. Nonetheless, each study provides support that

1286 cannabis use may be associated with subtle alterations to cardiac mechanics, which

1287 may or may not have eventual functional consequences. Unlike Khanji and colleagues,

1288 we did not demonstrate differences in LV or RV volumes between groups. Their study

1289 population was older than those recruited in the present thesis and they were not able

1290 to accurately quantify cannabis use. Thus, age, sample size, and cannabis exposure

1291 may underscore the differences in findings between the two studies.

1292 Our characterization of the hearts of cannabis users allows for comparison to the

1293 cardiac function of healthy cigarette users. Cigarette users have frequently been

1294 reported to have reductions in diastolic function compared to controls.76–80 We did not

1295 identify any reductions in measures of diastolic function in cannabis users, and this may

1296 highlight an important distinction between detrimental cardiac effects between cannabis

1297 and cigarettes. Different effects on the heart may also help to explain the differences in

1298 long-term associations with different forms of CVD. As previously discussed, cigarette

1299 use is correlated with an increased risk of coronary artery disease while cannabis is

1300 not,93 and some studies report no associations between cannabis use and CVD.94

1301 However, it is important to note that there is no measure of equivalency for cannabis

1302 and cigarette exposure, so it is difficult to compare the two smoking habits.

1303 Actions of cannabinoid receptors within the myocardium must also be considered

1304 in understanding the effects of cannabis on the heart. CB1 is present in

1305 cardiomyocytes216 and its signaling has been studied with endogenous and synthetic

1306 cannabinoids. Activation of CB1 in isolated human atrial tissue by the endogenous

1307 cannabinoid anandamide results in reductions in contractility,84 as does activation by the

1308 synthetic cannabinoid HU210 in vivio.217 CB1 is also implicated in pro-

1309 apoptotic/hypertrophic pathways in cardiomyocytes.216 Chronic activation of CB1 in

1310 cannabis smokers by THC could be associated with either of these processes and

1311 partially explain the reduced apical rotation we report in this study. It should be noted,

1312 however, that THC is a weak partial agonist of CB1 in comparison to endogenous

1313 cannabinoids and synthetic cannabinoids, and therefore may not exert the same effects.

1314 As well, the acute effects of THC on the heart have not been directly studied in humans,

1315 as the only studies performing echocardiography after THC exposure have utilized

1316 cannabis smoking as a mode of administration.121,129,130

1317 Our study provides preliminary evidence that cannabis use is associated with

1318 alterations to cardiac function. Specifically, we show that apical rotation and rotation

1319 velocities are reduced in cannabis users. While these findings represent less dramatic

1320 differences than has been reported by other investigators,158 and differ from what is

1321 observed in cigarette smokers, they may still bare functional significance. The long-term

1322 functional consequences of these differences should be the focus of future research.

1323 Future work should also explore whether alterations in cardiac mechanics are greater in

1324 individuals with greater cannabis exposure.

1325 4.4 Limitations and Future Directions

1326 The present work has a number of limitations that should be acknowledged. First

1327 and foremost, the present study utilized a cross-sectional design, and is therefore not

1328 able to assess causality. While exclusion criteria and study restrictions were designed to

1329 limit factors that could confound our primary outcomes, we are not able to say cannabis

1330 use was the sole contributor to our findings.

1331 Our study also recruited young cannabis users with a minimum cannabis use

1332 requirement of only three years of use, once per week. It is possible that these criteria

1333 led to the recruitment of individuals with insufficient exposure to cannabis to

1334 demonstrate the effects of prolonged cannabis use on cardiac function. Therefore, null

1335 findings in traditional measures of cardiac function and FMD between groups should not

1336 be viewed as conclusive and should be the focus of future work. If cannabis exerts

1337 effects on these outcomes, it is possible they would only be evident in individuals with

1338 much greater exposure to cannabis.

1339 Although the effect of cigarette smoking on the primary outcomes have been

1340 published extensively, the present study did not include a cigarette smoking comparator

1341 group. The notion that cannabis use exerts measurable, detrimental cardiovascular

1342 effects is largely founded upon the broader evidence base concerning the known effects

1343 of cigarettes. Inclusion of a cigarette comparator group would allow for a better

1344 understanding of how cannabis use compares to cigarettes on our selected measures.

1345 A direct comparison between cannabis and cigarette users would also help to generate

1346 hypotheses for future study. 84

1347 In the present study all identified differences in cannabis users were observed at

1348 rest. We attempted to examine cardiac responses during an acute afterload challenge

1349 by having participants perform sustained isometric handgrip exercise; however, this

1350 model of exercise did not reveal any group differences, and in fact, abolished all

1351 significant differences observed at rest. Isometric exercise presents a distinct challenge

1352 to the heart in comparison to dynamic exercise.218 Therefore, we cannot rule out that

1353 different exercise stresses do or do not result in subtly different cardiovascular

1354 responses in cannabis users compared to non-users.

1355 At present, there are large knowledge gaps regarding the relationship between

1356 cannabis use and human cardiovascular physiology. Understanding the acute effects of

1357 cannabis use on arterial stiffness, endothelial function, and cardiac morphology and

1358 function is necessary in guiding further research on the chronic effects of use. Results

1359 from these studies can then guide studies exploring the mechanistic underpinnings of

1360 any effects of cannabis use on cardiovascular physiology in humans. Perhaps most

1361 urgently, there is a need for large prospective longitudinal trials which systematically

1362 examine the relationships between long-term cannabis use and cardiovascular health.

1363 These studies would overcome many of the limitations of the present study. Namely,

1364 they could better assess causality and could provide greater insight into possible dose-

1365 dependent effects of cannabis use. This research is extremely topical, as cannabis is

1366 the most used recreational drug in the world exclusive of alcohol, and recreational use

1367 has become legal in Canada, and numerous states in the USA.

1368 4.5 Conclusions

1369 The main findings of the present study were that healthy young cannabis users

1370 demonstrated greater arterial stiffness measured by PWV and reduced apical rotation

1371 compared to age-matched non-users. They did not, however, demonstrate any

1372 differences in endothelial function, cardiac structure, or cardiac function at rest or during

1373 isometric handgrip exercise. This study is the first laboratory-controlled study to

1374 compare examine these outcomes in cannabis users. These findings support previous

1375 evidence that suggests that cannabis users have advanced arterial stiffening and

1376 altered cardiac tissue mechanics. Our findings provide evidence that cannabis users

1377 demonstrate subclinical differences in arterial stiffness and cardiac mechanics that

1378 could be due to chronic effects of cannabis on the structure and function of the human

1379 cardiovascular system.

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