by Mostafa Hossam El Beheiry - University of Toronto T-Space · 2013-09-27 · Mostafa Hossam El...
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METOPROLOL IMPAIRS MESENTERIC AND POSTERIOR CEREBRAL ARTERY FUNCTION IN MICE by Mostafa Hossam El Beheiry A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto © Copyright by Mostafa Hossam El Beheiry 2010
Transcript of by Mostafa Hossam El Beheiry - University of Toronto T-Space · 2013-09-27 · Mostafa Hossam El...
METOPROLOL IMPAIRS MESENTERIC AND POSTERIOR CEREBRAL ARTERY FUNCTION IN
MICE
by
Mostafa Hossam El Beheiry
A thesis submitted in conformity
with the requirements for the degree of Master of Science
Graduate Department of Physiology
University of Toronto
© Copyright by Mostafa Hossam El Beheiry 2010
ii
1 Abstract
METOPROLOL IMPAIRS MESENTERIC AND POSTERIOR CEREBRAL ARTERY FUNCTION IN
MICE Mostafa Hossam El Beheiry Master of Science, 2010 Department of Physiology, University of Toronto Background/Rationale: In addition to their established cardioprotective role, β-adrenergic
antagonists also increase the risk of stroke and mortality. We propose that a vascular mechanism
could contribute to cerebral tissue ischemia in β-blocked patients.
Methods: Cardiac output (CO), mean arterial pressure (MAP) and microvascular brain oxygen
tension (PBrmvO2) were measured in anesthesized mice treated with metoprolol (3mg·kg-1, i.v.).
Dose-response curves (DRCs) for adrenergic-agonists were generated in mesenteric resistance
arteries (MRAs; isoproterenol, clenbuterol) and posterior cerebral arteries (PCAs;
phenylephrine, isoproterenol) before and after metoprolol treatment.
Results: Metoprolol reduced CO, maintained MAP and increased systemic vascular resistance
(SVR) resulting in a decreased PBrmvO2 in mice. Metoprolol attenuated β-adrenergic mediated
vasodilation in both MRAs and PCAs.
Conclusions: Metoprolol reduced brain perfusion in mice. A decrease in CO contributed
however, metoprolol also inhibited β-adrenergic vasodilation of mesenteric and cerebral arteries.
This provides evidence in support of a vascular mechanism for cerebral ischemia in β-blocked
patients.
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2 Acknowledgements
First and foremost I must sincerely thank Drs. Gregory Hare and Steffen-Sebastian Bolz
for their invaluable guidance and support. They have helped me to understand and appreciate
the joys and the sacrifices involved in the scientific process. I am eternally thankful for being
given the opportunity to work with them.
Thanks as well to Dr. Scott Heximer for giving his time to be a part of my advisory
committee. His input was helped to steer this project in the right direction.
Additional thanks must be given to Sharon Klimosco at Department of Anesthesia at St.
Michael’s Hospital as well as the department itself for supporting my salary and funding my
travel and attendance at several scientific conferences. Thanks again to Dr. Bolz for providing
operating funds.
I would like to thank Drs. Jenny Zhang for her surgical expertise in collecting mean
arterial blood pressure data. Thanks as well to Drs. Golam Kabir and Kim Connelly for their
help in collecting and analyzing the left ventricular function of my β-blocked mice.
Special thanks to the people in the Bolz and Hare labs who made the last two years an
amazing and memorable experience.
Finally, thanks to my family and friends for their support outside of the laboratory.
Without these people I would have surely lost my sanity in the weeks where the science gods
were not on my side.
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3 Table of contents
1 ABSTRACT ..................................................................................................................................................... II
2 ACKNOWLEDGEMENTS .......................................................................................................................... III
3 TABLE OF CONTENTS .............................................................................................................................. IV
4 LIST OF TABLES ........................................................................................................................................ VII
5 LIST OF FIGURES .................................................................................................................................... VIII
6 OVERVIEW ..................................................................................................................................................... 1
7 INTRODUCTION ............................................................................................................................................ 3
7.1 CLINICAL EFFICACY OF BETA-BLOCKERS ........................................................................................................ 3
7.1.1 Beta-blockers alleviate angina symptoms and reduce morbidity and mortality ................................... 5
7.1.2 Beta-blockers reduce reinfarction and mortality post-myocardial infarction ...................................... 6
7.1.3 Beta-blockers reduce morbidity and mortality in chronic heart failure ............................................... 7
7.1.4 Beta-blockers reduce blood pressure and risk of stroke in hypertensive patients ................................ 9
7.1.5 Beta-blockers reduce the risk of MI in perioperative patients at cardiac risk ................................... 11
7.2 INCREASED RISK OF MORTALITY AND STROKE ASSOCIATED WITH BETA-BLOCKADE ..................................... 12
7.3 BETA-ADRENERGIC SIGNALLING IN REGULATING THE CARDIOVASCULAR SYSTEM ...................................... 14
7.3.1 Cardiac beta-adrenergic signal transduction pathways .................................................................... 14
7.3.1.1 Beta-adrenergic modulation of chronotropy .................................................................................................. 15
7.3.1.2 Beta-adrenergic modulation of contractility ................................................................................................... 18
7.3.2 Vascular beta-adrenergic signal transduction pathways ................................................................... 20
7.3.2.1 Distribution across vascular beds ................................................................................................................... 24
7.4 POTENTIAL OF A VASCULAR MECHANISM IN BETA-BLOCKER PATHOLOGY .................................................... 26
7.4.1 Vasodilators have better risk reductions for stroke and mortality than beta-blockers ....................... 26
7.4.2 Cardioselective beta-blockers may act on beta2-adrenergic receptors .............................................. 28
7.4.3 Metoprolol impairs cerebral oxygen delivery in acutely anemic rats ................................................ 29
8 HYPOTHESIS AND AIMS ........................................................................................................................... 31
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9 METHODS ...................................................................................................................................................... 32
9.1 EFFECT OF METOPROLOL ON BRAIN O2 TENSION AND HEMODYNAMICS IN VIVO ............................................ 32
9.1.1 Animals ............................................................................................................................................... 32
9.1.2 Experimental protocol ........................................................................................................................ 32
9.1.3 Heart rate and mean arterial blood pressure ..................................................................................... 32
9.1.4 Microvascular brain oxygen tension .................................................................................................. 34
9.1.5 Cardiac responsiveness and left ventricular function ........................................................................ 34
9.2 EFFECT OF METOPROLOL IN THE MESENTERIC RESISTANCE ARTERY IN VITRO ............................................... 35
9.2.1 Animals ............................................................................................................................................... 35
9.2.2 Mesenteric resistance artery isolation ............................................................................................... 35
9.2.3 Pressure myography ........................................................................................................................... 37
9.2.4 Effect of metoprolol on beta1,2-adrenergic mediated vasodilation ................................................... 39
9.2.5 Effect of metoprolol on beta2-adrenergic mediated vasodilation ...................................................... 39
9.3 EFFECT OF METOPROLOL IN THE POSTERIOR CEREBRAL ARTERY IN VITRO .................................................... 40
9.3.1 Animals ............................................................................................................................................... 40
9.3.2 Posterior cerebral artery isolation ..................................................................................................... 40
9.3.3 Pressure myography ........................................................................................................................... 40
9.3.4 Effect of metoprolol on adrenergic mediated vasomotor function ..................................................... 41
9.4 DRUGS AND SOLUTIONS ................................................................................................................................ 41
9.5 STATISTICAL ANALYSIS ................................................................................................................................ 42
9.5.1 Effect of metoprolol on brain O2 tension and hemodynamics in vivo ................................................. 42
9.5.2 Effect of metoprolol in mouse arteries ............................................................................................... 42
10 RESULTS ........................................................................................................................................................ 44
10.1 EFFECT OF METOPROLOL ON BRAIN O2 TENSION AND HEMODYNAMICS ................................................... 44
10.1.1 Metoprolol injection reduced brain O2 tension and heart rate ...................................................... 44
10.1.2 Metoprolol injection reduced CO and increased SVR ................................................................... 45
10.2 METOPROLOL INHIBITS ISOPROTERENOL MEDIATED VASODILATION MRAS ............................................ 51
10.3 METOPROLOL INHIBITS ISOPROTERENOL MEDIATED VASODILATION INPCAS .......................................... 58
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11 DISCUSSION .................................................................................................................................................. 63
11.1 BETA-ADRENERGIC VASODILATION IS REQUIRED TO MAINTAIN VITAL ORGAN PERFUSION ...................... 64
11.2 METOPROLOL INHIBITS ADRENERGIC VASODILATION IN MESENTERIC AND CEREBRAL ARTERIES ............ 67
11.3 METOPROLOL MAY BE ASSOCIATED WITH INCREASED MORBIDITY AND MORTALITY ............................... 67
11.4 DIFFERENCES BETWEEN MESENTERIC AND CEREBRAL ARTERIES ............................................................. 69
11.5 LIMITATIONS ............................................................................................................................................ 70
12 SUMMARY ..................................................................................................................................................... 73
13 APPENDIX A – BETA-BLOCKADE: A HISTORICAL PERSPECTIVE ............................................... 74
14 APPENDIX B – CHARACTERIZATION OF PCA PHENYLEPHRINE RESPONSES ........................ 79
14.1 DETERMINING OPTIMAL PHENYLEPHRINE PRECONSTRICTION DOSE IN PCAS ........................................... 79
14.1.1 Methods ......................................................................................................................................... 79
14.1.2 Results ........................................................................................................................................... 80
14.2 EFFECT OF METOPROLOL ON PHENYLEPHRINE DOSE-RESPONSE CURVE IN PCAS ..................................... 83
14.2.1 Results ........................................................................................................................................... 83
14.2.2 Interpretation: Phenylephrine responses are time-dependent in the PCA .................................... 86
15 REFERENCE LIST ....................................................................................................................................... 88
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4 List of Tables
TABLE 7.1 PHARMACOLOGICAL PROPERTIES OF CLINICALLY USED BETA-BLOCKERS .................................................... 4
TABLE 10.1 HALF MAXIMAL CONCENTRATIONS FOR MRA DOSE-RESPONSE CURVES. ................................................ 53
TABLE 10.2 HALF MAXIMAL CONCENTRATIONS OF ISOPROTERENOL IN PCAS. ........................................................... 62
TABLE 13.1 ORIGINAL CHARACTERIZATION OF ADRENERGIC RECEPTOR SUBTYPES .................................................... 76
TABLE 13.2 STRUCTURES OF BETA-ADRENERGIC RECEPTOR LIGANDS ........................................................................ 77
TABLE 14.1 HALF MAXIMAL CONCENTRATIONS OF PHENYLEPHRINE IN PCAS. ........................................................... 85
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5 List of Figures
FIGURE 7.3.1 SUMMARY OF CARDIAC BETA-ADRENERGIC SIGNALLING. ..................................................................... 17
FIGURE 7.3.2 SUMMARY OF VASCULAR BETA-ADRENERGIC SIGNALLING. ................................................................... 21
FIGURE 9.1.1 EXPERIMENTAL PROTOCOL FOR ASSESSING MAP AND PBRMVO2 WITH METOPROLOL ............................ 33
FIGURE 9.2.1 PHOTOGRAPHS OF ARTERY ISOLATION AND CANNULATION. .................................................................. 36
FIGURE 9.2.2 SET UP OF THE PRESSURE MYOGRAPHY APPARATUS .............................................................................. 38
FIGURE 10.1.1 TIME COURSE OF HYPOXIC CHALLENGE PROTOCOL WITH SALINE/METOPROLOL. ................................. 46
FIGURE 10.1.2 MEAN HEART RATE, MAP AND PBRMVO2 IN HYPOXIC CHALLENGE WITH SALINE/METOPROLOL. ......... 47
FIGURE 10.1.3 DELTA HEART RATE, MAP AND PBRMVO2 IN HYPOXIC CHALLENGE WITH SALINE/METOPROLOL. ........ 48
FIGURE 10.1.4 HEMODYNAMIC CHANGES FOLLOWING SALINE AND SUBSEQUENT METOPROLOL INJECTIONS. ............ 49
FIGURE 10.1.5 REPRESENTATIVE PRESSURE-VOLUME LOOP BEFORE AND AFTER METOPROLOL TREATMENT. ............. 50
FIGURE 10.2.1 REPRESENTATIVE TRACING OF MRA EXPERIMENTAL PROTOCOL. ....................................................... 52
FIGURE 10.2.2 EFFECT OF METOPROLOL ON ISOPROTERENOL DOSE-RESPONSE CURVES IN MESENTERIC ARTERIES. .... 54
FIGURE 10.2.3 PERCENT DILATION TO EC50 DOSE (30µM) OF ISOPROTERENOL IN MESENTERIC ARTERIES. ................ 55
FIGURE 10.2.4 PERCENT DILATION AT EMAX OF ISOPROTERENOL DOSE-RESPONSE CURVES. ......................................... 56
FIGURE 10.2.5 EFFECT OF METOPROLOL ON CLENBUTEROL MEDIATED VASODILATION. ............................................. 57
FIGURE 10.3.1 REPRESENTATIVE TRACING OF PCA PROTOCOL. ................................................................................. 60
FIGURE 10.3.2 EFFECT OF METOPROLOL ON ISOPROTERENOL DOSE-RESPONSE IN PCAS. ............................................ 61
FIGURE 14.1.1 PHENYLEPHRINE DOSE-RESPONSE CURVES IN MESENTERIC AND CEREBRAL ARTERIES. ....................... 81
FIGURE 14.1.2 EFFECT OF CONSECUTIVE DOSES OF PHENYLEPHRINE IN POSTERIOR CEREBRAL ARTERIES. ................. 82
FIGURE 14.2.1 EFFECT OF METOPROLOL ON PHENYLEPHRINE DOSE-RESPONSE IN PCAS. ........................................... 84
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6 Overview
The β-blocker class of antihypertensive drugs is routinely used in the treatment of
chronic disease (hypertenstion, heart failure, coronary artery disease). Additionally, it is
prescribed as a prophylactic intervention in patients at cardiovascular risk undergoing surgery.
Their efficacy in reducing cardiac morbidity and mortality was established in conditions of
myocardial ischemia and infarction. Without clear testing, their scope of treatment broadened
and they were used more liberally due to the perception that β-blockers had broad efficacy with
limited toxicity. In 2009 alone there were 130 million prescriptions for β-blockers in the US
placing it in the top 5 most prescribed drug class.
In addition to their ability to promote cardioprotection by reducing myocardial oxygen
demand, β-blockers became a first line therapy to treat high blood pressure. However, despite
their ability to control blood pressure, β-blockers were not as effective as Ca2+ channel blockers
(CCBs) and angiotensin converting enzyme inhibitors (ACE-Is) at reducing stroke and
mortality. In perioperative medicine, initial trials recommended their use as they coincided with
a reduction in adverse cardiac events (eg. nonfatal myocardial infarction or ischemia and cardiac
death). A recent randomized controlled trial in perioperative medicine (POISE) revealed that β-
blockade with the common β1-antagonist, metoprolol, reduced the incidence of myocardial
infarction. However, metoprolol administration was also associated with an increased risk of
stroke and death during the perioperative period. This emphasized that in certain clinical settings
(surgery, acute blood loss) the inhibition of cardiac responsiveness may have the consequence of
decreased vital organ perfusion In support of this assumption, a growing body of evidence has
demonstrated an increase in cerebral ischemia and mortality associated with β-blocker therapy
in a diverse number of clinical settings. Defining the mechanisms contributing to these negative
2
outcomes is essential in informing clinical practice to develop effective preventative therapeutic
strategies.
The general premise of this in vivo and in vitro experimental study is that a vascular
mechanism could contribute to β-blocker induced cerebral ischemia and mortality. This premise
is based on the following lines of reasoning: 1) Anti-hypertensive therapy with primary
vasodilators (ACE inhibitors, Ca2+ channel blockers) are associated with improved survival and
fewer strokes when compared with β-blockers; 2) β-blockers with vasodilatory capacity
(carvedilol) are associated with a lower stroke incidence and mortality when compared to β-
blockers that do not cause vasodilation (metoprolol); 3) β-blockers with greater vascular β2-
adrenergic receptor cross reactivity (metoprolol) are associated with increased mortality when
compared to more β1-cardioselective β-blockers (atenolol, bisoprolol); 4) There is evidence that
shows that stimulation of both β1- and β2- adrenergic receptor subtypes initiate vasodilatory
mechanisms in vascular smooth muscle. Thus β-blockade may have a negative impact on the
ability of the resistance vasculature to dilate, either through a direct β1-effect or by cross-
reacting with the β2-receptor. We therefore propose the following hypothesis:
Cardioselective β1-adrenergic antagonists impair resistance artery vasodilation and increase
the risk of organ ischemia.
The β-blocker metoprolol was chosen as our study drug because of its extensive clinical
use in North America and its specific pharmacology which may promote increased morbidity
and mortality within this class of drugs.
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7 Introduction
7.1 Clinical efficacy of beta-blockers
The β-adrenergic receptor (β-AR) antagonist class of drugs has been growing in
popularity as a cardiovascular intervention over the last half century. With 128.3 million
prescriptions in 2009 they were the 5th most dispensed class of medicine in North America (IMS
Health). Though they are currently used for the treatment of a wide range of cardiovascular
ailments (hypertension, myocardial infarction, chronic heart failure) and various non-
cardiovascular morbidities (glaucoma, migraine headaches, essential tremors, alcohol
withdrawal)1, β-blockers were initially developed as a specific treatment for angina pectoris2.
By inhibiting sympathetic activation of the heart through the β-adrenergic receptor, these drugs
are able to limit myocardial oxygen demand in order to match supply. In this way they protect
the heart from ischemia and their use subsequently expanded to post-MI and chronic heart
failure treatments. In addition to an expansion of their clinical indications, β-blockers also
developed from first generation, non-selective β1/2-antagonists, such as propranolol, to the
second generation β1-, “cardioselective”, antagonists, such as metoprolol, and are now in their
third generation which includes both nonspecific and β1-specific antagonists that have
vasodilating capacity, such as carvedilol and nebivolol respectively (Table 7.1). Because of their
efficacy in treating ischemic heart disease, their use further expanded to the treatment of
hypertension and as a prophylactic against myocardial ischemia and infarction in the
perioperative setting.
4
Drug Receptor Selectivity*
Current Clinical
Use*
Additional Actions* Fold Selectivity for β1-AR vs. β2-AR**
Nadolol β1/2 -
Penbutolol β1/2 -
Pindolol β1/2 -
Propranolol β1/2 + -
Timolol β1/2 -
Acebutolol β1 + - 2.4
Atenolol β1 ++ - 4.7
Bisoprolol β1 ++ - 13.5
Esmolol β1 + -
Metoprolol β1 +++ - 2.3
Nebivolol β1 Stimulates eNOS
Labetalol β1/2 α1-AR antagonist
Carvedilol β1/2 ++ α1-AR antagonist
Celiprolol β1 Partial β2-AR agonism
Table 7.1 Pharmacological properties of clinically used beta-blockers *Adapted from Pulido and Kor 20083. **Adapted from Baker 20054.
5
7.1.1 Beta-blockers alleviate angina symptoms and reduce morbidity and mortality
Beta-blockers were initially developed as a targeted treatment for stable angina pectoris
which is characterized by myocardial ischemia due to an increase in oxygen demand without the
consequent increase in supply. Stable angina manifests in patients as severe chest pains during
periods of activity which disappear once activity has ceased. Propranolol was first indicated in
the management of stable angina in 1964, shortly after its discovery by Sir James Black (See
Appendix A). In a small trial (n=20), the authors were encouraged by a trend of increased
number of days without symptoms, reduced number of angina attacks and increased level of
patient satisfaction with β-blockade5. In another small (n=19) double blind crossover trial there
was a significant reduction in the number of angina attacks and in the number of nitrate tablets
(to treat angina) taken during the 8 weeks of study (4 weeks of placebo, 4 weeks propranolol)6.
The 306 patient double-blind placebo controlled Atenolol in Silent Ischemia Study Trial
(ASIST) remains to date the largest randomized controlled trial (RCT) assessing β-blocker
efficacy in angina7. Atenolol treatment resulted in a 56% reduction in adverse events (death, MI,
unstable angina, aggravation of angina, revascularization) during the 1 year long follow up
period7.
Larger trials assessing the efficacy of β-blockade in managing angina have been reserved
for studies of its effectiveness in comparison to other monotherapies and in combination
therapies. Two large trials (TIBET and APSIS) demonstrated that β-blockade alone resulted in
no better outcomes than Ca2+ channel blockade alone or in combination8;9. Without placebo
controls it is difficult to assess the efficacy of β-blockade in the long-term prognosis of angina
in these two trials.
With a lack of large randomized controlled trials, the indication for β-blockade in
patients with angina pectoris has been extrapolated from RCTs of related cardiovascular
6
morbidities. As will be discussed further, β-blockers have been proven to improve outcomes in
post-MI and heart failure patients 10-12. For this reason they are currently suggested as first line
therapy by the European Society of Cardiology and the American Heart Association in post-MI,
acute coronary syndrome and heart failure patients diagnosed with stable angina pectoris13;14.
7.1.2 Beta-blockers reduce reinfarction and mortality post-myocardial infarction
Early experimental studies found that β-blockade, with propranolol, was able to reduce
myocardial infarct (MI) size by 56 and 28% before and 3 hours after left anterior descending
coronary artery occlusion15. In the clinical setting, β-blocker prophylaxis for acute MI is next to
impossible; trials have therefore assessed the effectiveness of β-blockade soon after MI or
suspected MI. In these trials MI is defined a rise in plasma troponin in addition to at least one of
the following: 1) ischemic symptoms (chest, epigastric, arm wrist or jaw pain or dyspnea); 2)
development of pathologic Q-waves on an ECG; 3) ST segment elevation or depression or T
wave inversion.
The earliest clinical trials revealed that immediate (within 12 hours after the onset of MI
symptoms) and sustained β-blockade until patient discharge was associated with a 50%
reduction in risk of developing definite infarct or reinfarction after 10 days and a 36% reduction
in mortality after 90 days16-18. In the much larger Norwegian β-Blocker Heart Attack Trial
(BHAT), patients with suspected MI were treated orally with placebo (n=1921) or propranolol
(n=1916) within 5 to 21 days post-MI. The BHAT demonstrated that propranolol reduced total
mortality by 28% and total fatal and nonfatal coronary heart disease by 23% over a 3 year
follow up period19;20. Since BHAT did not address the effect of immediate β-blocker
intervention, nor short-term patient outcomes, an even larger RCT, Metoprolol in Acute
Myocardial Infarction (MIAMI), was conducted. Patients eligible for the MIAMI trial
(presenting in hospital within 24 hours of the onset of MI symptoms) were randomized to
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placebo (n=2901) and metoprolol (n=2877) treatments. In high risk patients, metoprolol
treatment reduced 15 day post-MI mortality by 29% 21. The first International Study of Infarct
Survival (ISIS-1) was conducted in 16207 patients with 8037 receiving early intravenous
atenolol and 7990 receiving no treatment within 5 hours after the onset of suspected MI for 2
weeks. ISIS-1 found that there was a significant 15% reduction in mortality up to 7 days
following treatment with atenolol versus no treatment22. ISIS-1 also demonstrated that the
benefits of β-blockade are most prominent during the first 24 hours of treatment22 following MI,
prompting the question of whether early β-blockade is beneficial versus delayed β-blockade.
The Thrombolysis in Myocardial Infarction Phase II (TIMI-II) trial found a significant 60%
reduction in the incidence of death and reinfarction after 6 weeks in a subgroup of patients
treated with immediate metoprolol (n=185) versus those in which β-blockade was deferred until
6 days after symptom onset (n=190)23. In the largest RCT of β-blockers to date, the Clopidogrel
and Metoprolol in Myocardial Infarction Trial (COMMIT) showed a 25 and 20% relative risk
reduction of reinfarction and ventricular fibrillation, respectively, following metoprolol (n=22
929) vs. placebo (n=22 923) treatment within 24h of suspected MI24.
A clear benefit has therefore been associated with early β-blockade in post-MI patients.
The American Heart Association has consequently recommended β-blockade as a management
strategy in all patients without contraindications due to the apparent early and long-term
mortality risk reductions in post-MI patients10.
7.1.3 Beta-blockers reduce morbidity and mortality in chronic heart failure
Chronic heart failure (CHF) is generally characterized by ventricular dysfunction leading
to an impairment of cardiac output and consequently a reduced capacity of the heart to supply
blood throughout the systemic circulation. In order to compensate for the reduction in pumping
capacity of the heart, the sympathetic nervous system and renin-angiotension-aldosterone
8
system are recruited to preserve systemic blood flow25. These initial benefits of sympathetic
activation (ie. positive inotropy) following myocardial insult led to the contraindication of β-
blockers (negative inotropic agent) in CHF. However, in the mid-1990s, a neurohormonal
hypothesis for the progression of CHF proposed that the increased concentration of circulating
catecholamines and angiotensin II eventually leads to long-term deleterious effects on cardiac
tissue and therefore the worsening of CHF26. This theory paved the way for the first-line usage
of angiotensin converting enzyme (ACE) inhibitors in CHF patients and eventually led to the
reconsideration of β-blockade as a therapeutic approach26.
The US Carvedilol Heart Failure Study Group was among the first large RCTs to
examine the effect of β-blockade with carvedilol on mortality in CHF patients. The study
enrolled patients with a left ventricular ejection fraction (LVEF) less than 35%. The overall
relative risk reductions with carvedilol treatment after 12 months was 65% in mortality and 27%
in risk of hospitalization for cardiovascular reasons27. In the Carvedilol Prospective Randomized
Cumulative Survival Study Group (COPERNICUS) trial, the efficacy of carvedilol in CHF was
tested specifically in severe HF patients (LVEF <25%). There was a significant 34% relative
risk reduction in 10 month mortality with carvedilol (n=1156) treatment compared to placebo
(n=1133)28. It has been suggested that carvedilol, a non-selective β-blocker, may be especially
beneficial due to its α1-adrenergic blocking ability and its antioxidant effect which could help to
reduce the loss of cardiomyocytes while the disease progresses. However, substantially larger
trials using β1-selective blockers have shown similar profound survival benefits to CHF patients.
The Cardiac Insufficiency Bisoprolol Study II (CIBIS-II) examined the effect on 12
month survival of the β1-selective antagonist bisoprolol in patients (placebo n=1320; bisoprolol
n=1327) with an LVEF less than 35%. CIBIS-II demonstrated 32, 15 and 25% relative risk
reductions in all cause mortality, all cause hospital admissions and all cardiovascular deaths
9
respectively29. Similar findings were revealed by the much larger Metoprolol CR/XL
Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF). Metoprolol (n=1990)
treatment was associated with a 38% reduction in total mortality as well as cardiovascular death
and a 48% reduction in risk of death from worsening heart failure compared to placebo
(n=2001)30. The mechanisms of cardioprotection remain unclear, however an anti-fibrillatory
effect of β1-blockade as well as reduced myocardial oxygen demand and apoptosis may
contribute29;30.
Despite early reservations, β-blockers have emerged as being overwhelmingly successful
in the management of CHF. In fact, in each of the RCTs outlined above, the respective data
monitoring committees were obligated to terminate the studies earlier than anticipated due to the
substantial reduction in mortality compared to placebo treatments27-30. Consequently, β-blockers
have been recommended by both the American College of Cardiologists/American Heart
Association and the European Society of Cardiology as first-line treatment in all CHF patients
without contraindications to the treatment11;12.
7.1.4 Beta-blockers reduce blood pressure and risk of stroke in hypertensive patients
Hypertension, defined as a systolic blood pressure over 140mmHg and a diastolic blood
pressure above 90mmHg, has become one of the more prevalent disease in the western world
and, if uncontrolled, can lead to future cardiovascular complications (stroke, MI, CHF, angina).
Few placebo-controlled trials in hypertension exist due to the ethical dilemma of
withholding anti-hypertensive treatment from hypertensive control patients. One of the largest
of such trials, The International Propsective Primary Prevention Study in Hypertension
(IPPPSH), tested the non-selective β-blocker oxprenolol (n=3185) against placebo (n=3172)
over a 3-5 year follow up period in patients 40-64 years old with diastolic blood pressures above
100mmHg31. Oxprenolol brought about a significant reduction in blood pressure; however it
10
was no more effective at reducing stroke rates, MI and mortality than placebo treated patients31.
The Medical Research Council in England studied the effectiveness of propranolol (n=2285)
against placebo (n=4525) in managing mild hypertension (diastolic blood pressure between 90-
109mmHg) in 35-64 year olds. Propranolol treatment reduced systolic and diastolic blood
pressures by 10 and 6mmHg respectively after a 5 year follow up period32. They also reported a
27% relative risk reduction in all strokes (fatal and non-fatal) in patients treated with
propranolol32. In a study of elderly patients (60-79 years old; mean systolic and diastolic blood
pressures 196 and 99mmHg respectively), Coope et al. (1986) reported the antihypertensive
benefits of atenolol (n=419) versus placebo (n=469) during a mean 4.4 year follow up period.
Atenolol treatment reduced systolic and diastolic blood pressures by 20 and 11mmHg
respectively33. Additionally, atenolol treatment was associated with a 41% relative risk
reduction in the incidence of fatal and non-fatal strokes but did not confer any significant
reductions in adverse cardiovascular events33. In another study of atenolol as an anti-
hypertensive in the elderly (ages 65-74), the Medical Research Council found a similar
reduction in mean systolic and diastolic pressures (20 and 10 mmHg respectively) after a mean
5.6 year follow up period 34. Like in Coope’s trial, there was a significant reduction in the risk of
stroke (though only by 17%) and no apparent benefits in reducing adverse cardiovascular
events34.
These placebo controlled trials identified β-blockers as effective anti-hypertensives
despite their inability to improve long-term prognosis. Until recently β-blockers were accepted
as a first-line therapy, however they have fallen out of favour due to their inferiority to other
common cardiovascular pharmaceuticals (ACE inhibitors, Ca2+ channel blockers)35;36. The
reasons for their change in status from 1st line to 3rd line antihypertensive agents will be further
explored in section 7.4.1.
11
7.1.5 Beta-blockers reduce the risk of MI in perioperative patients at cardiac risk
Following the success of β-blockade in various cardiovascular morbidities, the treatment
quickly moved into prophylactic use in surgical patients at cardiac risk. Two small early studies
guided the practice of perioperative β-blockade. In 1996, treatment with atenolol (n=99) was
tested against placebo (n=101) in patients with or at risk of coronary artery disease undergoing
non-cardiac surgery. Atenolol treatment was associated with significantly higher 2-year survival
rates (90 vs. 79%) and 2-year event free survival (83 vs. 68%) than placebo37. In 1999, similar
findings were found in patients with 1 or more cardiac risk factors and a positive dobutamine
stress test treated with bisoprolol (n=59) or placebo (n=53). Bisoprolol significantly reduced
death by cardiac causes (80% relative reduction in risk) and incidence of non-fatal myocardial
infarction (100% relative risk reduction)38. In a 2-year follow-up of these patients, there was a
63% relative risk reduction in cardiac events (cardiac death or non-fatal MI) with bisoprolol
treatment39. Though these were very small trials (n<200 patients), perioperative β-blockade
quickly became a standard clinical practice40. Future studies, however, would call into question
the value of this treatment strategy.
The 2005 POBBLE study tested metoprolol (n=55) versus placebo (n=48) in elderly
patients undergoing vascular surgery. Metoprolol treatment offered no significant survival or
cardioprotective benefits than placebo treatment41. In the metoprolol after vascular surgery
(MaVS) trial, there was no observed benefit in reducing the primary outcome (non-fatal MI,
angina, CHF, atrial or ventricular dysrhythmia or cardiac death)42. In the Diabetic Post-
Operative Mortality and Morbidity (DIPOM) trial, there was again no significant benefit to
metoprolol treatment for reduction in the incidence of mortality, non-fatal MI, angina or CHF
after an 18 month follow up period43. In the Swiss Beta-Blocker in Spinal Anesthesia (BBSA)
trial, patients undergoing non-cardiac surgery with spinal block were treated with bisoprolol
12
(n=110) or placebo (n=109). Again, there was no observed benefit in reducing cardiac death,
non-fatal MI, angina or CHF in β-blocked patients44. In 2008, the Perioperative Ischemic
Evaluation (POISE) study group published the results of its multicenter, 8000 patient strong
prospective analysis of perioperative β-blockade. The POISE trial is the largest in perioperative
medicine and tested the effect of metoprolol (n=4174) against placebo (n=4177) in patients with
or at risk of atherosclerosis undergoing non-cardiac surgery. They found that metoprolol
treatment resulted in a significant 16% reduction in risk of reaching the primary outcome
(cardiovascular death [death due to cardiac arrest, MI, pulmonary embolus, stroke, hemorrhage
or following any cardiovascular procedure], non-fatal MI or non-fatal cardiac arrest) and that
this risk reduction was driven by a significant 26% reduction in the relative risk of non-fatal
MI45. Reduced cardiovascular morbidity risk was confirmed in a recent RCT demonstrating a
65% reduction in the relative risk of cardiac death and non-fatal MI46. A meta-analysis of
perioperative β-blocker trials further supports the cardioprotective effect of β-blockade47,
however, the POISE findings were controversial because of two alarming findings; increased
risks of all cause mortality and stroke in the metoprolol group45.
7.2 Increased risk of mortality and stroke associated with beta-blockade
Despite the proven cardioprotective benefits of β-blocker treatment in a wide range of
cardiovascular diseases, their use in certain patient populations is debatable. Specifically, in the
perioperative setting, where β-blocker therapy has seen a recent proliferation over the last two
decades, there is growing evidence of increased risk of harm in treated patients. The landmark
2008 POISE trial demonstrated that metoprolol treated patients experienced a 100% increase in
relative risk of developing fatal or non-fatal strokes45. 82% of these strokes were ischemic
strokes in nature while the remaining 18% were hemorrhagic or undefined. Of the ischemic
strokes, the investigators did not define whether they were hypo-perfusion or thrombosis related.
13
Additionally, the POISE investigators reported a 35% increase in the relative risk of all-cause
mortality (this includes cardiovascular and non-cardiovascular deaths) in metoprolol treated
patients. Development of a stroke resulted in an 18-fold increase in risk of death45. Shortly after
the publication of POISE, a meta-analysis of 33 randomized placebo-controlled perioperative β-
blockade trials revealed an alarming 116% increase in the relative risk of developing a non-fatal
stroke with β-blocker therapy as well as a 12.5% relative risk increase in all-cause mortality47.
The POISE study also reported that increased harm was associated with β-blockade in low
cardiac risk patients (revised cardiac risk scores ≤ 2)45. This finding was previously reported in a
large retrospective analysis of over 300, 000 propensity matched patients undergoing major non-
cardiac surgery. Lindenauer and colleagues reported that in patients with an RCRI score of 0 or
1 there was an increased risk of mortality with β-blocker treatment48. Furthermore, in the MaVS
trial where the large majority of patients were classified with an RCRI of ≤ 2 there was no
observed benefit of perioperative β-blockade in reducing primary outcomes42. Similarly, in
DIPOM and POBBLE there were again no observed benefits to metoprolol treatment41;43.
Increased risk of negative outcomes persists outside of the perioperative setting. In the
CIBIS-II trial in patients with heart failure there was a 91% increased risk of stroke with
bisoprolol treatment compared to placebo29. In the COMMIT trial, despite the benefits in
reducing the risk of reinfarction and ventricular fibrillation, there was a 28% increase in the risk
of cardiogenic shock when post-MI patients were treated with metoprolol24. In hypertension, β-
blockers are associated with 26% increased relative risk of stroke49 when compared to other
commonly used cardiovascular pharmaceuticals (CCBs, ACE-Is)50-55. These findings suggest
that patient selection is crucial in optimizing the benefits and minimizing the harm with β-
blocker therapy. There may also be a large population of patients being subjected to unnecessary
risks through treatment with β-blockers. It is therefore important to elucidate the mechanism by
14
which β-blockade could promote organ ischemia (ie. stroke) in order to better inform clinical
practice. In a recent meta-analysis and review, the risk of stroke and mortality associated with
prerioperative β blocker therapy was attributed to: 1) Degree of hepatic metabolism, 2) Dose
titration and 3) Relative degree of β1/2-AR selectivity56. Thus, a clearer understanding of the beta
receptor physiology is required.
7.3 Beta-Adrenergic signalling in regulating the cardiovascular system
Lands and colleagues, in 1967, discovered the two subtypes responsible for the
physiologic effects of β-AR activation. Using isolated cardiac, adipose, bronchiole and vascular
tissue, Lands et al. observed different orders of activity to stimulation with isoproterenol,
norepinephrine and epinephrine within these samples57;58. It was discovered that heart rate,
myocardial force of contraction and lipolysis in adipose tissue had a potency order of (most to
least potent) isoproterenol > norepinephrine = epinephrine and consequently designated these
responses as being β1-mediated58. Bronchodilation and vasodilation responses were found to
follow an activity order of isoproterenol > epinephrine >> norepinephrine and consequently
were designated as β2-mediated responses. For the purposes of this report, cardiac and vascular
β-adrenergic signalling will be of particular focus.
7.3.1 Cardiac beta-adrenergic signal transduction pathways
Sympathetic activation of the β-ARs expressed on the heart result in an increase in heart
rate (chronotropy), contractility (inotropy) and relaxation rate (lusitropy). By modulating these
latter two characteristics, β-AR activation results in increased stroke volume. Coupled with an
increase in heart rate, β-adrenergic stimulation increases cardiac output in order to maintain
adequate tissue perfusion in the typical sympathetic fight or flight response59. At the cellular
15
level, the downstream effectors of β-AR activation must therefore modulate two fundamentally
different physiologic responses; pacemaking and myogenic force production.
The β-ARs are Gs-protein coupled receptors expressed on both atrial and ventricular
cardiomyocytes. The receptors are composed of 7 transmembrane regions with an extracellular
N-terminus and intracellular C-terminus. They are made up of approximately 400-500 amino
acids and the β1- and β2-ARs show 48.9% sequence homology60.The ratio of expression of the
β1-AR to β2-AR subtypes is approximately 80:20% in ventricular myocytes and 70:30% in atrial
myocytes61. When bound by the endogenous catecholamines (norepinephrine and epinephrine),
both β-AR subtypes catalyze the transfer of a phosphate from guanosine triphosphate (GTP) to
the Gαs subunit of the heterotrimeric G-protein complex. This causes the dissociation of the Gαs
subunit from the βɣ subunits. The Gαs subunit then goes on to activate membrane bound
adenylyl cyclase which is responsible for catalyzing the conversion of adenosine triphosphate
(ATP) to cyclic adenosine monophosphate (cAMP)62. The ubiquitous second messenger, cAMP,
binds to protein kinase A (PKA) allowing the dissociation of its catalytic units from its
regulatory units. The catalytic units of PKA go on to phosphorylate various downstream
molecules that consequently regulate chronotropy, inotropy and luistropy59. The signalling
pathways are summarized in Figure 7.3.1.
7.3.1.1 Beta-adrenergic modulation of chronotropy
The heart is unique in its ability to generate spontaneous contractile activity through the
specialized pacemaker myocytes localized within the sinoatrial node (SAN)63. Action potentials
generated within the SAN are propagated through the left and right atria to the atrioventricular
node (AVN) where the electrical signal is transmitted to the left and right ventricles through the
bundle of His and Purkinje fibres. With its high intrinsic rate of action potential generation, the
SAN serves as the primary pacemaker of the heart, although the AVN and Purkinje fibres also
16
display automaticity, albeit at a slower rate64. It is understood that diastolic depolarization, a
feature distinctly lacking in the force generating cardiomyocytes, is responsible for the
spontaneous activity of pacemaker cells. Specifically, the inward “funny” current (If), activated
during diastole, is responsible for the slow depolarization of a pacemaker cell following the
termination of the previous action potential. As the cell is slowly depolarized by the If it will
eventually reach the threshold potential (-55mV) and subsequently cause the generation of a
new action potential63;64.
17
AdenylylCyclase
ATPcAMP
Sarcoplasmic Reticulum
PLB
[Ca2+]sr
[Ca2+]i
SERCA
Gαs Gβγ
GTPGDP
Protein Kinase A
β1/2-AR
RyR
[Na+]ec If [Na+]i
[Ca2+]ec
Cav1.2
[Ca2+]ec
[Ca2+]i
Cav1.2
Figure 7.3.1 Summary of cardiac beta-adrenergic signalling. See text for details. β1/2-AR = β1/2-adrenergic receptor; If = funny current channel; [Na+]ec = extracellular Na+; [Na+]i = cytoplasmic Na+; [Ca2+]ec = extracellular Ca2+; [Ca2+]i = cytoplasmic; Ca2+; [Ca2+]sr = sarcoplasmic reticulum Ca2+; Cav1.2 = voltage gated L-Type Ca2+ channel; PLB = phospholamban; SERCA = sarcoplasmic/endoplasmic reticulum calcium ATPase; RyR = ryanodine receptor.
18
Stimulation of the β-adrenergic receptor in preparations of SAN cells has shown that the
increases in heart rate from sympathetic activation are likely caused by a shortening and
steepening of the slope of diastolic depolarization65. This causes the threshold potential to be
reached faster during diastole and consequently will cause the generation of more action
potentials per minute. Critical to this phenomenon is the modulation of If activity. In early
experiments, it was discovered that application of epinephrine in whole cell and single If
channel patch clamp preparations increases channel open probabilities and therefore If 66. Beta-
adrenergic modulation of If is mediated by direct binding of cAMP to the If channel as opposed
to phosphorylation by PKA. In patch clamp preparations of SAN cells, application of the
catalytic subunit of PKA alone did not activate If ; however channel activation was observed
when cAMP (alone or with PKA) was applied67. Therefore, β-AR stimulation increases heart
rate through direct action of cAMP on If channels by increasing open probability and therefore
activation of If.
7.3.1.2 Beta-adrenergic modulation of contractility
In regulating cardiac inotropy and lusitropy, β-adrenergic signalling primarily acts on L-
type Ca2+ channels (primarily the Cav1.2 channel), ryanodine receptors, phosphlamban and the
troponin complex. These former three molecules, when activated, modulate intracellular Ca2+
concentrations ([Ca2+]i) which in turn modulates the actions of troponin. Increases in [Ca2+]i
cause contraction whereas decreases in [Ca2+]i promote relaxation64.
In cardiomyocytes, the Cav1.2 channel is primarily responsible for conductance of the
contraction inducing calcium current (ICa)68. Treatment of isolated rat ventricular myocytes with
the non-selective β-AR agonist, isoproterenol, has shown 3-fold increases in ICa through
phosphorylation at the serine 1928 residue on the C-terminal end of Cav1.2 channel69.
Incubation of cardiomyocytes with β-AR specific-antagonists revealed that the β1- and β2-ARs
19
are responsible for 65% and 35% of the phosphorylation at S1928 respectively. Furthermore,
forskolin, an adenylyl cyclase agonist, mimicked the increases in phosphorylation and ICa,
implicating PKA in the phosphorylation and activation of Cav1.269. It is likely that the A kinase
anchoring protein 15 (AKAP15) is responsible for the differential phosphorylation patterns
observed with β1- or β2-AR activation. Via a leucine zipper motif, AKAP15 colocalizes with the
C-terminal end of the Cav1.2 channel, allowing targeted activation of PKA at the plasma
membrane and in close proximity to its intended effector molecule70. It is suggested that β1-AR
activation is responsible for activation of Cav1.2 throughout the cell by a general increase in
cytosolic cAMP whereas β2-AR activation produces increases in local cAMP at specific areas
near the cell surface69. These regions seem to be associated with cardiac t-tubules as well as
ryanodine receptors (RyRs).
The RyRs are integral in cardiac excitation-contraction coupling. Localized on the
membrane of the sarcoplasmic reticulum in cardiomyocytes, they are stimulated by cytoplasmic
Ca2+ to release sarcoplasmic Ca2+ thereby greatly increasing [Ca2+]i in a process known as Ca2+
induced Ca2+ release (CICR). The β-ARs therefore indirectly regulate RyRs through their
activation of L-type Ca2+ channels which are responsible for the initial increase in [Ca2+]i
necessary for CICR64. However, there is emerging evidence for a direct regulatory role of β-
AR/cAMP/PKA on RyR function. Using patch clamp techniques and confocal imaging in
isolated rat ventricular myocytes, it has been discovered that β-AR activation (isoproterenol) is
responsible for an increase in the number of RyRs that simulatenously respond to the Ca2+
transient of a single L-Type Ca2+ channel71. This synchronization of RyR activation is abolished
when cardiomyocytes were pretreated with a cAMP antagonist, implicating the importance of
PKA in this process. Modulation of the synchronization of RyR opening is suggested to be
integral in producing immediate increases in heart pumping power 71.
20
RyRs cause Ca2+ efflux from intracellular stores; however, the
sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) is responsible for pumping Ca2+
out of the cytoplasm and back into the sarcoplasmic reticulum. In so doing, the SERCA pump
reduces [Ca2+]i and therefore is important in cardiomyocyte relaxation64. Phosphlamban is a 52
residue sarcoplasmic reticulum membrane protein that is in direct contact with SERCA. In
resting conditions it is dephosphorylated and serves to inhibit the function of SERCA, thereby
reducing the rate at which [Ca2+]i is removed from the cytoplasm. Beta-adrenergic stimulation,
however, causes phosphorylation of phospholamban at its serine-16 residue by PKA72.
Phosphrylated phosphlamban no longer inhibits SERCA, allowing an increased rate of Ca2+
influx to the sarcoplasmic reticulum. The increased rate of [Ca2+]i efflux from the cytoplasm
allows for the increased rate of relaxation necessary for the heart to keep up with sympathetic
increases in chrontropy. Therefore by modulating Ca2+ cycling in cardiomyocytes, the β-ARs
are responsible for increasing cardiac output to meet the increased oxygen demand associated
with sympathetic nervous system activation.
7.3.2 Vascular beta-adrenergic signal transduction pathways
Lands et al. identified the β2-AR receptor as the primary subtype associated with smooth
muscle relaxation and specifically with vasodilation in vascular smooth muscle cells
(VSMCs)58. Though β2-vasodilation has been entrenched since Lands et al.’s findings, there is
evidence suggesting a tissue and species specific functional role of the β1-AR in promoting
vasodilation (see section 7.3.2.1). β-AR activation follows the same signalling pathways
regardless of receptor subtype being stimulated, and, similar to cardiomyocyte signalling, there
is a central role of cAMP/PKA in mediating VSMC relaxation. The vasodilatory β-adrenergic
signalling pathways, compared to the comparison to the vasoconstriction pathway of α1-
adrenergic signalling, are show in Figure 7.3.2.
21
AdenylylCyclase
ATPcAMPSarcoplasmic Reticulum
MLC20-P(vasoconstriction)
MLC20
(vasodilation)
PLCPIP2
IP3
Calmodulin
PLB
[Ca2+]sr
IP3R
[Ca2+]iSERCA
Gαs Gβγ
GTPGDP
Protein Kinase A
MLCP MLCK
Gαq
β1/2-AR
GTPGDP
Gβγ
α1-AR
LCC [Ca2+]i
[K+]i
KCa
[K+]ec
KATP
[Ca2+]ec
Figure 7.3.2 Summary of vascular beta-adrenergic signalling. See text for details. β1/2-AR = β1/2-adrenergic receptor; α1-AR = α1-adrenergic receptor; [K+]ec = extracellular K+; [K+]i = cytoplasmic K+; [Ca2+]ec = extracellular Ca2+; [Ca2+]i = cytoplasmic; Ca2+; [Ca2+]sr = sarcoplasmic reticulum Ca2+; LCC = voltage gated L-Type Ca2+ channel; KCa = Ca2+ sensitive K+ channel; KATP = ATP-sensitive K+ channel. PLB = phospholamban; SERCA = sarcoplasmic/endoplasmic reticulum calcium ATPase; IP3 = inositol trisphosphate; IP3R = inositol trisphosphate receptor; PLC = phospholipase C; PIP2 = phosphoinositol diphosphate; MLC20 = myosin light chain; MLC20-P = phosphorylated MLC; MLCP = MLC phosphatase; MLCK = MLC kinase.
22
As in cardiomyocytes, dissociation of the Gαs subunit from Gβɣ following β-AR
receptor activation causes stimulation of L-type Ca2+ channels (LCCs) in vascular smooth
muscle cells. It has been shown that in isolated rabbit vein myocytes, simultaneous PKA and
PKC inhibition completely abolishes isoproterenol induced LCC activation73. Partial inhibition
of the ICa is observed when myocytes are treated with one of the two protein kinase inhibitors.
Furthermore, when either Gβɣ or Gαs is inhibited, the ICa is reduced and is completely abolished
when both subunits are inhibited during isoproterenol treatment73. A PKC inhibitor blocks Gβɣ
increases in ICa and a PKA inhibitor blocks ICa increases by Gαs implicating these two second
messengers in the activation of LCCs.
LCC activation causes an influx of Ca2+ into the cell, and raises the question of how
increased [Ca2+]i by β-AR stimulation can promote vasodilation when increased Ca2+ is required
for vasoconstriction. The answer lies within the downstream effects of Gαs in both PKA-
independent and dependent processes. Gαs has been discovered to potentiate the efflux of K+
through the calcium activated K+ channel (KCa) by both PKA independent and dependent
mechanisms74. This causes membrane hyperpolarization which limits further increases in [Ca2+]i
by closing voltage gated Ca2+ channels75. Additionally, it has been shown that PKA activates the
vascular ATP-sensitive K+ channel (KATP) which could contribute to membrane
hyperpolarization and therefore VSMC relaxation76. In order to further limit [Ca2+]i, PKA,
similarly to cardiomyocytes, will also inhibit the actions of phospholamban on the SERCA
pump in VSMCs. SERCA pumps Ca2+ out of the cytoplasm and into the endoplasmic reticulum,
reducing global [Ca2+]i77. In addition to reducing [Ca2+]i, the increased SR Ca2+ load caused by
phospholamban inhibition causes an increased frequency of Ca2+ sparks which can activate
KCa77. This ultimately leads to increased hyperpolarization, and thus reduced [Ca2+]i. While
23
PKA has an important role in hyperpolarizing VSMCs and globally reducing [Ca2+]i, it is also
central in reducing the Ca2+ sensitivity of the cell to further promote vasodilation.
In VSMCs, the actual contractile force is generated by ATP dependent cross-bridge
formation of myosin with actin. Myosin slides a short distance along the actin filament before
releasing and completing the cross-bridge cycle78. This cross-bridge cycling is modulated by
myosin light chain (MLC20), a component of the myosin filament that, when phosphorylated at
serine-19, activates the ATPase of myosin allowing cross-bridge formation78. MLC20 is itself
regulated by an MLC kinase and phosphatase (MLCK; MLCP). MLCK is activated by the Ca2+-
calmodulin (Ca2+/CaM) complex, causing phosphorylation of MLC20 and consequently
vasoconstriction. PKA exerts an inhibitory role on MLCK by phosphorylating serine-1760 and
thereby reducing the affinity of MLCK for Ca2+/CaM79;80. Furthermore, PKA has been shown to
phosphorylate serine-695 of the myosin targeting subunit (MYPT1) of MLCP81. This prevents
the Rho-associated kinase phosphorylation of MLCP and its subsequent deactivation82;83. PKA
therefore inhibits MLCK activity while promoting MLCP activity. This shifts the balance within
the VSMC from constriction to vasodilation by increasing the concentration of
dephosphorylated MLC20.
There is evidence that β-adrenergic mediated vasodilation has a nitric oxide component.
Though the direct signalling pathways are yet to be elucidated, it has been demonstrated that
endothelial nitric oxide synthase (eNOS) is upregulated with β-AR receptor stimulation84-86.
Ferror and colleagues propose that activation of eNOS results from PKA activation and by
Akt/P13K. They suggested that the Gβɣ subunit may activate Akt/P13K and consequently cause
NO to be released from the endothelium86.
24
7.3.2.1 Distribution across vascular beds
Activation of the sympathetic nervous system fight or flight response involves
preferential distribution of blood flow to organs necessary for survival (brain, heart, skeletal
muscle). The endogenous adrenergic catecholamines (epinephrine, norepinephrine) can bind to
both the vasoconstricting α1-adrenergic receptor and the vasodilating β-ARs. The relative
expression and functionality of these receptor subtypes will therefore determine whether an
organ sees reduced or increased perfusion following sympathetic activation87.
The distribution of β-ARs has been well characterized in the cerebral circulation of large
mammals. Bovine anterior, internal carotid and middle cerebral artery strips demonstrate α1-
adrenergic activity (ie. contraction) when stimulated by norepinephrine while posterior cerebral,
posterior communicating and basilar arteries (caudal arteries) are mediated by the β-AR (ie.
relaxation with norepinephrine treatment)88. Furthermore, relaxation in these latter three arteries
can be reversed to contraction when the isolated artery strips are treated with propranolol. Using
metoprolol (β1-specific antagonist) and butoxamine (β2-specific antagonist) this same group
found that each of the caudal arteries were predominantly under the functional control of the β1-
adrenergic receptor89. In humans, radioligand binding assays revealed both β1- and β2-AR
expression in human basilar and middle cerebral arteries in a 40:60% ratio90;91. Interestingly,
pharmacological studies of isolated human pial artery strips show a predominant role of the β1-
AR in mediating vasodilation based on low sensitivity of arteries to the β2-selective agonist
tertbutaline92. In swine basilar artery rings, radioligand binding has demonstrated a 65:35% β1-
/β2-AR expression ratio as well as a functionally dominant role of the β1-AR in the vasodilatory
response of these arteries93. In feline middle cerebral artery rings, there is expression of both β-
AR subtypes with the β1-AR being predominantly functional based on reduced potency to
tertbutaline94. Radioligand binding in rat middle cerebral arteries reveal expression of both β-
25
ARs91 and vasodilation in isolated rat middle cerebral, posterior cerebral and basilar artery rings
showed equivalent responses to β1- and β2-AR stimulation85. There is no reported functional
data on the primary β-AR involved in the vasodilatory response of mouse cerebral arteries.
Since sympathetic activation increases cardiac responsiveness and therefore myocardial
oxygen demand, there must be a consequent increase in oxygen supply to the tissues of the
heart. The coronary circulation therefore shows β-AR mediated vasodilation to ensure adequate
perfusion and oxygen delivery to the heart during times of increased oxygen demand95;96. In
helical strips of human left anterior coronary artery branches there is a predominant contribution
of the β1-AR subtype in relaxation as determined by metoprolol blockade of isoproterenol
mediated vasodilation as well as a low potency of β2-specific dilation by tertbutaline97. These
researchers also found the same relationship in Japanese monkey and mongrel dog left anterior
coronary artery strips97. These findings further confirmed a previous report that there is a
predominant role of β1-mediated vasodilation in dog coronary arteries98. Similarly, it has been
shown that there is a dominant functional role of the β1-AR in swine anterior descending
coronary artery strips and, through radioligand binding, there is a 68:32% ratio of β1-/β2-AR
expression in these strips99. In rodent models, rings of the rat left anterior coronary atery exhibit
predominantly β1-AR mediated vasodilation100 while isolated and pressurized, mouse
ventricular septal arteries display β-AR mediated vasodilation, though the subtype responsible is
unreported101. Despite the questions remaining in the mouse coronary circulation, it appears that
across species there is predominance for β1-AR mediated vasodilation in coronary arteries.
The evidence is less extensive in skeletal muscle arteries as to which β-AR subtype
promotes vasodilation. Based on potency orders to epinephrine and norepinephrine and on
isoproterenol mediated vasodilation in the presence of practolol (β1-AR antagonist), it was
established that helical segments of rat femoral artery branches are predominantly under β2-
26
adrenergic receptor control98. Evidence in rodents is unclear; there is evidence for the expression
of both β-AR subtypes in rat gastrocnemius, soleus and vastus lateralis arterioles, though
receptor functionality is unknown102. In mice, using a β1- or β2-AR knockout model, the β1-AR
was discovered to be functionally predominant in femoral artery wire myograph preparations103.
Beta-AR signalling is therefore an important mediator of cardiovascular function. As
evidenced, β-blockers are very useful pharmacological tools for physiologists; however as
discussed, they are very valuable medications for clinical interventions. Their use proliferated
rapidly and only now are they being reconsidered in certain cardiovascular conditions. The
predominance, especially, of β1-AR receptor mediated vasodilation in vital organs suggests that
β-blockers may have important vascular effects that were not previously considered.
7.4 Potential of a vascular mechanism in beta-blocker pathology
Beta-blocker therapy reduces myocardial oxygen demand, a benefit which is invaluable
in diseased hearts where supply is pathologically limited. As described in section 7.3.2.1,
however, the cardioselective (ie. β1-AR specific) designation of β-blockers may be ambiguous
given that various vascular beds display a functional role of β1-AR mediated relaxation. If β-
blockers interfere with the vasodilatory capacity of blood vessels within vital organs (ie. brain)
they could consequently limit tissue perfusion during times of stress when circulating
catecholamines are at their zenith. Several lines of evidence point towards a potential vascular
effect contributing to the pathology of β-blocker administration.
7.4.1 Vasodilators have better risk reductions for stroke and mortality than beta-blockers
Clinical evidence comes from randomized trials assessing the efficacy of
“cardioselective” β-blockers against other cardiovascular drug treatments known to promote
vasodilation. In hypertension, specifically, there have been several trials of β-blockers versus
27
calcium channel blocker (CCB) or renin-angiotensin system inhibitors (RAS-I) in improving the
long term outcomes of patients. A significantly stronger stroke signal has consistently been
found with β-blocker treatment50-55.
In the European Lacidipine Study on Atherosclerosis (ELSA), patients with systolic
blood pressures between 150 and 210 mmHg were treated and monitored over a 4 year period
with atenolol (n=1157) or the CCB lacidipine (n=1177). Atenolol treatment was associated with
a 33 and 36% increased relative risk of non-fatal stroke or mortality, respectively, compared to
lacidipine52. In the International Verapamil-Trandolapril Study (INVEST), hypertensive patients
were treated with atenolol (n=11309) or verapamil (n=11267) over a 2 year period. Atenolol
treatment was associated with a small 15% increase in relative risk of non-fatal stroke and no
appreciable difference in mortality53. In the Anglo-Scandinavian Cardiac Outcomes Trial
(ASCOT), treatment of atenolol (n=9618) was again tested against a CCB, amlodipine (n=9639)
in hypertensive patients over a mean 5.5 year follow up period. β-blockade was associated with
a significant 33% increase in relative risk of non-fatal stroke with no appreciable difference in
mortality51. Pooling all these results together, a Cochrane meta-analysis reported a significant
24% increase in relative risk of stroke with β-blocker versus CCB treatment in hypertensive
patients49. Similar findings were reported in patients treated with RAS-Is versus β-blockers.
In the Losartan Intervention For Endpoint (LIFE) reduction in hypertension trial, patients
with systolic blood pressures between 160 and 200mmHg were treated with atenolol (n=4588)
or the angiotensin II receptor blocker losartan (n=4605). Atenolol treatment was associated with
a 34% increased relative risk of fatal and non-fatal stroke54. Pooling these results with a smaller
trial (n=758) showing no differences between atenolol and the angiotensin coverting enzyme
inhibitor captopril55, a Cochrane meta-analysis found a significant 30% increase in the risk of
stroke in β-blocker treated patients49. These findings explain why β-blockers are no longer used
28
as a first-line therapy in the treatment of hypertension36. However, the less favourable side effect
profile of β-blockers against vasodilators is not limited to its use in hypertension.
In the Carvedilol or Metoprolol European Trial (COMET), the 3rd generation,
vasodilating β-blocker, carvedilol (non-selective β-blocker with α1-AR antagonism) was tested
against metoprolol (2nd generation, β1-AR specific) in the treatment of chronic heart failure.
Patients with heart failure were given either carvedilol (n=1511) or metoprolol (n=1518) over a
5 year treatment and follow up period. Patients treated with metoprolol were at a significant 18
and 21% increased risk of all cause mortality and cardiovascular mortality respectively104.
Metoprolol treated patients were also more likely to experience a fatal stroke (190% increased
relative risk)105, a fatal or non-fatal MI (35% increased relative risk)106 and unstable angina
(35% increased relative risk)106. These findings all suggest a superior ability of the vasodilating
β-blocker against β1-specific blockade.
In the perioperative setting, acute β-blockade in preoperative anemic patients is
associated with a significant 116% increase in the relative risk of major adverse cardiac events
(MI, non-fatal cardiac arrest, in-hospital mortality) in a propensity matched retrospective
analysis107. Conversely, in a previous propensity matched retrospective analysis, it was reported
that ACE inhibitors and CCBs are associated with a significant reduction in the risk of
postoperative mortality in preoperative anemic patients 108. The inferiority of β-blockade
compared to vasodilating treatments is therefore of some concern and it may be a significant
mechanism of harm.
7.4.2 Cardioselective beta-blockers may act on beta2-adrenergic receptors
The COMET trial as well as Beattie’s retrospective analysis of preoperative anemic
patients raises an interesting question: are some β-blockers better than others? COMET showed
that metoprolol consistently did worse than carvedilol in reducing adverse events in patients104-
29
106. In Beattie’s 2009 study, he found that metoprolol is associated with an increased risk of
death in anemic patients compared to treatment with atenolol or bisoprolol 108. A possible reason
for these discrepancies may have to do with the relative selectivity of these β-blockers at their
target receptors. In addition to directly inhibiting β1-adrenergic mediated vasodilation, a β-
blocker that cross-reacts with the β2-AR may compound the inhibitory action on vasodilation
thereby limiting tissue perfusion. Interestingly, bisoprolol and carvedilol, both showing better
outcomes than metoprolol, are 10 times more selective at the β1-AR than the reference β-blocker
propranolol109. Metoprolol shows a comparable potency to propranolol at the β1-AR109. In fact,
using radioligand binding in CHO cells expressing human β1- or β2-ARs, it was found that
metoprolol has the lowest selectivity for the β1-AR of all clinically used β-blockers tested4. This
is an alarming finding when considering that metoprolol is widely used based on its β1-AR
selectivity; it is currently the number one most prescribed β-blocker in North America with over
40 million prescriptions dispensed in 2009 (IMS Health).
7.4.3 Metoprolol impairs cerebral oxygen delivery in acutely anemic rats
Our lab examined the effect of the β-AR antagonist metoprolol in acutely anemic
(hemodiluted) rats. Administration of metoprolol preceding hemodilution resulted in no
significant change in mean arterial pressure (MAP) between β-blocked and control animals,
however, it significantly impaired the ability of rats to increase their cardiac output in response
to the stress associated with hemodilution110. When metoprolol administration preceded
hemodilution, there was a significant reduction in cerebral tissue and microvascular O2
tension110. These findings suggest that when the global blood supply is blunted by β-blockade,
there is reduced perfusion to the brain during anemia. The extent of the reduction in brain O2
tension, however, suggests that there could be a second mechanism contributing to reduced
30
perfusion. Based on the evidence presented above, this other effect may be related to the
inhibition of the vasodilatory capacity of cerebral blood vessels.
Altogether, these observations suggest that a possible vascular role of β-blockers may
promote tissue ischemia. The effect of metoprolol administration in a mouse model has not been
previously assessed. A vascular mechanism of β-blockade in limiting tissue perfusion could be
indicated by examining the cerebral oxygen tension of β-blocked mice during hypoxic stress.
Acute hypoxia in mice causes a significant drop in mean arterial pressure but not CO suggesting
that there is global vasodilation (reduced systemic vascular resistance) in response to the
reduction in FiO2 (unpublished data). Cerebral tissue oxygen tension also drops in acute
hypoxia. If β-blockade with metoprolol inhibits cerebral vasodilation there may be a greater
drop in cerebral tissue PO2 in hypoxia due to an inability of the animal to adequately maintain
cerebral blood flow. This would provide a good rationale to examine how metoprolol affects
isolated resistance arteries. β-AR function has not been previously assessed in isolated mouse
cerebral arteries, though some studies have characterized their expression in small mesenteric
arteries. In the absence of β-blockade, the endogenous catecholamines will bind to both the
vasoconstricting α-adrenergic receptors and the vasodilating β-adrenergic receptors. If β-
blockers inhibit β-AR mediated vasodilation they could potentially shift the balance towards α-
AR vasoconstriction. This could therefore limit tissue perfusion during conditions of increased
oxygen demand. There is therefore a strong rationale to pursue the effects of the β-blocker
metoprolol on the function of these arteries.
31
8 Hypothesis and Aims
Cardioselective β1-adrenergic antagonists impair resistance artery vasodilation and increase
the risk of organ ischemia.
Specific Aim #1
To determine the effect of a clinically utilized cardioselective β-blocker (metoprolol) on cardiac
output, systemic vascular resistance and cerebral tissue oxygen tension in mice.
Specific Aim #2
i) To determine the effect of metoprolol on β-adrenergic mediated vasodilation in mouse
mesenteric arteries.
ii) To characterize the response of mouse posterior cerebral arteries to adrenergic stimulation
(phenylephrine, isoproterenol) before and after metoprolol treatment.
Specific Aim #3
To determine the β-adrenergic receptor (β1 or β2) selectivity of metoprolol in mesenteric
arteries.
32
9 Methods
9.1 Effect of metoprolol on brain O2 tension and hemodynamics in vivo
9.1.1 Animals
Wild type C57BL6/J mice were purchased from The Jackson Laboratory at 2-3 months
old (Bar Harbor, Maine, USA) and housed under normal husbandry conditions with food and
water ad libitum. All animal protocols were approved by the St. Michael’s Hospital Animal
Care Committee.
9.1.2 Experimental protocol
After establishing a baseline (FiO2=21%) for 10min, anesthetised (3% isoflurane) mice
were given an i.v. injection of vehicle (saline) or metoprolol (3mg·kg-1)111 and allowed to
achieve a post-drug baseline for an additional 10min. In order to test whether β-blockade
impaired the physiologic response to a stressor, mice were given a hypoxic challenge
(FiO2=15%) for 15min and were subsequently exposed to an FiO2 of 21% during a 10 min
recovery phase (Figure 9.1.1). Following the recovery phase, mice were euthanized by cervical
dislocation.
9.1.3 Heart rate and mean arterial blood pressure
Anesthetised mice had their femoral artery and vein exposed. The artery was cannulated
with PE10 tubing (427401 Intramedic, BD Biosciences, Mississauga, ON, Canada) filled with
heparinised saline and connected to a PowerLab pressure transducer (TCB-600, Millar
Instruments, Inc., Houston, TX, USA). The femoral vein was cannulated with PE10 tubing
attached to syringe to allow the injection of saline or metoprolol. PowerLab ECG electrodes
(ML224, ADInstruments, Colorado Springs, CO, USA) were inserted subcutaneously to
33
PMOD
PowerLab
PBrmvO2
HR, Temp
Vein
Artery
PowerLabHR
MAP
Baseline Post-Drug
Saline/Metoprolol
HypoxiaFiO2 =15%
RecoveryFiO2=21%
10 min 10 min 15 min 10 min
Oxyphor G2Saline or Metoprolol
Saline or Metoprolol
Figure 9.1.1 Experimental protocol for assessing MAP and PBrmvO2 with metoprolol One group of mice was assigned to microvascular cerebral oxygen tension (PBrmvO2) measurements and a separate group was assessed for mean arterial pressure (MAP).
34
measure heart rate. PowerLab data acquisition systems (Chart, ADInstruments) were used to
record these physiological outcomes during the protocol outlined in the previous section.
9.1.4 Microvascular brain oxygen tension
The tail veins of anesthetised mice were cannulated to allow injection of phosphorescent
dye and saline or metoprolol. PMOD 5000 LED emitting and phosphorescent detecting probes
(Oxygen Enterprises, Philadelphia, PA, USA) were placed approximately 0.5 cm apart directly
above the exposed skulls of mice. Mice were injected with Oyxphor G2-phosphorescent dye
(Pd-tetra-(4-carboxyphenyl) tetrabenzoporphyrin dendrimer) allowing measurements of
phosphorescence quenching by oxygen through the PMOD 5000 probes. Using
phosphorescence lifetime, the PMOD data acquisition system can calculate absolute measures of
microvascular oxygen tension. ECG probes were also inserted subcutaneously in the left and
right front paws and left hind leg of these mice. After the phosphorescent dye was injected, mice
went through the hypoxic challenge protocol outlined above in section 9.1.2. (n=5 for saline
treatment, n=6 for metoprolol treatment).
9.1.5 Cardiac responsiveness and left ventricular function
In a separate group of animals (n=6) we measured left-ventricular function during
metoprolol treatment. Animals were placed on a warming pad (37˚C), intubated, and ventilated
using positive pressure (110 beats/min) with 2% (v/w) isoflurane admixed with 100% O2. Mice
were secured in a recumbent position and the right jugular vein was cannulated. Pressure was
calibrated after warming the catheter in 0.9% NaCl at 37˚C for 30 minutes. The right internal
carotid was then identified and ligated cranially. The 1.2F miniaturized combined conductance
catheter-micro-manometer (#FT112B Scisense Inc, London, Canada) was inserted into the right
carotid artery and advanced into the left ventricle until stable PV loops were obtained. After 10
35
minutes, 0.9% heparized saline was injected via the right jugular vein and a steady state
achieved. Following saline, 3mg·kg-1 of metoprolol was administered and another steady state
was reached. All loops (pre and post beta blockade) were obtained with the ventilator turned off
for 5 -10 seconds and the animal apnoeic. Data were then acquired under steady state
conditions.
Using the pressure conductance data, a range of real-time functional parameters were
then calculated using the ADVantage systemTM (Scisence Inc). These included: end diastolic
pressure (EDP), end systolic pressure (ESP), end diastolic volume (EDV), end systolic volume
(ESV), cardiac output (CO), the time constant of relaxation (Tau ), arterial elastance (Ea) and
systemic vascular resistance (SVR).
9.2 Effect of metoprolol in the mesenteric resistance artery in vitro
9.2.1 Animals
Wild type C57BL6/J mice were purchased from Charles River Laboratories at 2-3 months old
(Montreal, QC, Canada) and housed under normal husbandry conditions (12:12h light-dark
cycle, chow and water ad libitum). All animal protocols were approved by the University
Animal Care Committee (University of Toronto, Toronto, ON, Canada).
9.2.2 Mesenteric resistance artery isolation
Mice were euthanized via cervical dislocation and the small and large intestines were
removed and placed in MOPS-buffered salt solution (pH 7.4, NaCl 145, KCl 4.7, CaCl2·2H2O
1.5, MgSO4·7H2O 1.17, NaH2PO4.2H2O 1.2, pyruvate 2.0, EDTA 0.02, MOPS 3.0, glucose 5.0
mmol/L) on ice. The large intestine distal to and including the cecum were dissected and
discarded as was the duodenum. The remaining portion of the intestine were then carefully
36
A B
C
Figure 9.2.1 Photographs of artery isolation and cannulation. Mesenteric resistance artery (A) and posterior cerebral artery (B) locations within their respective organs. C) Posterior cerebral artery mounted on glass micropipettes and pressurized to 45mmHg.
37
spread in a circular pattern and held in place by pins to expose the mesenteric circulation (Figure
9.2.1A). Second generation mesenteric resistance arteries (MRAs; diameter≈200µm) were
carefully dissected and cleaned and transferred to the MOPS containing organ baths of the
pressure myography chambers.
9.2.3 Pressure myography
Pressure myography chambers were fitted with a coverglass bottom and could
accommodate up to 5mL of buffer solution within the organ bath. Vessels were imaged with an
inverted light microscope (Leica, Richmond Hill, ON, Canada). A monochrome CCTV camera
(Panasonic, Mississauga, ON, Canada) was attached to the microscope and the output signal was
sent to a TV monitor (Living Systems Instrumentation, Burlington, VT, USA). A video
dimension analyzer (V-94; Living Systems Instrumentation) was used to accurately measure
real time changes in vessel lumen diameter by sensing optical density alterations in the vessel
image displayed on the monitor (Figure 9.2.2). Output from the dimension analyzer was
recorded by data acquisition systems (DI-720, DATAQ Instruments, Akron, OH, USA and
MP100, BIOPAC Systems Inc, Goleto, CA, USA).
Isolated arteries were tied to two glass cannulae in a pressure myography vessel chamber
(LS-C1-SH; Living Systems Instrumentation) and held at a constant transmural pressure using a
pressure servo controller with peristaltic pump (PS-200; Living Systems Instrumentation).
Mounted arteries were pressurized with no flow to 45mmHg and warmed to 37°C for 30
minutes (Figure 9.2.1C). After warming, MRAs were brought to a pressure of 60mmHg and
equilibrated for 10 min before undergoing a viability check. Vessels were constricted with 1µM
of the α1-adrenergic agonist phenylephrine for 4 minutes or until a steady state was reached.
Viable vessels (30-50% constriction from baseline) continued through their respective protocols,
38
diameter
time
Figure 9.2.2 Set up of the pressure myography apparatus Mounted vessels in myography chambers are imaged using an inverted light microscope. The image is displayed on a TV monitor attached to data acquisition systems which measure changes in lumen diameter by detecting differences in contrast ratios of the vessel wall. This data is recorded on a computer.
39
otherwise they were discarded. In all protocols, doses were added by removing 1mL of solution
from the organ bath and replacing it with 1mL of the desired dose.
9.2.4 Effect of metoprolol on beta1,2-adrenergic mediated vasodilation
Doses are applied by replacing 1mL of solution from the 5mL organ bath with the
desired dose of agonist. Viable vessels were initially preconstricted with 1 µM phenylephrine
for 2 min to induce steady state tone and then exposed to increasing concentrations of the non-
selective β1,2-adrenergic receptor (β-AR) agonist isoproterenol (0.3µM-200µM). Each individual
dose of isoproterenol contained a maintenance dose of phenylephrine (1µM) and was added to
the organ bath until a steady-state was reached (2 min). The vessel diameter after every given
dose was recorded. This isoproterenol dose-response curve served as the control curve.
Following the final dose of isoproterenol, the organ bath was replaced with 37°C MOPS buffer
and the vessel was allowed to equilibrate for 10 min before changing the bath with 0, 5, 10 or
50µM metoprolol in MOPS for 30 min incubation period. These concentrations were chosen as
they correspond to the plasma concentration of 3mg·kg-1 metoprolol (i.v.) measured by HPLC in
rats111. Following incubation, vessels were preconstricted and the isoproterenol dose-response
was repeated. During this second isoproterenol dose-response, phenylephrine (1µM) and
metoprolol (5, 10 or 50µM) concentrations were maintained in the organ bath. The organ bath
was then changed and vessels were equilibrated for 10 min before being incubated in a Ca2+-free
MOPS buffer for 30 min to determine the maximum passive diameter.
9.2.5 Effect of metoprolol on beta2-adrenergic mediated vasodilation
The above protocol in section 0 was repeated to assess the effect of 50µM metoprolol on
the vasodilatory dose-response curve of clenbuterol (0.3 µM-300µM), a β2-selective agonist,
instead of isoproterenol.
40
9.3 Effect of metoprolol in the posterior cerebral artery in vitro
9.3.1 Animals
Wild type C57BL6/J mice were purchased from Charles River Laboratories at 2-3
months old (Montreal, QC, Canada) and housed under normal husbandry conditions (12:12h
light-dark cycle, chow and water ad libitum). All animal protocols were approved by the
University Animal Care Committee (University of Toronto, Toronto, ON, Canada).
9.3.2 Posterior cerebral artery isolation
Mice were euthanized via decapitation. The skull was exposed and then removed; the
brain was carefully dissected and placed in MOPS solution on ice. Ventrally, portions of the
posterior cerebrum, the cerebellum and the medulla were cut such that the resulting piece of
brain tissue encompassed the posterior cerebral artery (PCA; diameter≈150µm; Figure 9.2.1B).
The segment of cerebellum was carefully removed, exposing the PCA. The PCA was carefully
pulled free from the cerebral tissue and any remaining connective tissue was removed by blunt
dissection. This process was repeated for both left and right PCAs. Arteries were then
transferred to the MOPS filled organ baths of the perfusion myography chambers.
9.3.3 Pressure myography
Refer to section 9.2.3. Note that PCAs were maintained at a transmural pressure of
45mmHg and were checked for viability immediately after the 30 min warming period. Because
of reduced phenylephrine induced tone, the viability dose of phenylephrine was increased to
10µM in PCAs as opposed to 1µM in MRAs.
41
9.3.4 Effect of metoprolol on adrenergic mediated vasomotor function
The preconstriction and maintenance dose of phenylephrine in PCAs was 3µM PE (See
Appendix B). In this series of experiments, PE dose-response curves (0.03-30µM) were first
generated in viable vessels. Each dose was held for 2 minutes to ensure a steady state was
reached. Following the PE dose-response, the bath was changed and the vessels were allowed to
equilibrate for 10 minutes. ISO dose-response curves (0.03-3000µM) were then generated with
each dose again being held for 2 minutes. The organ bath was then changed and after 10 minutes
was replaced with unaltered warm MOPS buffer (0µM metoprolol; n=5) or 50µM metoprolol
(n=7) in MOPS buffer and held for 30 minutes. Following this incubation period, PE and ISO
dose-response curves were repeated as above. During these dose-response curves, metoprolol
(50µM) concentrations were maintained in the organ bath. Additionally, phenylephrine (3µM)
concentration was maintained in the second ISO dose-response. The bath was changed after the
ISO curve and held for 10 minutes before being replaced with Ca2+ free MOPS buffer.
9.4 Drugs and solutions
The following drugs were used; L-phenylephrine hydrochloride (P6126), ±-metoprolol
(+)-tartrate salt (M5391), DL-isoproterenol hydrochloride (I5627), and clenbuterol
hydrochloride (C5423; Sigma-Aldrich, St. Louis, MO, USA).
Metoprolol tartrate solution as Betaloc® 1mg/mL (Prod. No. 1332, AstraZeneca Canada
Inc., Mississauga, ON, Canada) was used in whole animal studies.
42
9.5 Statistical analysis
9.5.1 Effect of metoprolol on brain O2 tension and hemodynamics in vivo
Analysis of differences in heart rate, mean arterial pressure and cerebral microvascular
oxygen tension were performed by two-way repeated measures ANOVA. Analysis of cardiac
responsiveness and left ventricular function were performed by paired t-test. All data is
presented as mean ± SEM. Statistical analyses were done in SigmaPlot 11 (Systat Software Inc.,
Chicago, IL, USA).
9.5.2 Effect of metoprolol in mouse arteries
Acute diameter measurements (diameasured, representing steady state diameter following
dose of agonist) were normalized to represent either tone or percent dilation. Tone represents the
proportion of constriction relative to the Ca2+ free diameter (diamax). Tone is calculated as
follows:
Equation 9.5.1
100 dia
diadia)dia of (% tone
max
measuredmaxmax
An increase in tone corresponds to an increased degree of vasoconstriction in the vessel. A
reduction in tone would correspond to a reduced degree of vasoconstriction.
Percent dilation represents the percent change in diameter from the minimum diameter
observed (diamin) normalized to diamax. Percent dilation is calculated as follows:
Equation 9.5.2
100dia-dia
dia-diadilation %
minmax
minmeasured
An increase in percent dilation is indicative of increased vasodilation.
43
Data are presented as mean ± SEM, n-values indicated number of animals or number of
vessels tested. Dose-response data of agonist before and after incubation with metoprolol (or
MOPS buffer) are tested by two-way repeated measures ANOVA. The concentration of agonists
that elicited half the maximal response of a dose-response curve is expressed as the EC50 value
and is represented as the log[EC50]. Additionally, dose-response curves will be assessed
quantitatively by their Emax value which represents the magnitude of the response at the
uppermost plateau of the dose-response curve. This value is extrapolated when it is not reached
experimentally. Mean EC50 and Emax values are tested by paired t-test where both curves were
generated in the same artery otherwise they were tested by unpaired student’s t-test.
Vasoconstrictor (tone) and vasodilator (% dilation) responses to any one dose of agonist before
and after metoprolol (or MOPS buffer) incubation are tested by paired t-tests. Statistical
analyses were performed in Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Differences
were significant at an alpha value less than 0.05.
44
10 Results
10.1 Effect of metoprolol on brain O2 tension and hemodynamics
10.1.1 Metoprolol injection reduced brain O2 tension and heart rate
Metoprolol injection dropped heart rate and microvascular brain oxygen tension
(PBrmvO2) while mean arterial pressure (MAP) was maintained immediately after drug injection
(Figure 10.1.1). Hypoxic challenge increased the heart rate in saline treated animals which was
subsequently decreased during the recovery phase. β-blocked mice maintained a depressed heart
rate throughout the protocol. MAP and PBrmvO2 were decreased in both treatments following
hypoxic challenge and increased during the recovery phase (Figure 10.1.1).
Metoprolol treatment significantly depressed the heart rate compared to baseline (469 ±
13.2 vs. 548 ± 13.0 bpm; n=12, p<0.05) throughout the experimental protocol while saline
treated animals had a significant increase in heart rate compared to baseline (600 ± 32.7 vs. 552
± 20.2 bpm; n=11, p<0.05) only during hypoxic challenge (Figure 10.1.2A). There was no
difference in MAP between treatment groups (Figure 10.1.2B). Hypoxic challenge significantly
dropped MAP in both groups compared to baseline (saline: 62.0 ± 3.8 vs. 73.2 ± 1.7 mmHg;
n=6, p<0.05; metoprolol: 67.0 ± 3.0 vs. 71.7 ± 0.9 mmHg; n=6, p<0.05). Directly following
metoprolol injection, PBrmvO2 (Figure 10.1.2C) was significantly decreased compared to
baseline (60.8 ± 2.5 vs. 68.9 ± 1.6 mmHg; n=6; p<0.05) and compared to saline treatment (60.8
± 2.5 vs. 69.7 ± 2.1 mmHg; n=6, 5; p<0.05). There was no difference between metoprolol and
saline treatment during hypoxia or during the 21% FiO2 recovery phase. Hypoxic challenge
significantly dropped PBrmvO2 in both groups compared to baseline (p<0.05) and recovery at
21% FiO2 did not return PBrmvO2 to baseline levels (p<0.05) in both groups. Figure 10.1.3
depicts the degree of change from baseline of each condition in both treatment groups.
45
10.1.2 Metoprolol injection reduced CO and increased SVR
Metoprolol injection significantly reduced cardiac responsiveness compared to saline
treatment in the same mouse (Figure 10.1.4). A representative tracing of a pressure-volume loop
is shown in Figure 10.1.5. The P-V loop indicates that preload (the lower right hand corner of
the curve, representing left ventricular end-diastolic pressure and volume) is increased following
metoprolol injection. The slope of the end-systolic pressure-volume relationship is smaller
following metoprolol injection, suggesting reduced ventricular contractility. The slope of the
end-diastolic pressure-volume relationship is steeper following metoprolol injection which
suggests decreased left ventricular compliance. Additionally, the area under the curve,
representing left ventricular stroke work, is smaller following metoprolol treatment. Heart rate
was significantly decreased 10 minutes after metoprolol injection compared to 10 minutes after
saline injection (399 ± 23.9 vs. 485 ± 23.3 bpm; n=6, p<0.05; Figure 10.1.4A). Similarly, stroke
volume was reduced following metoprolol compared to saline (19.7 ± 1.8 vs. 26.3 ± 1.5
µL/beat; n=6, p<0.05; Figure 10.1.4B). As a result of decreases in heart rate and stroke volume,
cardiac output was also significantly decreased following metoprolol treatment (7.96 ± 0.8 vs.
12.8 ± 1.1 mL/min; n=6, p<0.05; Figure 10.1.4C). Systemic vascular was consequently
increased in metoprolol treated animals (9.5 ± 0.6 vs. 5.3 ± 0.2 dynes/cm5 ; n=6, p<0.05; Figure
10.1.4D). There were no differences between left ventricular end-systolic and end-diastolic
pressures (Figure 10.1.4E, F) and volumes (Figure 10.1.4G, H).
46
He
art
Rat
e, b
pm
300
400
500
600
700
Mea
n A
rter
ial P
ress
ure,
mm
Hg
40
50
60
70
80
90
100
40
60
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100
Time, min
0 10 20 30 40 50
Mic
rova
scu
lar
Bra
in O
2 T
ensi
on,
mm
Hg
20
40
60
80
100
A
B
C
SalineMetoprolol (3 mg kg-1)
Saline/Metoprolol
FiO2=15% FiO2=21%
Saline/Metoprolol
FiO2=15% FiO2=21%
Saline/Metoprolol
FiO2=15% FiO2=21%
Figure 10.1.1 Time course of hypoxic challenge protocol with saline/metoprolol. Changes in heart rate (A), mean arterial pressure (B) and microvascular brain O2 tension (C) over time during before and after beta-blocker or saline administration, during hypoxic stress (FiO2=15%) and during recovery at FiO2=21%. Data points represent mean ± SEM.
47
Hea
rt R
ate,
bpm
0
200
400
600
800
Mea
n A
rter
ial P
ress
ure,
mm
Hg
0
20
40
60
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100
Baseline Drug FiO2 15% FiO2 21%
Mic
rova
scul
ar B
rain
O2
Ten
sion
, m
mH
g
0
20
40
60
80
Saline
Metoprolol (3 mg kg-1)
A
B
C
*, #
*
**
*, #
*, #
* *
*, #
*
*
Figure 10.1.2 Mean heart rate, MAP and PBrmvO2 in hypoxic challenge with saline/metoprolol. Graph of mean heart rate (A), MAP (B) and PBrmvO2 (C) during each experimental condition in hypoxic challenge protocol. Error bars represent SEM. * p<0.05 from baseline, # p<0.05 between treatment group; two-way repeated measures ANOVA.
48
H
ea
rt R
ate
, bp
m
-150
-100
-50
0
50
100
150
200
MA
P,
mm
Hg
-20
-10
0
10
20
30
Drug FiO2 15% FiO2 21%
PB
r mvO
2,
mm
Hg
-60
-40
-20
0
20
40
SalineMetoprolol (3 mg kg-1)
A
B
C
*
*
*
#
*
**
Figure 10.1.3 Delta heart rate, MAP and PBrmvO2 in hypoxic challenge with saline/metoprolol. Mean change (Δ) in heart rate (A), MAP (B), and PBrmvO2 (C) from baseline condition. Error bars represent SEM. * p<0.05 between treatment groups, # p<0.05 within treatment group; two-way repeated measures ANOVA.
49
He
art
Ra
te,
bpm
0
100
200
300
400
500
600
Str
oke
Vo
lum
e,
uL
/be
at
0
5
10
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30
Car
dia
c O
utp
ut,
mL
/min
0
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End
Sys
tolic
Pre
ssur
e, m
mH
g
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En
d D
iast
olic
Pre
ssu
re,
mm
Hg
0
2
4
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14
Saline Metoprolol
En
d S
ysto
lic V
olu
me,
uL
0
10
20
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50
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Saline Metoprolol
End
Dia
stol
ic V
olu
me
, u
L
0
20
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* *
*
A B
C D
E F
G H
SV
R,
dyn
es/
cm5
0
2
4
6
8
10
12
*
Figure 10.1.4 Hemodynamic changes following saline and subsequent metoprolol injections. Heart rate (A), stroke volume (B), cardiac output (C) and systemic vascular resistance (SVR; panel D) are significantly reduced following metoprolol (3mg·kg-1) injection End-systolic and diastolic pressures and volumes (E, F, G, H) are not affected by metoprolol treatment. Error bars represent SEM. n=6; * p<0.05 by paired t-test.
50
0
20
40
60
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100
120
0 10 20 30 40 50 60 70 80
Left
ven
tric
ula
r pre
ssu
re, m
mH
g
Volume, uL
Saline Metoprolol (3mg/kg)
Figure 10.1.5 Representative pressure-volume loop before and after metoprolol treatment. Tracing of a pressure-volume loop recorded in a single mouse after 10 minutes of i.v. saline followed by 10 minutes of i.v. metoprolol.
51
10.2 Metoprolol inhibits isoproterenol mediated vasodilation MRAs
A representative tracing of the entire time course of the experimental protocol is shown
in Figure 10.2.1. Isoproterenol (ISO) dose-response curves generated before incubation with
metoprolol are considered controls. There was no effect of time on the ISO dose-response curve
as incubation in 0µM metoprolol was no different from its control (Table 10.1; Figure 10.2.2A).
In MRAs the LogEC50 values of ISO in 5 and 10M metoprolol were not significantly different
than their respective control curves or from each other (Table 10.1; Figure 10.2.2B, C). A
significant right-shift in the ISO dose-response curve was observed at a metoprolol
concentration of 50µM (-4.1 ± 0.1 vs -4.4 ± 0.1; n=6; p<0.05; Figure 10.2.2D). The LogEC50 of
ISO at 50M metoprolol was significantly higher than at 0, and 5M metoprolol (-4.1 ± 0.1 vs -
4.6 ± 0.1 and -4.6 ± 0.04; n=6, 3, 7, p<0.05). In addition, the percent dilation at the control EC50
dose of isoproterenol (30µM or LogEC50=-4.5) was significantly attenuated by metoprolol at 10
(31 ± 4.2 vs. 40 ± 1.8%; n=7; p<0.05; Figure 10.2.3C) and at 50µM (17 ± 2.3 vs. 34 ± 2.0%;
n=6; p<0.05; Figure 10.2.3D). At all concentrations of metoprolol, dose-response curves
reached equivalent Emax plateaus (Figure 10.2.4).
The clenbuterol (β2-specific agonist) dose-response curve is shown in Figure 10.2.5A.
The LogEC50 values are not statistically different, though there is a slight increase at 50µM
metoprolol (from -4.9 ± 0.04 to -4.7 ± 0.1; Table 10.1). The percent dilation at the control EC50
dose (10µM or LogEC50=-5) is significantly attenuated at 50µM metoprolol (25 ± 1.6 vs. 34 ±
1.0; n=5, p<0.05; Figure 10.2.5B).
52
0 2500 5000 7500 10000100
150
200
250
3001M PE
ISO DRC
50M Metoprolol
1M PECa2+ Free
ISO DRC
-6.5
-6
-5.5
-5.0
-4.5
-4.0
-6.5
-6 -5.5-5.0
-4.5
-4.0
time, sec
dia
met
er,m
Figure 10.2.1 Representative tracing of MRA experimental protocol. Time course of experimental protocol in mesenteric resistance arteries; the first dose-response curve (DRC) is considered the control to which the second dose-response curve (with metoprolol in the bath) is compared to. Dose numbers represent 10xµM of ISO. PE=phenylephrine; ISO=isoproterenol.
53
Control (- Metoprolol) + Metoprolol
ISO ± 0µM Metoprolol (n=3) -4.5 ± 0.1 -4.6 ± 0.08
ISO ± 5µM Metoprolol (n=7) -4.6 ± 0.05 -4.6 ± 0.04
ISO ± 10µM Metoprolol (n=7) -4.6 ± 0.06 -4.4 ± 0.1
ISO ± 50µM Metoprolol (n=6) -4.4 ± 0.07 -4.1 ± 0.08*,#
CLEN in 50µM Metoprolol -4.9 ± 0.04 -4.7 ± 0.1
Table 10.1 Half maximal concentrations for MRA dose-response curves. LogEC50 values for isoproterenol (ISO) in 0, 5, 10 and 50µM metoprolol and clenbuterol (CLEN) in 50µM metoprolol. Control values are EC50s in the absence of the stated dose of metoprolol. * p<0.05 between columns (paired t-test); # p<0.05 within column (one-way ANOVA).
54
-8 -6 -40
20
40
60
80 Control, n=3
0M Metoprolol, n=3
log [isoproterenol (mol/L)]
% d
ilat
ion
-7 -6 -5 -4 -30
20
40
60
80 Control, n=7
5M Metoprolol, n=7
log [isoproterenol (mol/L)]
% d
ilat
ion
-7 -6 -5 -4 -30
20
40
60
80 Control, n=7
10M Metoprolol, n=7
log [isoproterenol (mol/L)]
% d
ilat
ion
-7 -6 -5 -4 -30
20
40
60
80 Control, n=6
50M Metoprolol, n=6
* ***
**
log [isoproterenol (mol/L)]
% d
ilat
ion
A B
C D
Figure 10.2.2 Effect of metoprolol on isoproterenol dose-response curves in mesenteric arteries. Isoproterenol (ISO) dose-response curves were generated before (control) and after incubation with metoprolol in mesenteric resistance arteries. The ISO dose-response curve was unaffected by both time (A) and 5µM of metoprolol (B). There is a small rightward shift of the ISO dose-response curve at 10uM metoprolol, though the difference in EC50s is not significant (log[EC50]=-4.6 ± 0.06 vs -4.4 ± 0.1; n=7; panel C). There is a significant rightward shift in the ISO dose-response curve from –4.4 ± 0.07 to -4.1 ± 0.08 at 50µM metoprolol (paired t-test, p<0.05, n=6; panel D). Data points represent mean ± SEM. * p<0.05 by two-way repeated measures ANOVA.
55
Control 5M Metoprolol0
20
40
60
% d
ilat
ion
at
ISO
EC
50
(30
M)
Control 0uM Metoprolol0
20
40
60%
dil
atio
n a
t IS
O E
C5
0 (3
0 M
)
Control 10uM Metoprolol0
20
40
60
*
% d
ilat
ion
at
ISO
EC
50 (
30M
)
Control 50uM Metoprolol0
20
40
60
*
% d
ilat
ion
at
ISO
EC
50 (
30M
)
A B
C D
Figure 10.2.3 Percent dilation to EC50 dose (30µM) of isoproterenol in mesenteric arteries. The percent dilation at the dose of isoproterenol (ISO) which elicits half the maximal response (30µM) from mesenteric resistance arteries is affected by time (A) or 5uM metoprolol (B). At 10uM metoprolol there is a significant reduction in the percent dilation to 30µM ISO (40 ± 1.8% vs. 31 ± 4.3%; paired t-test, p<0.05, n=7; panel C). There is a significant reduction in the percent dilation to 30µM ISO from 34 ± 2.0% to 17 ± 2.3% at 50uM metoprolol (paired t-test, p<0.05, n=6; panel D). Data presented as mean ± SEM.
56
Control 0M Metoprolol0
20
40
60
80
100IS
O E
ma
x,
% d
ilat
ion
Control 5M Metoprolol0
20
40
60
80
100
ISO
Em
ax,
% d
ilat
ion
Control 10M Metoprolol0
20
40
60
80
100
ISO
Em
ax,
% d
ilat
ion
Control 50M Metoprolol0
20
40
60
80
100
ISO
Em
ax,
% d
ilat
ion
A B
C D
Figure 10.2.4 Percent dilation at Emax of isoproterenol dose-response curves. The % dilation to isoproterenol at Emax of the best-fit curves is not affected by time (A), 5 (B), 10 (C) or 50µM(D) metoprolol. Data presented as mean ± SEM. * p<0.05 by paired t-test.
57
A
-7 -6 -5 -4 -30
20
40
60
80
100 Control, n=5
50M Metoprolol, n=5
*
*
log [clenbuterol (mol/L)]
% d
ilat
ion
Control 50uM Metoprolol0
20
40
60
80
100
*
% d
ilat
ion
at
CL
EN
EC
50
(10
M)
Control 50M Metoprolol0
20
40
60
80
100
CL
EN
Em
ax,
% d
ilat
ion
CB
Figure 10.2.5 Effect of metoprolol on clenbuterol mediated vasodilation. The percent dilation at intermediate doses of clenbuterol (β2-specific agonist) is significantly attenuated at 50µM metoprolol (panel A; * p<0.05 by two-way repeated measures ANOVA). The percent dilation to the EC50 dose of clenbuterol (10µM or LogEC50=-5; panel B) is significantly attenuated at 50µM metoprolol (25 ± 1.6 vs. 34 ± 1.0; n=5, p<0.05). Data presented as mean ± SEM.
58
10.3 Metoprolol inhibits isoproterenol mediated vasodilation inPCAs
The effect of phenylephrine in PCAs has not been previously assessed; therefore it was
necessary to determine optimal phenylephrine preconstriction and maintenance doses before
generating isoproterenol dose-response curves. See Appendix B for work up of posterior
cerebral artery to determine optimal phenylephrine preconstriction dose in addition to the effect
of metoprolol on phenylephrine dose-response curves.
Isoproterenol (ISO) dose-response curves were generated before and after metoprolol
incubation (0µM metoprolol represents a time control, Figure 10.3.1). There was a significant
time effect on the logEC50 value of isoproterenol; the second ISO dose-response curve was
shifted to the right compared to the first (LogEC50=-4.1 ± 0.18 vs. -4.5 ± 0.10; n=5, p<0.05;
Table 10.2). However, two-way repeated measures ANOVA of the ISO dose-responses before
and after 0µM metoprolol showed no significant treatment or interaction effect (Figure
10.3.2A). Additionally, time had no effect on the percent dilation induced by the EC50 dose of
ISO (-4.5 or 30µM), as seen in Figure 10.3.2C, or on the Emax value of the ISO DRCs before and
after 0µM metoprolol (Figure 10.3.2D).
There was a significant treatment and interaction effect before and after incubation with
50µM metoprolol (two-way repeated measures ANOVA, n=7, p<0.05; Figure 10.3.2B). The
LogEC50 was right shifted at 50µM metoprolol (-4.0 ± 0.13 vs. -4.5 ± 0.24; n=7, p<0.05; Table
10.2). The % dilation at the EC50 dose of ISO was reduced at 50µM metoprolol, but the effect
was not significant (23 ± 4.8 vs. 37 ± 6.6 %; n=7; Figure 10.3.2C). The Emax of ISO was
significantly reduced at 50µM metoprolol (61 ± 5.5 vs. 79 ± 4.0 %; n=7, p<0.05; Figure
10.3.2D).
Taking into account a possible effect of time on the isoproterenol dose-response, the
DRC generated at 50µ metoprolol was compared to its time matched control (ie. the curve
59
generated at 0µM metoprolol). LogEC50 values and the percent dilation at this dose were not
significantly different, however there was a significant reduction in Emax at 50µM metoprolol
compared to its time matched control (61 ± 5.5 vs. 79 ± 3.7; n=7, 5, p<0.05; Figure 10.3.2D).
60
0 2500 5000 7500 10000 125000
50
100
150
20010M PE
PE DRC
10M PE
ISO DRC
0uM Metoprolol
10M PE
Ca2+ Free
PE DRC ISO DRC
-7.5-6.5
-5.5-4.5
-7.5 -5.5
-3.5
-7.5-6.5
-5.5
-7.5 -5.5
-3.5
-4.5
time, sec
dia
met
er,m
Figure 10.3.1 Representative tracing of PCA protocol. Time course of a single PCA showing dose-response curves (DRC) before and after metoprolol (0 or 50µM) incubations. Dose numbers represent 10xµM of PE or ISO. PE=phenylephrine; ISO=isoproterenol.
61
-8 -6 -4 -20
20
40
60
80
100 Control, n=5
0M Metoprolol, n=5
log [isoproterenol (mol/L)]
% d
ilat
ion
-8 -6 -4 -20
20
40
60
80
100 Control, n=7
50M Metoprolol, n=7 **
log [isoproterenol (mol/L)]
% d
ilat
ion
M M
et
- 0
M M
et
+
0M
Met
- 5
0M
Met
+
50
0
20
40
60
80
100
, #
Em
ax,
% d
ilat
ion
M M
et
- 0
M M
et
+
0M
Met
- 5
0M
Met
+
50
0
20
40
60
80
100
EC
50
(30
M)
dil
atio
n,
%A B
C D
Figure 10.3.2 Effect of metoprolol on isoproterenol dose-response in PCAs. Isoproterenol (ISO) dose-response curves were generated before (control) and after incubation with metoprolol in posterior cerebral arteries The ISO dose-response curve is not affected by time (panel A) but is significantly blunted at high doses by 50µM metoprolol (panel B, D). Emax of isoproterenol at 50µM metoprolol is significantly lower than its internal control as well as its time control. Data presented as mean ± SEM. * p<0.05 by two-way repeated measures ANOVA; ɣ p<0.05 by paired t-test; # p<0.05 by unpaired t-test.
62
Control (-Metoprolol) + Metoprolol
± 0µM Metoprolol (n=5) -4.5 ± 0.1 -4.1 ± 0.2*
± 50µM Metoprolol (n=7) -4.5 ± 0.2 -4.0 ± 0.1*
Table 10.2 Half maximal concentrations of isoproterenol in PCAs. LogEC50 values for ISO dose-response curves in PCAs. Data presented as mean ± SEM. *p<0.05 between columns (paired t-test); # p<0.05 within columns (unpaired-test).
63
11 Discussion
The novel findings of this research demonstrate the dual impact of metoprolol on CO,
cerebral perfusion and adrenergic mediated vasodilation in isolated mesenteric and cerebral
arteries. Our experimental results support the hypothesis that metoprolol may impair vital organ
perfusion by limiting cardiac output (global ischemia) and adrenergic mediated vasodilation at
the level of the small resistance artery (organ specific ischemia). When metoprolol was
administered to anesthetized mice in vivo, MAP was maintained while HR, SV and CO were
reduced. The associated increase in SVR resulted in decreased brain perfusion as indicated by a
reduction in cerebral microvascular oxygen tension. However, the mechanism by which SVR
was increased could not be determined in a whole animal experiment. It may have been due to a
reflex increase in sympathetic tone, or as a direct action of metoprolol on resistance arteries. In
order to explore the latter mechanism, we performed studies in isolated mesenteric resistance
arteries. Results demonstrated that metoprolol inhibited isoproterenol (β1/2) mediated
vasodilation in a dose dependent manner. This was at least partially mediated via the β2-
adrenergic receptor confirmed by data demonstrating that metoprolol also inhibited clenbuterol
mediated vasodilation. Metoprolol had a more profound effect on shifting the EC50 of the
isoproterenol dose-response curve, suggesting an additional β1-AR blocking effect. Since
clinical studies have implicated metoprolol therapy as a risk factor for increased stroke, we then
assessed the impact of metoprolol on isoproterenol mediated vasodilation in cerebral arteries.
Our results demonstrated that metoprolol also impaired adrenergic mediated vasodilation in
isolated posterior cerebral arteries. Because adrenergic stimulation in isolated mouse posterior
cerebral arteries has not been previously characterized, we assessed the effect of time and
metoprolol administration on the phenylephrine DRC. Our results demonstrated that the
64
posterior cerebral artery has a reduced phenylephrine dose-response over time which is not
affected by metoprolol.
Collectively, these data suggest that metoprolol could directly impair cerebral artery
dilation under conditions which require increased cerebral blood flow to maintain oxygen
delivery. The combination of reduced cardiac output coupled with organ specific inhibition of
resistance artery dilation may explain in part the mechanism of increased stokes and mortality
which occur in β-blocked perioperative patients.
11.1 Beta-adrenergic vasodilation is required to maintain vital organ perfusion
Immediately following metoprolol administration, mean arterial pressure as well as left-
ventricular pressures were maintained, CO was decreased and SVR was increased.
Subsequently, there was a reduction in PBrmvO2. Importantly, the reduction in brain perfusion
that was observed in this study occurred in β-blocked mice under resting conditions with low
metabolic requirements (ie. anesthetised). These results can be correlated to the clinical
condition of hypertension. During hypertension the health of the vasculature is altered and an
increased risk of organ injury is observed (stroke, MI)36. Beta-blockers concurrently reduced
MAP and the incidence of cardiovascular complications including stroke in hypertensive
patients49. However, vasodilatory anti-hypertensive medications (ACE-I, CCB) demonstrate a
further reduction in the incidence of stroke, relative to hypertensive patients treated with β-
blockers49. These clinical data suggested that anti-hypertensives with direct vasodilatory action
on vascular smooth muscle may improve cerebral perfusion. This argument is further supported
by the clinical finding that patients treated with β-blockers that possess vasodilatory capacity
(carvedilol) have fewer ischemic events (stroke, MI) than those treated with non-vasodilatory β-
blockers (metoprolol)104. A vascular mechanism for increased stroke incidence in hypertensive
patients treated with β-blockers is supported by our whole animal data. Mice treated with
65
metoprolol had a reduced HR, SV and CO and increased SVR. This hemodynamic pattern
resulted in a reduction in cerebral microvascular oxygen tension. In addition to limiting global
cardiac output, the direct effect of metoprolol to reduce adrenergic mediated vasodilation of
mesenteric and cerebral arteries might also increase the risk of brain ischemia. Additional
studies in hypertensive animal models would be required to test this hypothesis.
The impact of metoprolol on artery function may be further exacerbated during times of
increased oxygen demand (sympathetic activation) and/or reduced oxygen supply. During these
conditions, the brain and heart, organs with high metabolic requirements, are preferentially
perfused by active vasodilatory mechanisms112;113. In the heart, sympathetic activation results in
parallel increases in myocardial oxygen consumption and delivery95;96. The latter is regulated by
β-adrenergic receptor mediated coronary vasodilation. In swine and dogs, coronary vasodilation
was mediated by both β1- and β2-ARs during times of increased oxygen demand (exercise)95;96.
Inhibition of these receptors during exercise led to reduced oxygen delivery to the myocardium
and a greater mismatch of coronary oxygen supply and consumption95. We have not assessed
the impact of metoprolol on coronary arteries. However, clinical studies suggest that β-blockade
paradoxically increased the incidence of MI in patients who experience acute blood loss and
fluid resuscitation (hemodilution)107;114. The cardioprotective benefit associated with β-blockade
may therefore be negated by their inhibitory effect on coronary vasodilation during conditions
of increased myocardial oxygen demand and limited supply.
In the brain, cerebral blood flow is increased under conditions of increased cerebral
metabolic rate (increased neuronal activity)115 and reduced oxygen supply (hemodilution)116.
These responses are mediated in-part by β-adrenergic mechanisms110;117. In the clinical setting
of acute hemodilutional anemia, reduced blood oxygen content (reduced O2 supply) has been
associated with an increase in the incidence of stroke118;119. In a subset of the POISE trial,
66
significant blood loss (surgical anemia) in β-blocked patients was associated with a 2-fold
increased risk of stroke45. This suggests that β-blockers may increase the risk of ischemic organ
injury during times of reduced blood oxygen content. In parallel studies, our lab has previously
assessed the effect of β1-blockade (metoprolol) and β2-blockade (ICI 118, 551) in a model of
acute hemodilutional anemia in rats. Hemodilution causes an increase in oxygen demand to vital
tissue in order to maintain organ perfusion: CO is increased, MAP is maintained and SVR
decreases110. In animals pretreated with metoprolol, MAP was maintained, however the CO
response was blunted suggesting a global reduction in oxygen supply (in addition to reduced
blood O2 content)110. Animals treated with the β2-antagonist also maintained MAP during
hemodilution, however the heart rate response was no different from control (CO was not
measured)117. Metoprolol treatment reduced cerebral blood flow and oxygen tension and
increased cellular markers of tissue hypoxia (HIF-1α)110. This was due in part to the reduction in
cardiac output; however the magnitude of the reduction in oxygen tension suggests that a
cerebrovascular mechanism may have also contributed. This is supported by evidence from the
β2-blocked anemic rats. These animals also had reduced cerebral blood flow and tissue oxygen
tension during hemodilution, despite having similar HR responses to control117. As in the
coronary circulation, there may be a dual role for β1- and β2-AR mediated vasodilation in
maintaining cerebral perfusion. This assertion is supported by our findings in isolated arteries.
In the current study, we observed an inhibitory effect of metoprolol on β1/2-AR mediated
vasodilation (isoproterenol) in cerebral arteries. In addition, we demonstrated that metoprolol
can inhibit β2-specific mediated vasodilation (clenbuterol) in mesenteric arteries. Metoprolol
shifted the isoproterenol dose-response more profoundly than it did the clenbuterol dose-
response, suggesting a contribution of β1-AR mediated vasodilation. We have provided direct
evidence that metoprolol impairs cerebral artery dilation by a β-AR mediated mechanism in
67
response to an adrenergic stimulus This may explain the observed increased incidence of stroke,
MI and mortality in patients treated with metoprolol who experience acute blood loss45;107.
11.2 Metoprolol inhibits adrenergic vasodilation in mesenteric and cerebral arteries
In the mesenteric and posterior cerebral arteries there was a significant inhibitory effect
of metoprolol on the isoproterenol dose-response curve. This confirms that in our isolated vessel
model, a cardioselective β-blocker can impair the normal vasodilatory response of blood vessels
during increased demand (ie. sympathetic stimulation).
The relevance of our isolated vessel studies are supported by our lab’s previous animal
studies in anemic rats110;117 and by clinical findings. As described in 11.1, impaired cerebral β-
AR mediated vasodilation could contribute to the increased risks of stroke in β-blocked
perioperative and hypertensive (relative to ACE-I and CCB treatment) patients50-55;120. In
addition to studying vessels from our organ of interest (brain), we also studied vessels from the
mesenteric vascular bed due to their being well characterized as well as their ease of access. Gut
ischemia is not a commonly reported outcome measure in most β-blocker clinical trials.
However, a trial in post-MI patients, identified an almost 200% increase in relative risk of
developing gastrointestinal problems (event rate 1 vs 0.3 % in treatment vs. control group)19
with propranolol treatment. It is unknown whether these events were ischemic in nature; future
trials should therefore consider the effect of β-blockade on other, non-vital organ systems.
11.3 Metoprolol may be associated with increased morbidity and mortality
This study demonstrated that metoprolol can cross-react with the β2-AR to inhibit
vasodilation. This finding is supported in cell culture studies where metoprolol was identified as
having the lowest β1-selectivity compared with other β-blockers. Metoprolol has 2.3 fold
selectivity for the β1-AR compared to the β2-AR while bisoprolol shows 13.5 fold β1-AR
68
selectivity4. The low selectivity of metoprolol can explain why there is evidence of increased
morbidity compared to other cardioselective β-blockers. In a retrospective analysis of the impact
of anemia on mortality risks, metoprolol was associated with an increased risk of death while
atenolol and bisoprolol were not108. In a recent meta-analysis of perioperative β-blocker trials,
Badgett et al. found a significant negative correlation of mortality with increasing β1-
selectivity56. Patients treated with metoprolol were at a significantly greater risk of dying
perioperatively than a patient treated with bisoprolol. The authors did not find a significant
correlation between stroke and β-blocker selectivity. There is, however, a trend towards this
relationship. In POISE, metoprolol treatment was associated with increased stroke45 while the
DECREASE IV trial found that there was no significant difference between placebo and
bisoprolol treated patients in stroke incidence46. These are two different outcomes with two β-
blockers with significantly different β1-AR selectivity. However, they also differ in their
mechanisms of metabolism, which may also contribute to negative patient outcomes.
Certain β-blockers (metoprolol, carvedilol, propranolol, labetalol and timolol) are
metabolized by the cytochrome P450 CD6 (CYP2D6) enzyme121. The gene for CYP2D6 is
highly polymorphic resulting in differing effectiveness of the enzyme in individuals. Those who
are poor metabolizers tend to experience increased adverse outcomes with β-blockade121. There
is consequently a significant correlation of increased perioperative mortality risk with β-
blockers that rely on CYP2D6 metabolism56. In addition, a short titration period up to the target
dose is associated with increased mortality56. In POISE, patients were treated with metoprolol
(CYP2D6) over a short titration period (2-4 hours preoperatively)45 while in DECREASE-IV,
patients were given bisoprolol (non-CYP2D6) over a long titration period (34 days
preoperatively)46. These could potentially explain the negative outcomes associated with
metoprolol; however receptor selectivity is still a relevant mechanism. A patient with a CYP2D6
69
polymorphism making them a poor metabolizer of metoprolol would be overdosed by the
standard β-blocker dosage. The overdose could lead to cross-reactivity with β2-AR4 in addition
to occupation of the β1-AR to limit cerebral and coronary vasodilation95;96;110;117. Our results
with clenbuterol support the hypothesis that cross-reactivity with the β2-AR may be a significant
mechanism of increased harm in patients.
11.4 Differences between mesenteric and cerebral arteries
This study used 3rd order mesenteric arteries which makes them more similar to
resistance arteries122. These arteries are integral in the control of blood pressure within the body
due to their location as precapillary arteries as well as their intrinsic ability to constrict in
response to increases in pressure (the myogenic response)123. The resistance arteries
consequently have more vascular smooth muscle cells in their tunica media layer than do the
elastic conduit arteries124;125. A larger smooth muscle layer, and therefore larger population of
VSMCs, would result in a greater force of vasoconstriction. Our results support this assertion
given that the posterior cerebral artery is a 1st order artery and is closer to an elastic conduit
artery than a resistance artery. Consequently, the PCAs demonstrated a reduced magnitude of
contractile response (Emax) to phenylephrine compared to the mesenteric arteries. The sensitivity
(EC50) to phenylephrine was not different between the two arteries suggesting similar α-AR
densities. However, previous experimental studies have demonstrated that receptor density dose
not determine the maximal response to phenylephrine stimulation in VSMCs126. This is in line
with our findings that differential phenylephrine response in mesenteric and cerebral arteries
may be due to structural rather than receptor population differences.
The myogenic response is important in maintaining blood flow and tissue perfusion as
well as protecting the capillaries from large increases in pressure127. In our model, we observed
no change in MAP with metoprolol administration, suggesting that myogenic autoregulation
70
may not have been a very important factor in our results. However, it is possible that β-blockade
may inhibit this important autoregulatory mechanism which, in the brain, is an integral in
maintaining cerebral blood flow during times of increased demand or reduced supply128. The
importance of PKA (a downstream signalling molecule of β-AR stimulation) in the myogenic
response has not been extensively studied. However one study in isolated rat tail arteries has
reported that a PKA inhibitor attenuates the myogenic response at a transluminal pressure of
80mmHg129. Future work on the effect of β-blockade on not just the autonomic nervous
system’s role in autoregulation but also the myogenic contribution would be necessary to further
understand how β-blockade could affect tissue perfusion.
We found that the sensitivities and magnitudes of response to isoproterenol were
comparable in both mesenteric and posterior cerebral arteries (no differences in EC50 and Emax).
We also demonstrated that metoprolol inhibits dilation of resistance and conduit arteries which
could be relevant in regulating organ perfusion. In unpublished data in our lab, we have
demonstrated that CHF increases the myogenic response in mouse posterior cerebral arteries,
effectively making them important mediators of cerebral perfusion. A β-blocker effect on
conduit arteries could limit blood supply to the resistance arteries and could therefore be
additionally detrimental, especially with the presence of cardiovascular disease. This effect may
explain the reduced incidence of stroke with vasodilator (ACE-I, CCBs) treatment versus β-
blockers in hypertension49. This suggests, along with our findings, that a mechanism of conduit
and resistance artery dysfunction with β-blockade may play an important role in ischemic
injury.
11.5 Limitations
We assessed the impact of metoprolol in a mouse model which has a much higher
intrinsic heart rate than humans. Attempts were made to use a clinically relevant dose of
71
metoprolol which reduced heart rate by about 20%. This reduction is comparable to the intended
effect size in the clinical setting. Though mouse cardiovascular physiology is not the same as in
humans, our model supports current clinical findings and is therefore valuable in elucidating the
mechanisms of increased morbidity and mortality with β-blockade.
Due to methodological constraints, we were unable to measure MAP and CO in the same
animals. However, in addition to measuring a reduction in CO, our flow-volume loops
demonstrated that left ventricular systolic pressures were maintained following metoprolol
treatment. This is consistent with our MAP data and therefore allowed an accurate measurement
of increased systemic vascular resistance following β-blockade.
Isoflurane anesthesia was used in all whole animal protocols. Isoflurane is a vasodilator
and increases cerebral blood flow when administered130. Isoflurane was maintained at 1.5% in
21% O2 in both control and treatment groups, therefore any differences in CBF and in PBrmvO2
were due to the difference in treatment groups rather than isoflurane.
In our hypoxic challenge experiments, mice were spontaneously breathing and under no
mechanical ventilation. Hypoxia causes hyperventilation due to a reduced arterial PO2 and this
consequently causes a reduction in arterial PCO2. CO2 is an important metabolic vasodilator128,
therefore low arterial PCO2 will limit tissue perfusion. This effect may have been responsible the
observation that there was no appreciable difference in PBrmvO2 between saline and β-blocker
treated mice during hypoxic challenge. We did not measure arterial blood gases and therefore
cannot confirm that there was a reduction in PCO2 in our animals, however this stimulus may
have been great enough to mask the effect of β-blockade on cerebral blood flow during hypoxia.
Mechanically ventilating our mice during hypoxic challenge would have been a possible
solution to this methodological limitation. More importantly, however, our finding that
metoprolol injection significantly lowered resting brain microvascular PO2 would not have been
72
affected by this limitation and therefore still gave us a strong rationale to examine the effect of
β-blockers on isolated arteries.
Our isolated arteries are denervated and therefore lack the input from autonomic and
other perivascular nerves. All pharmacological agents are applied through the superfusate (ie.
external from the vessel lumen); which mimics prejunctional neurotransmitter release to our
postjunctional receptor targets. Although our agents’ pharmokinetics may differ in vivo than
what we see in vitro, our whole animal data support our findings in isolated arteries and provide
a closer tie to data from clinical studies.
We did not extend our study to determine the functionally predominant β-AR receptor
subtype in our two vessel types. Though we can make inferences based on sensitivities to
isoproterenol and clenbuterol, the assessment of a β1-AR agonist in these tissues would allow a
more complete understanding of the physiology at play. Additionally, assessing the
isoproterenol dose-response curve in the presence of a β2-specific antagonist (such as
butoxamine), would also greatly help the interpretation of our observed results. Our primary
aim, however, was to determine if metoprolol had an effect on mouse vascular tissue, and this
was clearly demonstrated.
The β-AR and α-AR have competitive signalling pathways. This may have been why we
observed a reduced phenylephrine dose-response following an isoproterenol dose-response
curve. However, since endogenous norepinephrine and epinephrine act on both α- and β-ARs in
vivo, our protocol would similarly mimic this dual receptor activation. These data therefore
remain relevant in linking our whole animal findings with those found in vitro.
Finally, we used different order of arteries from gut (3rd) and brain (1st). While the
mesenteric arteries are thought to be true resistance arteries, the cerebral vessels may have more
of a combined conduit/resistance artery function. The resistance arteries ultimately control tissue
73
perfusion. However, we demonstrated that there is a significant metoprolol effect on larger
arteries which also can control blood flow to the precapillary arteries.
12 Summary
We have provided novel evidence that metoprolol can limit cardiac output and increase
systemic vascular resistance in a mouse model in vivo. This was associated with a reduction in
brain microvascular oxygen tension in anesthetized mice suggesting that basal brain perfusion
can be impaired by metoprolol. Isolated mesenteric and cerebral arteries were utilized to
demonstrate a direct effect of metoprolol to inhibit β-adrenergic mediated vasodilation. These
data support both a cardiac and vascular mechanism for the clinical observation of increased
incidences of organ ischemia and mortality in patients treated with metoprolol.
74
13 Appendix A – Beta-blockade: A historical perspective
Sir James Black, working at Imperial Chemical Industries (ICI), invented the first
clinically used β-blocker, propranolol, in an era where knowledge of sympathetic nervous
transmission was in its infancy. Walter Cannon and Arturo Rosenblueth were the fathers of the
prevalent “sympathin theory” of adrenergic mediators131. They postulated that two circulating
molecules, sympathins E (excitatory) and I (inhibitory), when combined with epinephrine were
responsible for adrenergic activity in cells131. Though they could not actually prove the
existence of the sympathins in the body, their theory was held as physiologic dogma for almost
a decade until it was challenged by Raymond Ahlquist in 1948.
In an elegant series of experiments, across species and organ types, Ahlquist proposed
that there was only one adrenergic mediator. This was epinephrine (adrenaline), purified in 1901
by a Japanese chemist, Jokichi Takamine, and the first ever hormone isolated132. Ahlquist
proposed that epinephrine, an adrenal extract which strongly affected blood vessels, heart tissue
and skeletal muscle, bound to two different adrenergic receptors expressed on the target tissue.
Ahlquist used 6 different analogues of epinephrine (ethanolamine, isopropanolamine, dl- and l-
epinephrine, methyl isopropanolamine and isopropyl ethanolamine) and determined the relative
activity of each in producing a response in different organ systems from dogs, cats, rabbits and
rats. From the cardiovascular system, Ahlquist determined the order of activities of these amines
on vasoconstriction, vasodilation and myocardial excitation. He also determined how the
intestines, uterus, ureters, dilator papillae and nictitating membranes reacted to his
pharmacological manipulations. Ahlquist found that two different orders of activity emerged
based on both observed function and tissue type examined. For example; vasoconstriction in
renal, mesenteric, femoral and skin blood vessels followed an order of activity of l-epinephrine,
75
dl-epinephrine, ethanolamine, isopropanolamine, methyl isopropanolamine and isopropyl
ethanolamine133 while vasodilation in skeletal muscle and coronary blood vessels followed an
order of isopropyl ethanolamine, l-epinephrine, methyl isopropanolamine, dl-epinephrine,
isopropanolamine and ethanolamine133. These two orders of activity were consistent across
tissue types leading Ahlquist to arbitrarily designate one order of activity as representative of the
alpha receptor and the other as the beta receptor. His findings are summarized in Table 13.1. It
is now known that the α-receptors are sub classified as α1A, α1B, α1D, α2A, α2B, and α2C and that
the β-receptor has three subtypes; β1, β2, and β3134. For the purposes of this study, only the β1-
and β2-adrenergic receptors will be discussed in detail.
Although Ahlquist’s initial conclusions have survived over a half-century of scientific
scrutiny, his seminal discovery could not dislodge Cannon and Rosenblueth’s entrenched
sympathin theory. As luck would have it, Sir James Black came across Ahlquist’s theory of
adrenergic transmission while reading Drill’s Pharmacology in Medicine which, as he put it,
was “surprising, because at the time [Ahlquist’s] proposal had been published only with great
difficulty – it just wasn’t in the medical dogma.”2 As a clinician scientist, what was most
interesting to Black was Ahlquist’s finding that the β-receptor was responsible for the
adrenergic excitation of the myocardium and how this could apply to the treatment of angina
pectoris. He reasoned that instead of increasing myocardial oxygen supply, as most anti-angina
drugs (vasodilators: nitrates, calcium channel blockers)131 were designed to do at the time, he
could develop a drug that reduced myocardial oxygen demand by acting on Ahlquist’s β-
receptors2. Black and his colleague, chemist John Stephenson, recognized that the well known
β-receptor specific agonist isoproterenol (Table 13.2) has no α-selectivity because the amine
methyl group of epinephrine was modified to the larger isopropyl group2. With this in mind,
76
Alpha Receptors Beta Receptors Vasoconstriction
Renal Mesenteric Femoral
Vasodilation Coronary Skeletal muscle
Nictitating membrane Myocardium (excitatory) Uterus (excitatory)
Rabbit Dog
Uterus (inhibitory) Rat Cat Dog
Intestine (inhibitory) Dilator papillae
Table 13.1 Original characterization of adrenergic receptor subtypes Modified from Ahlquist, 1948133.
77
Drug Structure Selectivity
Epinephrine HO
OH
HN
OH
α and β
Isoproterenol HO
OH
HN
OH
β
Dichloroisoproterenol Cl
Cl
HN
OH
β (partial agonist)
Propranolol HN
OH
O β (antagonist)
Table 13.2 Structures of beta-adrenergic receptor ligands Evolution of the chemical modifications to epinephrine eventually leading to the first clinically used β-blocker, propranolol.
78
and knowing that the ring substituted dichloroisoproterenol was a partial β-agonist, Black and
Stephenson hypothesized that by further altering the ring structure of isoproterenol, they could
completely eliminate any agonist activity2. Propranolol, the world’s first β-blocker was
consequently invented by substituting a benzene group for the vicinal hydroxyl groups of
isoproterenol creating a double ring structure with complete antagonist activity at the β-
adrenergic receptor (Table 13.2). For this and other contributions to drug development, Sir
James Black was awarded the Nobel Prize in Medicine in 1988.
79
14 Appendix B – Characterization of PCA phenylephrine responses
14.1 Determining optimal phenylephrine preconstriction dose in PCAs
Phenylephrine preconstriction and maintenance doses have not been characterized
previously in the posterior cerebral artery. It was therefore necessary to determine the PE dose
which gives an adequate degree of initial tone while keeping a consistent degree of background
(maintenance) tone through several doses.
14.1.1 Methods
PCAs (n=12) were exposed to increasing concentrations of the α1-adrenergic receptor
agonist phenylephrine (PE; 0.03-30µM) to generate a dose-response curve. In order to match the
degree of preconstriction in MRAs, the concentration of PE which induced 30-50% tone in
PCAs was determined. This concentration (10µM) was found at the upper plateau of the PCA
PE dose-response whereas the MRA preconstriction dose is located along the slope of the MRA
PE dose-response. Therefore, two concentrations of PE were tested as potential preconstriction
and maintenance doses for the isoproterenol dose-response curves; 10µM and 3µM which lies
along the slope of the PCA PE dose-response.
Following a viability check, PCAs were treated with 9 consecutive doses of 10 (n=4) or
3µM (n=3) PE to determine their effectiveness as preconstriction and maintenance doses.
Following this first series, organ baths were exchanged for 50µM metoprolol in MOPS and
incubated for 30 minutes in order to determine the effect of β-blockade on consecutive
applications of these two doses. Each dose of 10 or 3µM PE was held for 2 minutes before
recording the vessel diameter.
80
14.1.2 Results
The phenylephrine dose-response curve in PCAs did not have significantly different
EC50 than in mesenteric resistance arteries (-6.0 ± 0.2 in MRAs vs -5.9 ± 0.08 in PCAs; n=5,
12). There was, however, a significant difference in the magnitude of response to PE in PCAs;
the degree of tone induced by the EC50 value of PE (1µM or LogEC50=-6.0) was significantly
lower in PCAs compared to MRAs (19 ± 3.0 vs. 37 ± 6.1%; n=12, 5, p<0.05; Figure 14.1.1B)
and the Emax % dilation was also significantly lower in PCAs (45 ± 1.7 vs. 60 ± 2.3; n=12, 5,
p<0.05; Figure 14.1.1C). This made the standard MRA preconstriction and maintenance dose
insufficient for the vasodilatory curves in PCAs.
Two potential concentrations of PE were tested for their ability to be effective
preconstriction and maintenance doses (Figure 14.1.2). Exposing PCAs to 9 consecutive doses
of 10µM PE resulted in a significant reduction in tone by the end of the series compared to the
tone induced by the first dose (from 40 ± 3.3 to 14 ± 6.4%; n=4, p<0.05; Figure 14.1.2A).
Consequently, consecutive doses of 10µM PE resulted in significant dilation of the PCA by the
end of the 9th dose (up to 66 ± 14%; Figure 14.1.2B). However, with 9 consecutive doses of
3µM PE, PCAs were able to maintain their initial degree of tone throughout the series and there
was no significant dilation (n=3; Figure 14.1.2C, D). Additionally, there was no difference in
these described responses at both concentrations PCAs before and after incubation with 50µM
metoprolol (Figure 14.1.2). With these findings, 3µM PE was chosen as the optimal
preconstriction and maintenance dose concentration in PCAs.
81
-8 -7 -6 -5 -40
20
40
60
log [phenylephrine (mol/L)]
ton
e (%
dia
ma
x 45
mm
Hg
)
-8 -7 -6 -5 -40
20
40
60
log [phenylephrine (mol/L)]
ton
e (%
dia
ma
x 6
0mm
Hg
)A B
MRA PCA0
20
40
60
80
*
PE
Em
ax,
ton
e (%
dia
ma
x)
D
MRA PCA0
20
40
60
80
*
ton
e (%
dia
ma
x)
at P
E E
C5
0(1M
)
C
Figure 14.1.1 Phenylephrine dose-response curves in mesenteric and cerebral arteries. Dose-response curves to phenylephrine in mesenteric resistance (MRA; A) and posterior cerebral arteries (PCA; B). The degree of tone elicited by the EC50 dose of phenylephrine (C) was significantly higher in MRAs than PCAs (37 ± 6.1% vs. 19 ± 3.0%; n=5, 12, p<0.05). Emax values (D) were significantly lower in PCAs than in MRAs (45 ± 1.7 vs 60 ± 2.3; n=12,5, p<0.05) Data presented as mean ± SEM.
82
0 2 4 6 8 100
20
40
60
80
100 Control, n=4
50M Metoprolol, n=4
* * * * * * *
# of 10M PE Doses
ton
e (%
dia
ma
x 45
mm
Hg
)
0 2 4 6 8 100
20
40
60
80
100 Control, n=4
50M Metoprolol, n=4
** * * * * *
# of 10M PE Doses
% d
ilat
ion
0 2 4 6 8 100
20
40
60
80
100 Control, n=3
50M Metoprolo, n=3
# of 3M PE Doses
ton
e (%
dia
ma
x 4
5mm
Hg
)
0 2 4 6 8 100
20
40
60
80
100 Control, n=3
50uM Metoprolol, n=3
# of 3M PE Doses
% d
ilat
ion
A B
C D
Figure 14.1.2 Effect of consecutive doses of phenylephrine in posterior cerebral arteries. In PCAs, tone is lost (A) and arteries dilate (B) in response to nine consecutive doses of 10µM phenylephrine. There is a significant dose effect, but there is no treatment and no interaction effect (two-way repeated measures ANOVA, p<0.05, n=4). Tone is maintained (C) and vessels do not dilate (D) in response to nine consecutive doses of 3µM phenylephrine. Data presented as mean ± SEM.
83
14.2 Effect of metoprolol on phenylephrine dose-response curve in PCAs
14.2.1 Results
Phenylephrine dose-response curves (PE DRC) were generated before and after
metoprolol incubation (0µM metoprolol represents a time control; Figure 10.3.1). There was no
difference between phenylephrine LogEC50 values over time in the PCA (Table 14.1). There
was a significant inhibition of the magnitude of the response over time in posterior cerebral
arteries(Figure 14.2.1A, D). The tone generated at the EC50 dose of PE (1µM or logEC50=-6)
was not significantly altered by time Figure 14.2.1C. However, the tone at Emax (ie. at the top of
the PE DRC best-fit curve) was significantly inhibited by time (43 ± 3.0 in the first curve vs. 35
± 4 % tone in the second, 0µM metoprolol curve; n=5, p<0.05; Figure 14.2.1D). The same
relationship was found with application of 50µM metoprolol; no affect on LogEC50 value or on
the tone generated at this value (Table 14.1; Figure 14.2.1C) but a significant inhibition of the
degree of tone at Emax (47 ± 1.9 vs. 25 ± 3.9 %; n=7, p<0.05; Figure 14.2.1B, D). When
comparing the time matched PE DRC (ie. 0µM metoprolol) vs. 50µM metoprolol, there was no
significant difference in LogEC50 value, the tone at this concentration or in the degree of tone at
Emax(Figure 14.2.1).
84
-8 -7 -6 -5 -40
20
40
60 Control, n=5
0M Metoprolol, n=5
* *
log [phenylephrine (mol/L)]
ton
e, %
dia
ma
x 4
5mm
Hg
-8 -7 -6 -5 -40
20
40
60 Control, n=7
50M Metoprolol, n=7
** *
log [phenylephrine (mol/L)]
ton
e, %
dia
ma
x 4
5mm
Hg
M M
et
- 0
M M
et
+
0M
Met
- 5
0M
Met
+
50
0
20
40
60
EC
50
(1M
)to
ne,
% d
iam
ax 4
5mm
Hg
M M
et
- 0
M M
et
+
0M
Met
- 5
0M
Met
+
50
0
20
40
60
Em
ax
ton
e,%
dia
ma
x 4
5mm
Hg
A B
C D
Figure 14.2.1 Effect of metoprolol on phenylephrine dose-response in PCAs. Phenylephrine (PE) dose-response curves were generated before (control) and after incubation with metoprolol in posterior cerebral arteries. PE mediated increases in tone at high doses are blunted with time (represented by 0µM metoprolol; panel A) and at 50µM metoprolol (panel B). The PE curve at 50µM metoprolol is not significantly different from its time matched control (panel C and D). Emax values (D) are significantly reduced by time (43 ± 3.0 vs. 35 ± 4% tone; n=5, p<0.05) and at 50µM metoprolol (47 ± 1.9 vs. 25 ± 3.9% tone; n=7, p<0.05). Data presented as mean ± SEM. * p<0.05 by two-way repeated measures ANOVA; ɣ p<0.05 by paired t-test; # p<0.05 by unpaired t-test.
85
Control (-Met) + Metoprolol
± 0µM Metoprolol (n=5) -5.8 ± 0.1 -6.0 ± 0.2
± 50µM Metoprolol (n=7) -5.9 ± 0.1 -6.2 ± 0.2 Table 14.1 Half maximal concentrations of phenylephrine in PCAs. LogEC50 values for PE dose-response curves in PCAs. Data presented as mean ± SEM. *p<0.05 between columns (paired t-test); # p<0.05 within columns (unpaired-test).
86
14.2.2 Interpretation: Phenylephrine responses are time-dependent in the PCA
The control phenylephrine dose-response (ie. the first dose-response in the protocol) is in
agreement with a previous study in mouse middle cerebral arteries (MCA)135. It is likely that
these vessels responded similarly to phenylephrine because of their proximity to the circle of
Willis as well as their 1st to 2nd order of branching.
There was a significant time-dependent inhibition of the magnitude of the phenylephrine
response in PCAs. In order to overcome this phenomenon, our analyses in PCAs were compared
to internal controls (ie. the same vessel) and to time-matched controls. Time-matched analyses
showed that the phenylephrine dose-response in the posterior cerebral artery was not
significantly affected by metoprolol.
The time-dependent decay of the phenylephrine dose-response may be due to a
desensitization of the α1-AR over the course of the experimental protocol. A study in rat
thoracic aorta has shown that prolonged exposure to phenylephrine reduces the responsiveness
to α1-AR mediated vasoconstriction136. This effect, however, was not due to receptor down-
regulation, but rather by an up-regulation of nitric oxide synthase (NOS) and consequently nitric
oxide (NO). As a potent vasodilator, NO could antagonize the vasoconstriction induced by
phenylephrine in the second dose-response curve (the time controlled curve).
It is also possible, considering the full protocol in PCAs (phenylephrine dose-response;
isoproterenol dose-response; phenylephrine dose-response with/without metoprolol;
isoproterenol dose-response with/without metoprolol), that the first isoproterenol curve may
influence the contractile response of the second phenylephrine dose-response curve. In addition
to a possible phenylephrine-induced increase in NOS, there may also be an isoproterenol-
induced increase in NOS and therefore NO. There is evidence that isoproterenol-mediated
vasodilation has a nitric oxide component, arising from attenuated isoproterenol responses in
87
either endothelium denuded/L-NAME (NOS inhibitor) treated or eNOS-KO vessels84-86.
Furthermore, it has been demonstrated that isoproterenol has a dose-dependent inhibitory effect
on the phenylephrine dose-response curve in isolated rabbit common carotid arteries137. This
could likely suggest that with increasing doses of isoproterenol, there is an increase in NO. If it
is not fully removed when changing the bath, the NO could subsequently affect the
responsiveness of phenylephrine in the posterior cerebral arteries.
In our isoproterenol dose-response curves, there was no difference in the magnitude of
tone induced by our preconstriction dose (3µM) of phenylephrine before and after metoprolol.
The time dependent decay described above only affected higher concentrations of the
phenylephrine dose-response curve. This could explain why consecutive doses of 10µM
phenylephrine showed a slow reduction in tone (increased dilation) in PCAs. The mechanism
may be α1-AR mediated increases in NO, which would consequently reduce tone and promote
vasodilation 136. This effect was observed in our vessels and could contribute to reduced
responsiveness when high doses of phenylephrine are applied over a long period of time.
Conversely, consecutive doses of 3µM phenylephrine did maintain tone in our PCAs. For this
reason, this concentration of phenylephrine was chosen as our preconstriction and maintenance
dose for the isoproterenol dose-response curves.
88
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