AN OFFICIAL PUBLICATION OF THE AF SYMPOSIUM, PUBLISHED …

Spring 2014AN OFFICIAL PUBLICATION OF THE AF SYMPOSIUM, PUBLISHED BY ADVANCED MEDICAL EDUCATION

Page 3: David Wilber examines the link between obesity, inflammation and AF.

Page 5: Structural remod-eling and calcium leak in AF.

Page 8: What are the underlying mechanisms of AF progression?

Page 10: The best basic science papers of 2013 laid bare.

Inside

W elcome to the first in a new series of publications by the AF Symposium that will provide a year-round expansion of the sessions, debates and cutting-edge presentations featured during our annual symposium.

Each edition of this special series of AF Symposium News will provide a focused exploration of a number of core topics.

For this first Spring Edition, we will take a in-depth look at fibro-sis, inflammation and the transition of science and medicine from cell to bedside – topics which were featured during a particularly-intriguing session held at the 2014 AF Symposium in Orlando.

Structural remodeling in AF is of high importance, and as such the session delved deeper into a number of facets of the underly-ing mechanisms that could affect the progression of AF, as well as exploring interventions that may have real power in its preven-tion. To that end, the following pages bring together reports from world-leading experts on fibrosis, inflammation and associated topics, all of which were exhibited at the symposium.

We hope you enjoy this new venture by the AF Symposium, and we look forward to bringing you more in-depth reports in our future editions, later this year.

T he 2014 AF Symposium played host to a session that explored inflamma-tion and fibrosis in atrial fibrillation, with a number

of invited experts gathering to offer their perspectives from the cellular level all the way to the bedside.

In his presentation, Eric Prystowsky (St. Vincent Hospital and Health Center, Indiana, Indianapolis, USA) gave an overview of treat-ment strategies for AF that fall outside mainstream ap-proaches, with an emphasis on just how much evidence warrants their consideration.

“It’s an interesting topic because it could mean anything, technically,

other than what we consider conven-tional approaches,” he said. “Con-ventional approaches would be direct antiarrhythmic therapy, either via drugs or ablation.”

After a comprehensive review of alternative therapies across several different categories, Dr Prystowsky narrowed his focus to several key

areas that have been more commonly discussed. One aspect that became im-mediately clear during the review was that many of these concepts – while initially very promising – can be largely

discarded once more data becomes available.

“I find a lot of these early concepts and studies to be what I call ‘teasers’,” said Dr Prystowsky, adding that often the preliminary data will be from super-selective patient groups, or is simply a good idea with somewhat positive early data. What follows can

then be somewhat of a rollercoaster ride, with initial enthusiasm for the results heightening rapidly, only to be slowed to a halt when larger or more

comprehensive studies come to the fore.

Nevertheless, a primary concept that Dr Prystowsky discussed in his lecture was upstream therapy – the meaning of which is not always im-mediately apparent. He commented: “I remember when I first heard it I thought ‘what are they talking about?’. It was a very clever term to put out a concept of not treating the actual arrhythmia, per se. Instead going upstream and doing something to modify the potential substrate i.e. the atria, before you ever get the ar-rhythmia. I and many people thought this was an interesting concept based in some form of reality.”

Initial core interests in the field of upstream therapy lied with inflamma-

Alternative therapies for atrial fibrillation

Continued on page 2

“Except for some very selected circumstances, non-traditional forms of treatment for atrial fibrillation have not proven useful.”

Eric Prystowsky (St. Vincent Hospital and Health

Center, Indiana, Indianapolis, USA)

A new venture

2 AF Symposium News Spring 2014

tion, and particularly atrial remodel-ling, thus ACE inhibitors and angio-tensin receptor blockers (ARBs) – with their pleomorphic (and anti-fibrotic) effects – became an interesting avenue of testing. However, once again the re-search would prove to be another so-called teaser. “Everything seemed to fit very nicely,” said Dr Prystowsky. “The idea was to take all of the people who have atrial fibrillation and assign them to an ACE or ARB. And again some of the early studies, which I’ll show, suggested that this was something that could be very positive. But then came the large studies – the thousands of patients-types of studies – and it’s been a negative ending to the story.”

Despite these types of outcomes, Dr Prystowsky cautioned that this does not mean upstream therapy is inac-curate, nor should it be abandoned, rather that we should be mindful of what we target. Referring to ACEs, ARBs and their role in hypertension, he said: “Once you have established atrial fibrillation with substrate issues, I do not think that adding drugs that may

alter a substrate are going to do any good... but it doesn’t, in my opinion, negate the concept of true upstream therapy, which is to minimize or elimi-nate hypertension.

“I think there are substantial data – good data – to say that the hyperten-sion over time will cause multiple changes that will make atrial fibrilla-tion more of a reality. So if you look at something like ACEs or ARBs or any really good anti-hypertensive therapy, I think the concept of upstream therapy makes sense, not necessarily to modify the atrium, but to get at the root cause.”

There are also other alternative therapies that have seen a lot of pub-licity both in the lay press and medical circles, such as polyunsaturated fatty acids (PUFAs) and omega-3s. However, they too have suffered from unsus-tainable enthusiasm: “They had some very early studies which were again a bit of a teaser – but the larger studies have proven to be negative,” said Dr Prystowsky.

“Then we also have things like statins... they weren’t really produced to do anything other than reduce LDL levels and cholesterol, and they do a fine job of doing that, but there were also some studies suggesting they might be beneficial for minimizing atrial fibrillation, and I think there are some realities there. There are data in people postoperatively after bypass surgery, and some for acute coronary syndrome, but I don’t consider those very worthwhile to the view of using statins to minimize atrial fibrillation, because there are very few cardiologists that I know that wouldn’t be giving statins anyway to people who had bypass graft surgery or acute coronary syndrome. The question for me is will statins work in non-acute coronary syndromes, or in bypass sur-gery? There I think there has been no substantial data to support that.”

Baring these in observa-tions in mind, in general terms, the more widely-discussed upstream therapies (e.g. ACEs, ARBs, statins and PUFAs) have been met with results that are largely disappointing: “Except for some very selected circumstances, non-traditional forms of treatment for atrial

fibrillation have not proven useful,” said Dr Prystowsky.

There are also additional novel upstream therapies under study that target changes in neuroactivity,

including renal denervation, yoga and acupuncture. For Dr Prystowsky, many of these upstream methods make a lot of sense because they alter the au-tonomic nervous system (and in turn, the heart rate). “They are getting to

a root cause, and that is my message throughout all of this,” he said. “I do think most atrial fibrillation, clearly not all, is substrate based. I’ve change my thinking over the years from triggers to

substrate. I think the data are just piling up.”

He continued: “If you look things such as the PUFAs, statins, ACEs and ARBs – all those sorts of therapies – they’re trying to affect things at the level of the atrial tissue. I think once you’ve got the substrate there it is going to be very difficult to do something like that, and that’s why at that point I would talk about antiarrhythmic drugs and ablation.”

Offering his take home message, Dr Prystowsky concluded: “I’m saying we shouldn’t pursue something to modify the substrate. We should be modifying and preventing the disease that causes the substrate: I’m taking it one step backwards.”

AF Symposium News

AF Symposium DirectorsJeremy Ruskin, MDMoussa Mansour, MDDavid Keane, MD, PhDPierre Jais, MDVivek Reddy, MD

Editor-in-ChiefPeter Stevenson

EditorsRysia BurmiczAlastair McQueen

DesignPeter Williams

Contacts: Muriel [email protected] © 2014: AF Symposium. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, transmitted in any form or by any other means, electronic, mechanical, photocopying, recording or otherwise without prior permission in writing of the AF Symposium. The content of AF Symposium News does not necessarily reflect the opinion of the AF Symposium Directors, the Committees or the Faculty.

Alternative therapies for atrial fibrillationInflammation and Fibrosis In Atrial Fibrillation: Cell to Bedside

Eric Prystowsky

“I’m saying we shouldn’t pursue something to modify the substrate. We should be modifying and preventing the disease that causes the substrate: I’m taking it one step backwards.”

Eric Prystowsky (St. Vincent Hospital and

Health Center, Indiana, Indianapolis, USA)

Continued from page 1

Spring 2014 AF Symposium News 3

D ata began to emerge around 10 years ago regarding the relationship of obesity with AF risk. With this in mind, David Wilber (Loyola

University Medical Center, Maywood, IL, USA) sum-marized the more recent findings on this extensive topic during the dedicated inflammation and fibrosis session, suggesting that pericardial and epicardial fat may be the source of local inflammatory mecha-nisms that lead to the evolution of AF pathology: a very encouraging pragmatic finding that has emerged from this work is that risk of AF progres-sion can be ameliorated by weight reduction.

Dr Wilber first spoke of the Framingham Heart Study, which looked at a community-based observa-tional cohort of over 5,000 with a mean follow-up of 13.7 years, identifying obesity as an important risk factor for AF.1 The study found that the inci-dence of new onset AF over the follow-up period had an adjusted hazard ratio of about 1.5, which equates to a 50% increased risk of AF with obesity. “This was independent of most of the clinical risk factors, such as hypertension and hyperlipidemia,” said Dr Wilber. “However, when the left atrial [LA] size was included and corrected in the model, the relationship with obesity no longer held. This sug-gested that in fact all of the effect of LA margin was really the mediator for the effect of obesity and its relationship to AF.”

This result has been replicated by several other analyses, including the work of Wanahita et al. (2008), a meta-analysis of five community-based cohorts, comprising 68,000 patients in total.2 Obese patients, defined as having BMI greater than 30kg/m2, had a 49% increased risk of new onset AF, explained Dr Wilber. “The other important thing that helps to confirm the relationship is the graded re-sponse, in that the risk of AF increased as you move from normal to overweight to obese.”

Describing the complexities of extricating the various factors seemingly involved with AF risk, Dr Wilber continued: “One of the problems with looking at the mediators of obesity’s effect is the end relationship between many variables. Particu-larly problematic is the LA volume. Some data from the MONICA [Monitoring Trends and Determinants

in Cardiovascular Disease] study,3 which is not directly related to AF per se, gives some perspective. These were patients that were part of a long term prevention study. They had a baseline echo and then another echo at 10 years. Both hypertension and obesity were very significant predictors of LA volume, after adjusting for all of the remaining im-portant clinical covariates. They were about equally the most important contributors to LA volume for prevalence of AF at the beginning of the study.

“But more interesting was that, as you followed patients over time, obesity was the predominant predictor of LA volume over that ten year period. Hypertension alone had very little influence over LA volume over time. The other interesting observation in this study was that there seemed to be a little bit of difference in terms of what predicted risk for hypertension and obesity mediation of LA volume. Obesity seemed to be more related to volume overload, whereas hypertension seemed to be medi-ated more through pressure overload, suggesting that there is a different contribution of these two variables.”

The longitudinal study of Tsang et al.4 looked at

the progression of paroxysmal to permanent AF (us-ing the traditional definition of AF that is persistent for more than six months and resistant to restora-tion of sinus rhythm), confirming the relationship between obesity and risk of AF (Figure 1). More importantly, the study confirmed an independent contribution of hypertension and obesity in terms of LA volume. “The two together were a much stronger risk, such that obesity had some independ-ent contribution beyond LA volume in terms of me-diating AF; LA volume was not the only explanation for the progression of AF.”

Describing evidence from the women’s health study,5 which characterized the relationship between changes in BMI and incident AF, Dr Wilber said: “Compared to those who stayed within BMI of less than 30, those whose BMI was reduced (that were initially obese and then became normal weight) had the same risk as those who were never obese at all. Those who remained obese, or who were newly obese in follow-up, had the same risk. This provides some hope that reduction in weight can ameliorate risk of atrial fibrillation. These same sorts of relationships are also shown in men, in terms of the BMI change from age 20 to 50. In fact, one sees a progressive increase in risk as there is more gain in BMI or weight over time.

“The effect of obesity seems to be at least partially independent of systemic factors. Cardiac remodeling, LA volume and diastolic dysfunction may be mediators. But that independent effect may be exerted by more local causes, being inflammation and pro-fibrotic cytokines, potentially due to the fact that immediately adjacent to the atrial myocardium is the epicardial fat. There is suggestion that this mechanism can be mediated by epicardial fat. Some of the inter-esting observations are that the epicardial fat is far more pro-inflammatory and a producer of potential pro-fibrotic cytokines than the subcutaneous fat.”

Dr Wilber then reasoned that, if it is the epicar-dial fat that is producing a local effect, it should be possible to see a stronger relationship of epicardial fat with AF than with other indices of body mass. “That is the case from some of the first data from

Obesity, inflammation and atrial fibrillation

Inflammation and Fibrosis In Atrial Fibrillation: Cell to Bedside

Figure 1. The relationship between obesity and progression of AF demonstrated in a cohort of 3248 patients. BMI predicted progression of paroxysmal to permanent AF,

independent of age, gender and clinical variables. Data courtesy of Tsang et al.3

Continued on page 4


the Framingham study,” he continued. “In this case, only the pericardial fat volume was significantly associated with AF risk. There was about a 10% increased risk for every 10ml of increased pericardial fat, and this was independent of everything else – BMI, all of the other sizes of fat depots.

“There was another study that we did years ago, looking at patients undergoing AF ablation with peri-cardial fat quantified by CT. Pericardial fat was again associated with age, gender, BMI and LA size. Despite that, it had an independent predictive value of all those. Similar to the Framingham study, it suggested about a 13% increase in risk for every 10ml increase in volume of pericardial fat. Although there is a cor-relation with LA volume, it is modest at best.”6

This relationship was demonstrated again by the Adelaide group, as Dr Wilber explained. “Again, there is a graded relationship between epicardial fat and the risk of AF. They looked at both atrial and ven-tricular components, and they found that they were equally predictive. All of them, again, are independ-ent of all the other risk factors, suggesting that there is something about epicardial fat that is uniquely related to risk of AF relative to other fat depots.”7

Dr Wilber noted that the finding that epicardial fat volume predicts AF, that it is a graded relation-ship, and that it is independent of BMI and LA volume, has been confirmed in five other studies. He said: “This suggests that there is a significant re-

lationship, which may have to do with the fact that there is no barrier for the diffusion of cytokines and pro-inflammatory molecules into the atrium. Several

of these studies attempted to look at more localized fat deposits around the LA, or at specific locations in the LA, and all of them were predictive but none was better than the others. Whether they used epi-cardial fat or more regional fat within the epicardial space, they all had the same predictive value.”

Describing some of the data that indicates that obesity has a negative impact on ablation outcomes, Dr Wilber then spoke of more proactive interven-tions, such as weight loss programs (Figure 2). “It appears as if epicardial fat is also a predictor of poor outcomes with a progressive increase in recurrences associated with increased epicardial fat. There is some hope for proactive intervention – a study of 150 patients randomized to active attempt at weight loss, showed that a greater loss of weight was as-sociated with both a reduction in the frequency of AF episodes and the symptoms associated with it. Obesity is a very strong impact on AF occurrence, including post-ablation outcomes and it is potentially modifiable.”8

References

1. Wang TJ et al. Obesity and the risk of new-onset atrial fibrillation. JAMA. 2004 Nov 24;292(20):2471-7.

2. Wanahita N et al. Atrial fibrillation and obesity--results of a meta-analysis. Am Heart J. 2008 Feb;155(2):310-5.

3. MONICA: Monitoring Trends and Determinants in Cardiovascular Disease. http://www.thl.fi/monica/

4. Tsang TS et al. Obesity as a risk factor for the progression of paroxysmal to permanent atrial fibrillation: a longitudinal cohort study of 21 years. Eur Heart J. 2008 Sep;29(18):2227-33.

5. Tedrow UB et al. The long- and short-term impact of elevated body mass index on the risk of new atrial fibrillation the WHS (women’s health study). J Am Coll Cardiol. 2010 May 25;55(21):2319-27.

6. Al Chekakie MO et al. Pericardial fat is independently associated with human atrial fibrillation. J Am Coll Cardiol. 2010 Aug 31;56(10):784-8.

7. Wong CX et al. Pericardial fat is associated with atrial fibrillation severity and ablation outcome. J Am Coll Cardiol. 2011 Apr 26;57(17):1745-51.

8. Abed HS et al. Effect of weight reduction and cardiometabolic risk factor management on symptom burden and severity in patients with atrial fibrillation: a randomized clinical trial. JAMA. 2013 Nov 20;310(19):2050-60.

Obesity, inflammation and atrial fibrillationInflammation and Fibrosis In Atrial Fibrillation: Cell to Bedside

Figure 2. Weight loss and AF burden. 150 patients with AF and BMI > 27 kg/m2 were randomized to a weight loss

program (intervention) or risk factor/lifestyle management alone (control). A 19% decrease in weight was shown

in the intervention group, versus 5% in the control group. A significantly greater decrease in AF burden occurred

in intervention versus control, along with greater declines in LA volume and mass. Data courtesy of Abed et al.8


“Compared to those who stayed within BMI of less than 30, those whose BMI was reduced (that were initially obese and then became normal weight) had the same risk as those who were never obese at all.”

David Wilber (Loyola University Medical

Center, Maywood, IL, USA)

David Wilber



F or his presentation taking place during the session, Stanley Nattel (Montreal Heart Institute,

University of Montreal, Quebec, Cana-da) spoke of the basic mechanisms of atrial structural remodeling and atrial fibrillation. “Given the fact that about two-thirds of the speakers so far have alluded to this, you could imagine that an exhaustive treatment of this subject would take several months, so I’ll try to restrict my comments to a study we were involved in recently that I find really fascinating,” he began.

First touching on what causes AF progression, Dr Nattel stressed that is has something to do with remod-eling that promotes transition from paroxysmal, to persistent, to per-manent AF. “In order to understand that we need to review very briefly the primary mechanisms by which remodeling leads to AF,” he said. “One is a shortening in refractory period, and that can be due to a decreased inward calcium current or an increased outward potassium current.

Stanley Nattel

Figure 1. A mouse model of AF progression,

showing progression from sinus rhythm to

atrial ectopy and atrial fibrillation

“The structural remodeling and conduction abnormalities of CREM mice are prevented by suppressing calcium leak, and these observations suggest that AF progression in this model is caused by structural modelling caused by ryanodine receptor calcium leak.” Stanley Nattel (Montreal Heart Institute, University of

Montreal, Quebec, Canada)

Structural remodeling and

calcium leak in AF

Continued on page 6


“Second is slowed conduction, which can result from impaired con-nexin function or tissue fibrosis. And the third is increased circuit path length of atrial dilatation. What are the main signals that lead to progres-sion of AF? We don’t know for sure, but we have an idea, and the study I’m going to talk to you about today fascinatingly supports that idea.”

Specifically, Dr Nattel pinpointed calcium levels as playing a key role, referring to supporting literature show-ing that, in cardiomyocytes, increased intracellular calcium activates calpains and other proteases to break down proteins. “It also increases nuclear calcium, changes gene programming, and promotes cardiomyocyte remod-eling,” he said. “In addition, increased fibroblast calcium causes proliferation and differentiation into myofibroblasts. These are the primary collagen-secreting form of the fibroblast, and they cause structural remodeling and fibrosis.”

He continued: “Increased diastolic calcium leak seems to be involved in paroxysmal AF and maybe occurs in persistent AF. The question one could ask is what about the effect of this increased calcium on the remod-eling program? Could the increased calcium itself be causing remodeling in cardiomyocytes, and through cardiomyocyte-secreted factors, could it be activating fibroblasts?”

Moving on to discuss his own work examining AF progression, Dr Nattel outlined the model his team had de-veloped in mice. “This model involves mice engineered to overexpress a repressor form of so-called cardiac response element modulator (CREM), which is a critical mediator of beta adrenergic responses,” he said. “These mice, very interestingly, develop atrial arrhythmias and atrial remodeling with normal ventricular function and virtu-ally no ventricular remodeling.”

Dr Nattel also touched upon pub-lished data showing that transgenic mice that over-expressed mutated


Figure 2. CREM mice have excessive Ca2+ leak

Felipe Aguel, PhD US Food and Drug Administration Silver Spring, MD

Antonio Bartorelli, MD Centro Cardiologico Monzino-University of Milan Milan, Italy

Hugh Calkins, MD Johns Hopkins Hospital Baltimore, MD

A. John Camm, MD St. George’s Hospital Medical School London, UK

Shih-Ann Chen, MD Veterans General Hospital Taipei, Taiwan

KR Julian Chun, MD Cardioangiologisches Centrum Bethanien Frankfurt, Germany

Phillip Cuculich, MD Washington University School of Medicine St. Louis, MO

Andre d’Avila, MD, PhD Hospital Cardiologico, Florianopolis Santa Catarina, Brazil

Luigi Di Biase, MD, PhD Albert Einstein College of Medicine Bronx, NY

Jun Dong, MD, PhD US Food and Drug Administration Silver Spring, MD

Patrick Ellinor, MD, PhD Massachusetts General Hospital Boston, MA

Gaetano Fassini, MD Centro Cardiologico Monzino-University of Milan Milan, Italy

David Haines, MD William Beaumont Hospital Royal Oak, MI

Michel Haissaguerre, MD Central Hospital University of Bordeaux Bordeaux, France

Kevin Heist, MD, PhD Massachusetts General Hospital Boston, MA

Gerhard Hindricks, MD University of Leipzig Leipzig, Germany

Meleze Hocini, MD Central Hospital University of Bordeaux Bordeaux, France

Pierre Jais, MD Central Hospital Univ of Bordeaux Bordeaux, France

Jose Jalife, MD University of Michigan Ann Arbor, MI

David Keane, MD, PhD St. Vincent’s University Hospital Dublin, Ireland

Young-Hoon Kim, MD, PhD Korea University Medical Center Seoul, Korea

Hans Kottkamp, MD Heart Center Hirslanden Zurich, Switzerland

Peter Kowey, MD Lankenau Hospital Wynnewood, PA

Karl-Heinz Kuck, MD St. George Hospital Hamburg, Germany

Dhanunjaya Lakkireddy, MD University of Kansas Hospital Kansas City, KS

Mark La Meir, MD, PhD Maastricht University Medical Center Maastricht, Netherlands

Chang Sheng Ma, MD Beijing Anzhen Hospital, Capital Medical Univ Beijing, China

Moussa Mansour, MD Massachusetts General Hospital Boston, MA

Francis Marchlinski, MD University of Pennsylvania Philadelphia, PA

Nassir Marrouche, MD University of Utah Salt Lake City, UT

Sanjiv Narayan, MD, PhD UCSD School of Medicine San Diego, CA

Andrea Natale, MD Texas Cardiac Arrhythmia Institute Austin, TX

Stanley Nattel, MD University of Montreal Montreal, Canada

Douglas Packer, MD Mayo Clinic Rochester, MN

Carlo Pappone, MD, PhD Villa Maria Cecilia Hospital Cotignola, Italy

Laurent Pison, MD Maastricht University Medical Center Maastricht, Netherlands

Evgeny Pokushalov, MD, PhD Research Institute of Circulation Pathology Novosibirsk, Russia

Eric Prystowsky, MD The Care Group Indianapolis, IN

Vivek Reddy, MD Mount Sinai School of Medicine New York, NY

Jeremy Ruskin, MD Massachusetts General Hospital Boston, MA

Boris Schmidt, MD Cardioangiologisches Centrum Bethanien Frankfurt, Germany

Daniel Singer, MD Massachusetts General Hospital Boston, MA

Claudio Tondo, MD, PhD Centro Cardiologico Monzino-University of Milan Milan, Italy

David Van Wagoner, PhD Cleveland Clinic Cleveland, OH

Albert Waldo, MD Case Western Reserve University Cleveland, OH

David Wilber, MD Loyola University Medical Center Chicago, IL

Faculty




CREM developed spontaneous AF, as well as demonstrating inducible AF. “If you do Holter monitoring on the mice, very interestingly they seem to follow progression much like the progression of AF in humans,” he said. “They start out at a young age in sinus rhythm, they then manifest atrial ectopy, and finally atrial fibrillation [Figure 1].” He added that optical mapping showed dramatic atrial dilation and conduction abnormalities in older CREM mice.

As can be seen in Holter data from wild type (WT) and CREM mice at three and seven months (Figure 2), CREM transgenic mouse showed atrial ectopy at three months, and atrial fibrillation at seven months. “There’s a larger ryanodine receptor calcium leak in these CREM mice, both at the individual experimental data level and at the mean data level,” said Dr Nattel.

He continued, presenting western blots and mean data plots showing ryanodine receptor phosphorylation by CaM kinase in the CREM mice (Figure 3). When CaM kinase was blocked with an organic blocker (KN-93) calcium leak was suppressed. However, KN-93 cannot be given orally, nor chronically, but following from other work in the field, it was possible to breed mice with a mutation in the ryanodine receptor itself. “The site at which CaM kinase phosphorylates the ryanodine receptor is serine 2814,” said Dr Nattel. “If you change the serine to an alanine, there’s nothing to phosphorylate, so the phosphorylation level of the ryanodine receptor goes to basically zero.

“So by crossing the CREM mice with these double-mutant mice – i.e. those with the S2814A muta-tion – you can now get CREM mice who have over activity of CaM kinase but no longer have phosphorylation of the ryanodine receptor, and presumably no longer have calcium leak.”

In confocal microscopy stud-ies of the different WT, CREM, KN-93-suppressed and double-mutant S2814A mice (Figure 4), it can be seen that the double transgenic mice with a non-phosphorylatable ryanodine receptor show no spontaneous leaks, and a dramatic reduction in the open probability of the ryanodine receptor.

“So now we can test the hypoth-esis that calcium leak is responsible for progression of AF in this mouse mod-el, which looks a lot like humans,” said Dr Nattel, adding that in the CREM mice there was a dramatic in-crease in the amount of AF over time. Specifically, looking at percentage of

mice with spontaneous AF, there were no cases at three months, but about 40% of CREM mice showed AF at five months, further increasing to 80% at eight months.

Dr Nattel continued: “In the double transgenic mice that have this non-phosphorylatable ryanodine receptor

mutation, and therefore don’t have the calcium leak, AF is completely eliminated. Similarly if you look at inducible AF using electrophysiological study, it is dramatically increased in

the CREM mice and eliminated in the double transgenic mice. So CREM ryanodine receptor S2814A mice lacking sponta-neous calcium releases do no develop spontaneous AF.”

He added: “The structural remodeling and conduction abnormalities of CREM mice are prevented by suppressing calci-um leak, and these observations suggest that AF progression in this model is caused by structural modelling caused by ryanodine receptor calcium leak.”

Offering his conclusions, Dr Nattel began by underlining that CREM mice mimic the progressive nature of human atrial arrhythmias which, again,

are mediated by calcium leaks due to CaM kinase hyperphosphorylated ryanodine receptors. “This is in many ways reminiscent of the calcium leak that we saw in human AF samples both from chronic and paroxysmal AF cases, albeit with somewhat different molecular mechanisms,” he said.

“Preventing ryanodine recep-tor phosphorylation eliminated the calcium leak, and by doing that elimi-nated atrial structural remodeling and AF progression. So this raises the very interesting idea that the same calcium signaling that underlies DADs [delayed afterdepolarizations] also causes structural remodeling and AF progression in the mice model. One can ask the question of whether the same mechanisms is operative in man, and is the apparently inexorable progression of AF in patients due to some underlying defect that we need to attack in order to prevent?”

“Is the apparently inexorable progression of AF in patients due to some underlying defect that we need to attack in order to prevent?”

Stanley Nattel (Montreal Heart Institute,

University of Montreal, Quebec, Canada)

Figure 3. Effects of non-phosphorylatable ryanodine receptor mutation on RyR function and Ca2+ leak

Figure 4. Confocal microscopy images showing the effects of reduced RyR Ca2+ leak on atrial structural remodeling and conduction


The underlying mechanisms of AF progressionInflammation and Fibrosis In Atrial Fibrillation: Cell to Bedside

T he effects of inflammatory markers and oxidative stress in the progression of atrial

fibrillation were placed under the spotlight during the fibrosis session, forming part of broader picture looking at how the disease develops at a cellular level and beyond.

With 20 years of experience focused on the mechanisms of atrial fibrillation behind him, David Van Wagoner (Cleveland Clinic, Cleveland, Ohio, USA) took to the podium to discuss how our understanding of the underlying causes of AF has evolved during this time.

“When I started, the main goal of antiarrhythmic therapy for treating AF was based on blocking ion channels – mostly sodium channels and potassium channels,” he told AF Symposium News. “The track record for an-tiarrhythmic drugs has been pretty dismal. We did cellular electro-physiology studies to characterize the changes that were associated with the electrical activity of atrial myocytes, from patients typically with coronary artery disease or atrial fibrillation. And we demon-strated that the electrical changes that we saw that were associated with AF were mainly observed only in the patients that had per-sistent AF, which suggested that the electrical changes were kind of secondary to whatever it was that was causing the AF.”

With this in mind, Dr Van Wag-oner stepped away from study of ion channels, instead focusing more of the underlying mechanisms and pathways that may lead to atrial fibrillation. Around the same time, studies were emerging that would suggest a link between inflammatory markers such as C-reactive protein (CRP) and atherosclerosis, and Dr Van Wagoner became interested in ex-ploring whether there may be similar changes in C-reactive protein levels in patients with atrial fibrillation.

“We knew that C-reactive protein increased in patients who had surgery or were sick, because inflam-mation is always associated with those kinds of trauma,” said Dr Van

Wagoner. “But what we weren’t sure was whether there was any link between inflammation and atrial fibrillation in regular patients who had AF. So, we did studies of blood CRP levels in patients who were healthy controls just coming in for a physical (i.e. no history of atrial fibrillation), compared to patients with paroxysmal atrial fibrillation, and patients with long-lasting persistent atrial fibrillation.

“What we found was that there was an association – which is not a mechanistic understand-ing, just a statistical association – between levels of C-reactive protein in the blood and persis-tence of atrial fibrillation. Patients who had paroxysmal AF tended to have higher levels of CRP than the controls. Those with persistent AF had higher levels than paroxysmal AF, and those with essentially permanent AF had higher still.”

In a 10-year study of over 5000 subjects aged 65 or over, Dr Van Wagoner and investigators were able to confirm that patients with higher levels of CRP had an

increased risk of developing atrial fibril-lation when compared to those with low CRP levels, suggesting that there is indeed prognostic significance in measuring inflam-matory cytokine levels.

Similarly, this strong association between inflammatory activation and the persistence of atrial fibrillation was also tested outside of CRP. “C-reactive protein is not a specific marker, it is only essentially a marker of inflammation,” said Dr Van Wagoner. “But inflammatory activity is often associated with increased production of oxidants by NADPH oxidase [nicotinamide adenine dinucleo-tide phosphate-oxidase] and other oxidant sources.”

To delve deeper, the next topic of study

Figure 1. Oxidant sources

in AF; potential targets

for intervention

David Van Wagoner

“If you have someone that has paroxysmal AF that is not long lasting, I think that might be a really effective time to catch it, and prevent it going further.”

David Van Wagoner (Cleveland Clinic, Cleveland,

Ohio, USA)



was to search for evidence of oxidant produc-tion, with a specific focus on protein nitration and altered regulation of nitric oxide. Results supported the initial hypothesis, with evidence of increased protein nitration in patients with atrial fibrillation, as well as patients who had underlying valvular heart disease. “This suggested that increased production of oxidants might both be related to the presence of atrial fibrillation, and perhaps also some of the risk factors that lead to atrial fibrillation,” explained Dr Van Wagoner.

He went on to highlight the oxidant sources in AF (Figure 1) that could act as potential targets for intervention. “In subsequent studies it has been shown that there is increased expression of some of the enzymes that create superoxide, i.e. the NADPH family, especially NADPH oxidase 4,” he said. With Figure 2, Dr Van Wagoner demonstrated how NADPH oxidase, present in cardiac myocytes and inflammatory cells, is modulated.

Dr Van Wagoner continued: “Superoxide, both directly and indirectly (when turned into hydrogen peroxide), can have electrophysiological effects. It

can affect the activity of calcium channels; it can affect the activity of ryanodine receptors that cause spontaneous release from intracellular stores of calcium; and it can affect the activity of sodium channels... that means that the cells have to struggle with handling more influx of calcium or sodium, and it makes them electrically unstable.”

Such observations in turn suggest that hemo-dynamic stress can cause atrial fibrillation, in part by altering the electrical activity of atrial myocytes, as well as the step-wise system of inflammatory re-sponse that occurs when stressed myocytes die (spe-cifically, recruited fibroblasts and extracellular matrix proteins that cause structural changes that then lead to altered conduction patterns in the atria). “Those are the things that we think are causing the transi-tion from paroxysmal episodes of atrial fibrillation to more long-lasting forms like persistent and perma-nent atrial fibrillation,” said Dr Van Wagoner.

Looking to the future, increased insight into the

sorts of mechanisms that trigger atrial fibrillation will ultimately need to be transposed into treatment strategies. “From my point of view the aspect that is really interesting is are there things we could do to prevent atrial fibrillation?” said Dr Van Wagoner. “I think what is needed is to try interventions that oc-cur earlier than the stage where someone is already presenting with persistent atrial fibrillation. If you have someone that has paroxysmal AF that is not long lasting, I think that might be a really effective time to catch it, and prevent it going further.”

In more recent avenues of research, Dr Van Wag-oner has investigated where inflammation is rooted within the overall disease. Specifically, observations that there are no alterations in the levels of inflam-

matory activation following successful treatment of atrial fibrillation have in turn suggested that inflam-mation stems from the underlying disease (i.e. the risk factors) as opposed to coming from the atrial fibrillation itself. “This suggests that treating the risk factors is as important, if not more important, than treating atrial fibrillation from the point of view of its progression,” said Dr Wagoner.

With this in mind, further study is now very much at the forefront in the quest for better understand-ing of what strategies would best help in combating the underlying mechanisms and causes that lead to atrial fibrillation. “Anti-fibrotics are interesting, and close to the top of the list of things to investigate,

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NADPH Oxidases (NOX)

NOX 2,4 are present in cardiac myocytes and inflammatory cells

Are activated by angiotensin-II, endothelin-1 (Gq-coupled receptor activation)

ACE-inhibitors and ARBs decrease NOX activity in an A-II dependent manner

NOX activity is modulated by the small G-protein, Rac; Rac activation is dependent on prenylation

Rac prenylation is prevented by HMG-Co-A reductase inhibitors (statins)

Figure 3. High plasma CRP is associated with increased areas of low voltage (fibrosis)

Figure 2. Modulation of NADPH oxidases

Continued on page 10


but we don’t have the answer to it yet,” said Dr Van Wagoner, adding: “We should also be focusing on the obvious things such as weight and blood pressure. Those are probably less appealing to the general electrophysiology community, but they are more important from a public health perspective.”

Dr Van Wagoner also described how increased CRP is associated with fibrosis (Figure 3). He went on to touch on how inflammatory mechanisms contrib-ute to risk of thrombosis as well as AF persistence, as detailed in Figure 4.

Summarizing his talk, Dr Van Wagoner ex-plained: “Oxidant stress promotes inflammatory cytokine production, it promotes atrial remodeling, it promotes atrial fibrosis, it promotes electrical remodeling and spontaneous calcium release and thus this may have some interesting targets that can be more specific than old thoughts about simple antioxidants. You can think about targeting oxidized CaM-kinase, you can think about targeting NADPH oxidase, you can think about targeting the Gq cou-pled receptors, and these provide new mechanistic ways of thinking about atrial fibrillation.

“In conclusion, oxidant and inflammatory mecha-nisms contribute to atrial fibrosis, persistent and risk of thrombosis.” Figure 4. Inflammatory mechanisms associated with AF and risk of thrombosis

The underlying mechanisms of AF progressionInflammation and Fibrosis In Atrial Fibrillation: Cell to Bedside


A s in previous years of the AF Sym-posium, the choice of best papers depends on the speaker and their

area of interest. For Dr Nattel, his best basic science selection related to the other presentations he gave that day, namely ‘New Insights into Mechanisms Underlying Focal Atrial Ectopic Firing in Paroxysmal AF’, and ‘Basic Mechanisms of Atrial Structural Remodeling in Atrial Fibrillation’.

Beginning with his first choice of paper, Dr Nattel said: “I will be talking about an in-teresting paper in the Journal of the Ameri-can College of Cardiology (JACC), looking at a specific mutation in a protein called junc-tophilin 2 (JPH2), which has not previously

been implicated in AF.1 What is interesting about this mutation is that it interferes with the stabilization of the ryanodine receptor (RyR) in its functional location in the heart cell. Normally, JPH2 anchors the RyR to the cell membrane, and this anchoring appears to be essential for its proper function.

“The authors looked at an individual proband with a specific JPH2 mutation associated with early-onset AF, and they found that this specific mutation impaired the interaction between JPH2 and the RyR. The RyR is the key control mecha-nism for the release of calcium (Ca2+) from the sarcoplasmic reticulum, which is the main cellular Ca2+ store. When the RyR leaks at inappropriate moments, it causes an excess of Ca2+ in the cell, which drives ectopic activity that we believe may underlie many cases of paroxysmal AF.”

The individual under investigation had par-oxysmal AF beginning at age 22, with further problems emerging at 24. Dr Nattel explained that the particular mutation in JPH2 interferes with the localization of RyR relative to the membrane and impairs the binding of JPH2 to the RyR, caus-

The fiber of the matter: Best basic science papers of 2013The best basic AF science papers of 2013 were featured in a standalone lecture that saw Stanley Nattel (Montreal Heart Institute, University of Montreal, Quebec, Canada) taking his pick of the crop of the past 12 months.

“What is very interesting to me is the ability to use MRI to quantify and localize atrial fibrosis. This technology may allow for a variety of potential future studies in terms of understanding how fibrosis may be contributing to AF and in terms of developing patient-specific therapies.”

Stanley Nattel (Montreal Heart Institute, University of

Montreal, Quebec, Canada)

Best basic science papers on AF for 2013


ing abnormal releases of Ca2+. Furthermore, this impairment could be eliminated by restoring JPH2 function.

While this particular mutation also causes hypertrophic cardiomyopathy, AF does not exactly share its underlying pathophysiology, as most people with other mutations in the JPH2 gene develop hypertrophic cardiomyopathy, but not AF. Therefore, this case provides an interesting window into the mechanisms of paroxysmal AF.

While Dr Nattel reasoned that it is a possibility that this case of paroxysmal AF could be due to factors other than the JPH2 mutation, a wealth of data obtained in mice and reported in the JACC paper support its role in conveying susceptibility to AF. “[The investigators] developed a knock-in mouse model with this mutation and showed that it was susceptible to AF. Then, they knocked down JPH2 in other mice and showed that this also produced an AF susceptibility. So everything points towards this notion that JPH2 regulates the RyR, and when JPH2 malfunctions it promotes AF.”

Dr Nattel also spoke on the mechanisms of par-oxysmal AF at the symposium, presenting the finding that abnormal RyR function contributes to spontane-ous atrial activity in paroxysmal AF patients.2 “The mechanism for that is not clear, but it is possible that it is due to a relative deficiency in JPH2, because RyR are overexpressed in paroxysmal AF and JPH is not, producing a reduction of the JPH2/RyR expression ra-tio. Taken together with the JACC paper, the Circula-tion paper2 gives us some insight into the molecular basis for spontaneous AF paroxysms, which has been very elusive until recently.”

Moving on to the reentrant substrate in AF, Dr Nattel cited a paper from his own research institute on the topic of exercise-related AF.3 AF promoted by endurance exercise is now a well documented and clinically relevant phenomenon, although its mecha-nisms remain elusive. By developing an animal model of endurance exercise-related AF in rats, Dr Nattel and colleagues were able to induce remodeling of the heart similar to that which occurs in endurance athletes, producing a substrate for AF induction.

Explaining the subsequent detailed evaluation on the remodeling underlying AF with endurance exercise, Dr Nattel said: “We found that there were three contributors to the substrate. The main one was vagal enhancement, and we studied in detail the mechanisms of this, which turned out to be both central and cardiac. There is increased release of ACh (the baroreflex is enhanced), and there is also an increased effect of ACh at the level of the heart. We also identified a potential molecular mechanism for the cardiac hypersensitivity to ACh, which was a downregulation of the regulatory G proteins that control vagal function. This was probably the most important mechanism, because when we blocked cholinergic tone with atropine we were able to stop AF in the exercised rats. We also found atrial dilatation and moderate fibrosis, which probably also contributed to the AF substrate.”

Dr Nattel then spoke about two novel phar-

Figure 1. Mutation E169K in JPH2 causes AF due to impaired RyR2 stabilization. E169K mice exhibited a higher

incidence of inducible AF than wild type (WT)-PKI mice, whereas A399S-PKI mice (expressing a hypertrophic

cardiomyopathy-linked JPH2 mutation not associated with atrial arrhythmias) were not significantly different

from WT-PKI (left). These changes were attributed to reduced binding of E169K-JPH2 to RyR2 (right). Data cour-

tesy of Beavers et al.1Continued on page 12


Stanley Nattel


macological approaches to treating AF that were published last year. “The first paper is on relaxin, a hormone produced in pregnancy that relaxes the uterus, but also turns out to have some cardiopro-tective effects.4 I am also going to mention a study that used an intravenous administration of a relaxin derivative [serelaxin, recombinant human relaxin-2] in acute heart failure, demonstrating a benefit for heart failure management.5

“This is a compound that has already been given in vivo to man with some benefits, so theoretically it could form the basis of an oral preparation for AF prevention. This paper4 used relaxin, not serelaxin, and they gave it by osmotic pump to spontaneously hypertensive rats. They showed that the spontane-ously hypertensive rats developed an AF substrate which was prevented by infusion with relaxin. Relaxin also prevented fibrosis of the atrium, which is also probably an important substrate of AF in these rats.”

Dr Nattel’s next paper reported on an investiga-tion targeting transforming growth factor beta (TGF-β),6 a protein that has been evidenced as a potential contributor to the AF substrate in a num-ber of studies. “The work was done by a Japanese group that used a molecule called tranilast, which is available for oral use,” explained Dr Nattel. “They administered this compound to dogs, and

they used atrial tachypacing to, in this case, induce both atrial-tachycardia remodeling and ventricu-lar dysfunction (because of a resulting ventricular tachycardiomyopathy). They found that this TGF-β inhibitor, tranilast, suppressed the AF substrate – again, apparently by preventing the development of tissue fibrosis.”

Dr Nattel’s final choice of paper is from the research group of Natalia A Trayanova (John Hopkins University, MD, USA),7 which used MRI to quantify fi-brosis in human atria and mathematical modeling to determine what component of the fibrosis promoted AF. The group investigated collagen composition, gap junction function changes, and the combination of these and myofibroblast coupling, concluding that all of these components were necessary for the promotion of AF (Figure 2).

Explaining this study’s appeal, Dr Nattel said: “The mechanistic part of it is mainly based on mathemati-

cal modeling, which involves a lot of assumptions. I wouldn’t necessarily conclude that the modeling is telling us what exactly is producing AF, but what is very interesting to me is the ability to use MRI to quan-tify and localize atrial fibrosis. This technology may allow for a variety of potential future studies in terms

of understanding how fibrosis may be contributing to AF and in terms of developing patient-specific thera-pies by defining where the fibrosis is and informing us better about how to treat the AF, both in terms of prevention of further fibrosis and in terms of guiding ablation procedures.”

The fiber of the matter: Best basic science papers of 2013


Figure 2. Modelling of fibrotic lesions represented with collagen deposition (left); gap junction remodeling (GJR)

and collagen deposition (center); and a combination of GJR, collagen deposition and myofibroblast coupling

(right). The occurrence of GJR and the subsequent conduction slowing in the fibrotic lesions was a necessary but

not sufficient condition for AF development, whereas myofibroblast proliferation and the subsequent electro-

physiological effect on neighboring myocytes within the fibrotic lesions was the sufficient condition necessary

for reentry formation. Collagen did not alter the arrhythmogenic outcome resulting from the other fibrosis

components. Reentrant circuits formed throughout the noncontiguous fibrotic lesions, without anchoring to a

specific fibrotic lesion. Data courtesy of McDowell et al.7


“[Serelaxin] is a compound that has already been given in vivo to man with some benefits, so theoretically it could form the basis of an oral preparation for AF prevention.”

Stanley Nattel (Montreal Heart Institute,

University of Montreal, Quebec, Canada)

References

1. Beavers DL et al. Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization. J Am Coll Cardiol. 2013 Nov 19;62(21):2010-9.

2. Voigt N et al. Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation. Circulation. 2014 Jan 14;129(2):145-56.

3. Guasch E et al. Atrial fibrillation promotion by endurance exercise: demonstration

and mechanistic exploration in an animal model. J Am Coll Cardiol. 2013 Jul 2;62(1):68-77.

4. Parikh A et al. Relaxin suppresses atrial fibrillation by reversing fibrosis and myocyte hypertrophy and increasing conduction velocity and sodium current in spontaneously hypertensive rat hearts. Circ Res. 2013 Jul 19;113(3):313-21.

5. Metra M et al. RELAX-AHF Investigators. Effect of serelaxin on cardiac, renal, and hepatic biomarkers in the Relaxin in Acute

Heart Failure (RELAX-AHF) development program: correlation with outcomes. J Am Coll Cardiol. 2013 Jan 15;61(2):196-206.

6. Nakatani Y et al. Tranilast prevents atrial remodeling and development of atrial fibrillation in a canine model of atrial tachycardia and left ventricular dysfunction. J Am Coll Cardiol. 2013 Feb 5;61(5):582-8.

7. McDowell KS et al. Mechanistic inquiry into the role of tissue remodeling in fibrotic lesions in human atrial fibrillation. Biophys J. 2013 Jun 18;104(12):2764-73.

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