ASSESSMENT OF SYSTOLIC AND DIASTOLIC FUNCTION VTA TISSUE DOPPLER ECHOCARDIOGRAPHY IN BOXERS
AFFECTED WITH ARVC COMPARED TO NORMAL BOXERS AND NON-BOXER CONTROL DOGS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
JEREMY ORR
In partial fulfilment of requirements
for the degree of
Doctor of Veterinary Science
February, 2011
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1+1 Canada ABSTRACT
ASSESSMENT OF SYSTOLIC AND DIASTOLIC FUNCTION VIA TISSUE DOPPLER ECHOCARDIOGRAPHY IN BOXERS AFFECTED WITH ARVC COMPARED TO NORMAL BOXERS AND NON-BOXER CONTROL DOGS
Jeremy Orr Advisor: University of Guelph, 2011 Dr. M. Lynne O'Sullivan
This thesis is an investigation of cardiac function in Boxer dogs affected by
Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC]. ARVC is the most common acquired cardiac disease reported in the Boxer breed. It is characterized by ventricular arrhythmia of right ventricular origin, resulting in clinical signs of syncope, exercise intolerance, congestive heart failure and sudden death.
Structurally, ARVC in the Boxer is characterized by infiltration of the right ventricular myocardium with fibrofatty tissue, which is believed to the arrhythmogenic substrate. Diagnosis of the disease currently relies on the use of
Holter monitors, however given the inherent variability associated with Holters, false negatives can occur. Therefore, adjunctive diagnostic modalities are needed to aid in the diagnosis of ARVC-suspected dogs that have inconclusive Holter recordings. Our objectives were to assess both systolic and diastolic function using
Tissue Doppler echocardiography (TDI) measured at the medial and lateral mitral valve annulus and the lateral tricuspid valve annulus in Boxers affected with ARVC compared to non-affected normal Boxers and non-Boxer control dogs. Signal averaged ECG (SAECG) was also performed in all dogs, and troponin I
concentrations were measured. Dogs were classified into five groups using Holter
monitors and measurement of frequency of ventricular arrhythmia (VPCs/hr).
Seventy dogs in total were evaluated for the study, with 15 Boxer dogs in group 1 (>
1000 VPCs/24 hrs), 10 Boxer dogs in group 2 (200-999 VPCs/24 hrs), 15 Boxer
dogs in group 3 (25-199 VPCs/24 hrs), 15 Boxer dogs in group 4 (< 24 VPCs/24 hrs)
and 15 non-Boxer control dogs in group 5 (< 24 VPCs/24 hrs). Two-dimensional
echocardiography, TDI, SAECG and troponin I concentrations were measured in all
dogs.
When comparing all groups, there were no significant differences in the TDI parameters measured between groups. This finding suggests that ventricular
dysfunction may be uncommon in affected Boxer dogs. Furthermore, no significant
correlations between TDI and SAECG variables were found. Significant differences
in troponin I concentrations were found between groups of dogs, namely Boxers
and the non-Boxer control dogs. However, a correlation between VPCs/hr and troponin I concentration was not found. ACKNOWLEDGEMENTS
I would like to extend my sincere appreciation and thanks to several key
individuals who provided so much of their time, effort, advice and support to
complete this project. First and foremost, I would like to thank my amazing mentor
Dr. Lynne O'Sullivan who has always believed in my abilities even when I doubted
them. Without her guidance, encouragement and support, I would not be where or who I am today. She has been a true role model for me, and I feel truly fortunate
that I was given the opportunity to learn under her wonderful guidance. Dr. Michael
O'Grady also deserves similar praise as a wonderful co-mentor during my D.V.Sc and
during the completion of this project. I truly feel that I had the two best cardiology
mentors on the planet (or as I called them, my "cardiology mom and dad"), and it
has been such a sincere pleasure to work and learn with both of them. I would also like to sincerely thank my other Graduate Advisory Committee members - Drs.
Sandra Minors and Robert Hamilton. Their continuous feedback and advice has
been instrumental in completing this project to date.
I would like to thank one of the largest driving forces of this project who worked so hard behind the scenes initially, and was "my right hand woman" so to speak during the data collection portion of the project - Cindy Walker. Her sincere dedication to this project was awe-inspiring, and she was absolutely instrumental in the recruitment of the majority of the Boxers for this project, as well providing technical support for me, especially with the analysis of thousands upon thousands of hours of Holter recordings. Her positive attitude and personality made working
i with her a pleasure and allowed the project to run so smoothly, and I can never thank her enough for all of her help both personally and professionally.
I thank Jim Mcintosh, technologist at the Guelph General Hospital, for performing the troponin analysis for this project. I also thank Dr. Jeff Caswell in the
Department of Pathobiology for generously donating several histopathology images to include in this manuscript. I especially thank William Spears in the Department of Population Medicine for his wonderful statistical expertise.
I also thank our other members of the wonderful cardiology team at the
Ontario Veterinary College, including Drs. Kimberly Hawkes and Maggie Schuckman, as well as Deborah Kingston for their constant support, laughter, and friendship. I feel honoured to have worked with such a wonderful group of people during my
D.V.Sc. and it would be difficult to put into words what their friendship has meant to me.
I thank the supreme generosity of the wonderful Pet Trust of the Ontario
Veterinary College program who provided the financial support of this project - without their support this project would not have been possible.
Finally, I would like to thank all those people of the Ontario Veterinary
College - fellow D.V.Sc. students, as well as the technical, administrative and front desk staff who provided much needed support to complete this project. I also thank my amazing family and friends who I am sure are now well educated about the ins and outs of arrhythmogenic right ventricular cardiomyopathy [whether they liked it or not!). And most importantly, all the wonderful Boxers, breeders and lovers of Boxers who cared enough about the well being of this wonderful breed to support the completion of this project. DECLARATION OF WORK PERFORMED
I declare that, with the exception of the items below, all work reported in this thesis was performed by the author.
Telephone interviews for owners of Boxers interested in study participation were performed by Cindy Walker. Assistance in the analysis of the Holter recordings was also performed by Cindy Walker. Laboratory support was provided by the Animal Health Laboratory at the University of Guelph as well as Guelph
General Hospital (serum troponin I concentrations). All statistical analysis was directed by William Sears.
IV TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS i
DECLARATION OF WORK PERFORMED iv
TABLE OF CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS USED x
CHAPTER 1 - LITERATURE REVIEW
1.0 Arrhythmogenic Right Ventricular cardiomyopathy in Humans 1 1.0.1 Definitions and Classification 1 1.0.2 Natural History of Disease 2 1.0.3 Prevalence of Disease 3 1.0.4 Pathological Changes Associated with ARVC 4 1.0.5 Etiology of ARVC 6 1.0.5.1 The Adhesion of cardiomyocytes: The Intercalated Disk 8 1.0.5.2 The Cell Adhesion Theory for ARVC: A Disease of the Desmosome 10 1.0.6 The Genetics of ARVC 13 1.0.7 Clinical Signs of ARVC 15 1.0.8 Etiology of Ventricular Arrhythmia in ARVC 16 1.0.9 Diagnostic Criteria for ARVC 17 1.0.9.1 Physical Examination Findings 20 1.0.9.2 Electrocardiographic Findings 21 1.0.9.3 SAECG ECG and ARVC 24 1.0.9.4 Correlating SAECG to other diagnostic modalities and risk for arrhythmia in patients with ARVC 27 1.0.9.5 Echocardiography and ARVC 29 1.0.9.6 Advanced Imaging in the diagnosis of ARVC: Computed Tomography (CT) and Cardiac Magnetic Resonance Imaging (cMRI) 34 1.0.9.7 Angiography and ARVC 37 1.0.9.8 Endomyocardial Biopsy 38 1.0.9.9 Electrophysiologic Studies and Electroanatomic Mapping in Patients with ARVC 41 1.0.10 New Options for the Diagnosis of ARVC 44 1.0.11 Risk Stratification for Patients with ARVC 45 1.0.12 Therapeutic Options for Patients with ARVC 47
v 1.1 Arrhythmogenic Right Ventricular Cardiomyopathy in Boxers 52 1.1.1 Identification and Natural History 52 1.1.2 Prevalence of Boxer ARVC 53 1.1.3 Pathologic Changes of Boxer ARVC 54 1.1.4 The Etiology of Boxer ARVC 56 1.1.5 The Genetics of Boxer ARVC 59 1.1.6 Other Species/Breeds Affected with ARVC 61 1.1.7 Clinical Signs of Boxer ARVC 65 1.1.8 Diagnostic Criteria for Boxer ARVC 66 1.1.8.1 Physical Examination Findings in Boxer ARVC 67 1.1.8.2 Electrocardiographic Findings in Boxer ARVC 68 1.1.8.3 Ambulatory Electrocardiographic Findings in Boxer ARVC 70 1.1.8.4 Signal Averaged Electrocardiogram in Boxer ARVC 73 1.1.8.5 Echocardiography in Boxer ARVC 74 1.1.8.6 Cardiac Magnetic Resonance Imaging in Boxer ARVC 76 1.1.8.7 The Utility of Biomarkers for Boxer ARVC 77 1.1.8.8 Genetic Tests for Boxer ARVC 80 1.1.9 Therapy for Boxer ARVC 81 1.2 Tissue Doppler Imaging Assessment of Systolic Function 84 1.3 Tissue Doppler Imaging Assessment of Diastolic Function 89 1.4 References 92
CHAPTER 2 - ASSESSMENT OF SYSTOLIC AND DIASTOLIC FUNCTION VIA TISSUE DOPPLER ECHOCARDIOGRAPHY IN BOXERS AFFECTED WITH ARVC COMPARED TO NORMAL BOXERS AND NON-BOXER CONTROL DOGS
2.1 Introduction 107 2.2 Statement of Objectives and Hypotheses 114 2.3 Materials and Methods 116 2.3.1 Study Design 116 2.3.2 Enrollment Criteria 117 2.3.3 Exclusion Criteria 118 2.3.4 Patients 118 2.3.5 Data Acquisition 120 2.3.6 Statistical Analysis 130 2.4 Results 134 2.5 Discussion 160 2.6 Conclusions 173 2.7 Limitations 175 2.8 References 179
APPENDICES
Appendix 3.1 Telephone Questionnaire for Boxer Recruitment 186
VI LIST OF TABLES
CHAPTER 1 PAGE
TABLE 1.1 Comparison of Original & Revised Task Force
Criteria for the Diagnosis of ARVC 19
CHAPTER 2
TABLE 4.1 Sample Characteristics and Descriptive Statistics 141 TABLE 4.2 Complex Analysis with Adjustment for Covariates - All Group Analysis 147
TABLE 4.3 Complex Analysis with Adjustment for Covariates - Boxer Group Analysis 150
TABLE 4.4 Analysis of Variables Correlated with VPCs/hr - All Dogs 152
TABLE 4.5 Analysis of Covariance Results Filtered QRS Duration 154
TABLE 4.6 Analysis of Covariance Results RMS Terminal 40 ms 156
TABLE 4.7 Analysis of Covariance Results HFLA<40uV 158
vn LIST OF FIGURES
CHAPTER 1 PAGE
FIGURE 1.1 Lead II ECG from Dog with Interpolated VPC of
Left Bundle Branch Block Morphology 21
FIGURE 1.2 ECG from ARVC Patient with Epsilon Waves 24
FIGURE 1.3 Normal and Abnormal SAECG from Two Boxer Dogs 27
FIGURE 1.4 Histological Section of RV Myocardium from an ARVC
Affected Boxer 55
FIGURE 1.5 9-Lead ECG from an ARVC Affected Boxer 69
FIGURE 1.6 Canine Pulsed-Wave Tissue Doppler Recording 87
CHAPTER 2
FIGURE 4.1 Canine Pulsed-Wave Tissue Doppler Recording 122
FIGURE 4.2 Placement of Electrodes per Frank Orthogonal Lead
System 123
FIGURE 4.3 Photograph of Boxer for Acquisition of SAECG 124
FIGURE 4.4 Template Report Generated Prior to SAECG Capture 127
vm ABBREVIATIONS
2D Two-dimensional
ACE-I Angiotensin-converting enzyme inhibitors
ARVC Arrhythmogenic right ventricular cardiomyopathy
ASD Atrial septal defect
BNP Brain natriuretic peptide
CHF Congestive heart failure cMRI Cardiac magnetic resonance imaging
CT Computed tomography cTnC Cardiac troponin C cTnl Cardiac troponin I cTnT Cardiac troponin T
Cx43 Connexin43
DCM Dilated cardiomyopathy
ECG Electrocardiogram
EDV End diastolic volume
EF Ejection fraction
EMB Endomyocardial biopsy
EP Electrophysiologic fQRSd Filtered QRS duration
GDV Gastric-dilation volvulus
HCM Hypertrophic cardiomyopathy
HFLA High frequency low amplitude HRV Heart rate variability
ICDs Implantable cardioverter-defibrillators
IMP Index of myocardial performance
IVS Interventricular septum
LPs Late potentials
LV Left ventricle
MLAP Mean left atrial pressures
MRI Magnetic resonance imaging
PKP2 Plakophilin-2
PLAX Parasternal long-axis view
PSAX Parasternal short-axis view
PVS Programmed ventricular stimulation
RA Right atrium
RCM Restrictive cardiomyopathy
RFA Radiofrequency catheter ablation
RMS Root mean square
RV Right ventricle
RVFW Right ventricular free wall
RVIT Right ventricular inflow tract
RVOT Right ventricular outflow tract
RyR2 Ryanodine receptor
SAECG Signal averaged electrocardiogram
SAS Subaortic stenosis SCD Sudden cardiac death
TDI Tissue Doppler imaging
TGF-P3 Transforming growth factor-(33
TMEM43 Transmembrane protein 43
UCM Unclassified cardiomyopathy
VA Ventricular arrhythmia
VM Vector magnitude
VPC Ventricular premature contraction
VPCs/hr Ventricular premature contractions per hour
WHO World Health Organization
XI Literature Review
The following literature review provides the background for the proceeding
investigation of myocardial annular velocities assessed by tissue Doppler imaging in
Boxer dogs affected by arrhythmogenic right ventricular cardiomyopathy (ARVC).
Initially presented is a review of the human literature highlighting the natural
history of ARVC, its histopathology, etiology, genetics, diagnostic criteria, prognosis
and risk stratification, and therapeutic options. A review of the disease as it occurs
in the Boxer dog follows, highlighting the similarities and some differences with the
human form, as well as discussion of other species affected by ARVC. Finally, a
review of tissue Doppler imaging and its utility in diagnosing subtle myocardial
dysfunction in patients with structural heart disease is presented.
1.0 Arrhythmogenic Right Ventricular Cardiomyopathy in Humans
1.0.1 Definition and Classification
ARVC is an inherited, progressive myocardial disorder which is characterized
by the partial to complete replacement of the right ventricular myocardium with
fatty or fibrofatty tissue.1 When first described in 1978 by Frank and Fontaine2, it was initially labeled a form of dysplasia due to the suspicion that the disease was a
result of a developmental defect of the right ventricle (RV). However, information
gained since that time has redefined the disease as a genetically determined
cardiomyopathy3, and in 1995, the World Health Organization (WHO] added ARVC
1 to the list of cardiomyopathies which had already included dilated cardiomyopathy
(DCM], hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy (RCM) and unclassified cardiomyopathy (UCM).4
1.0.2 Natural History of Disease
The earliest historical note of the disease was reported in 1736 by Giovanni
Maria Lancisi in the book "De Motu Cordis et Aneurysmatibus"4 where he described a family with a disease which occurred in four generations. The disease was characterized by palpitations, heart failure, dilation and aneurysms of the RV and sudden death.4 These characterizations described over two and a half centuries ago outline the natural history of ARVC very well. The palpitations refer to arrhythmias of right ventricular origin, namely, ventricular premature contractions (VPCs) and ventricular tachycardia (VT).5 These arrhythmias occur as a result of conduction block and the disruption of the normal wave of depolarization through the myocardium, resulting in macro re-entrant loops due to the infiltration of the right ventricular myocardium with fatty and fibrofatty tissue.6 As the replacement of the right ventricular myocardium with fatty and fibrofatty tissue is progressive, eventual loss of a critical mass of the RV results in dilation and eventual right-sided congestive heart failure.7 At any point, individuals affected with ARVC are always at risk for sudden cardiac death due to ventricular fibrillation.8 In the human population, it is currently estimated that the frequency of sudden cardiac death among individuals affected with ARVC is approximately 2-3.4% per year, with the age of onset generally being between 15 and 40 years of age.9 In fact, ARVC is the
2 leading cause of sudden cardiac death (SCD) in human patients under the age of 35, with 50% of the deaths estimated to occur prior to the age of 35.1-10
In some severe or long-standing cases of ARVC, bi-ventricular involvement can be noted with bilateral pump failure, resulting in signs attributable to both left- and right-sided congestive heart failure (CHF). These patients, who often have accompanying atrial enlargement, become at higher risk for supraventricular arrhythmias such as atrial fibrillation and supraventricular tachycardias, as well as for the development of atrial thrombosis.7
1.0.3 Prevalence of Disease
Due to the complexity of diagnosis of the disease, the true prevalence in the human population is difficult to estimate. However, it has been reported that the estimated prevalence is approximately 1 in every 2500 to 5000 individuals.10
However, prevalence in certain geographical locations is higher than others, namely in regions of Greece and Italy.1 The disease appears to be more expressed among men compared to women [71% versus 29%), however, gender has not been associated with a higher incidence of life threatening ventricular arrhythmias (VA) or with a worse clinical outcome.11
ARVC has been shown to be one of the leading causes of sudden death amongst competitive athletes, particularly in the Veneto region of Italy. In this particular region, where the disease and prevalence have been extensively studied, the incidence of sudden death due to ARVC is estimated to be 0.5 cases per 100,000
3 individuals per year, with all victims of sudden death being male with a mean age of
22.6 +/- 4 years.12
1.0.4 Pathological Changes Associated with ARVC
In human patients afflicted with ARVC, the dominant histopathological changes noted are a fibrofatty infiltration and replacement of the right ventricular myocardium in a focal or diffuse distribution.13 This infiltration typically begins in the subepicardial region of the RV free wall, and ultimately extends to the endocardial layer as the disease progresses.5 Amongst this fibrofatty infiltration is the remnant strands of normal or atrophied myocytes. This arrangement of surviving myocytes amongst the fibrofatty tissue leads to inhomogeneous intraventricular conduction, predisposing affected individuals to ventricular arrhythmia (VA) through re-entrant mechanisms.12 Accompanying this infiltration, inflammation has been identified in approximately 75% of afflicted hearts at autopsy. This is characterized by the presence of interstitial lymphocytes that surround areas of necrotic or degenerated myocytes.1-4 These changes within the
RV are most dramatic and apparent at the so-called "triangle of dysplasia", represented by the subtricuspid, apical and right ventricular outflow tract (RVOT) regions.14-15 The interventricular septum (IVS), which lacks an epicardium, is typically spared by the disease process, with histological changes only noted in approximately 20% of specimens from ARVC afflicted patients.5-16 The trabecular myocardium is also noted to be spared from the fatty/fibrofatty infiltration.16 In approximately 50% of affected individuals, similar histopathological changes are
4 present within the left ventricular myocardium, particularly the posterolateral free wall. This finding, in conjunction with the presence of biventricular failure in some patients, has prompted some to simply re-classify and refer to ARVC as simply
"arrhythmogenic cardiomyopathy".5'17
Isolated fat infiltration within the RV must be interpreted with caution, and on its own, is not pathognomonic for a diagnosis of ARVC.718 It has been noted in aging humans, as well as in those who are obese or taking long-term steroids, that infiltration of fat within the myocardium is a normal finding, particularly if it is observed in the anterior wall near the apex of the RV.15-19 Therefore, in addition to fatty infiltration, replacement type fibrosis and/or degenerative changes of the myocytes must also be observed in order to satisfy criteria to meet a diagnosis of
ARVC.16 Interestingly, it has been noted that the histopathological changes of ARVC vary with age, with fibrosis being the earliest hallmark noted in younger patients, and fatty infiltration being more prominent in older patients.20-21
The histopathological changes of ARVC that occur on a microscopic level result in gross pathological changes of the right heart. As a result of the myocyte replacement and loss, focal areas of thinning are often noted, with the RV free wall having a "parchment-like" appearance. This thinning may be so dramatic that transillumination through the RV wall is often possible, giving a 'Chinese Lantern' appearance.1 Other features which may be noted include ventricular cavity enlargement with accompanying aneurysms. RV aneurysms are observed in approximately 20-50% of autopsy cases of patients with ARVC, but are not specific as they are commonly observed as a result of other processes such as myocardial
5 infarction, HCM, sarcoidosis, Chagas disease and cardiac trauma.1 Intracavitary mural thrombi may also be noted, particularly in patients with RV aneurysms and those with RV wall motion abnormalities. Fine thrombus deposition and organization along the endocardial surface also results in a thickened appearance of the endocardium. The trabeculae are often noted to be shrunken in appearance with enlargement of the intertrabecular spaces, accounting for the characteristic appearance of deep fissures which are noted at angiography and are used as a pathognomonic finding in the diagnosis of ARVC.16
1.0.5 Etiology of ARVC
The exact etiology of ARVC is of great debate amongst the researchers who are investigating the disease in humans. Many believe that whatever the inciting cause, the result is myocyte cell death or apoptosis which triggers repair and replacement with fibrofatty tissue [regarded as a healing phenomenon], thus beginning the natural history of the disease.22 Due to presence of inflammation amongst the fibrofatty infiltration, others have theorized that a viral or immune mediated etiology may be causative. However, a viral analysis of RV biopsies from affected individuals only revealed that 15% of individuals had evidence for viral infection. This low percentage of viral identification therefore does not support a primary role for a viral cause for ARVC. However, the histopathological changes that occur with ARVC may predispose individuals to secondary viral myocarditis, or the viruses may simply represent innocent bystanders and are an incidental finding
6 in those affected individuals.47 Evidence is currently lacking to definitively support an infectious role.
Transdifferentiation of myocardial cells is another proposed mechanism for
ARVC. This hypothesis states that the myocytes undergo a switch to adipocyte cell fate due to altered cell signaling.1 This hypothesis stemmed from observed features of an explanted heart from an affected individual with ARVC. In this heart, ventricular myocytes exhibited partial replacement of myofibrils with lipid rich sarcoplasmic vacuoles.1 The intermediate filaments of the myocytes and the adipocytes both co-expressed desmin (a subunit of the intermediate filaments) and vimentin (a protein typically made by fibroblasts), suggesting a transitional state between two different cell fates. This would be in contrast to the normal heart, where only 1% of myocytes are replenished per year.1-5
The theory that appears to have gained the majority of acceptance amongst researchers is apoptosis leading to healing with fibrofatty replacement. In a majority of specimens (greater than 75%) from individuals with ARVC, expression of protease CPP-32 and fragmented DNA have been observed. These particular findings are a major indicator for cellular apoptosis.5 The etiology of the apoptosis has recently been supported by genetic studies of ARVC which have revealed mutations in several of the genes which code for the protein components of the desmosome, a cellular junction between myocytes. As the desmosome plays a pivotal role in the attachment of cells together through the linking of intermediate filaments of neighbouring cells at the intercalated disk, disruption of the integrity of the desmosome as a result of incorporation of a mutant protein or lack of a key
7 component protein could lead to detachment of myocytes thus leading to apoptosis.
22,23 To fate, mutations in genes encoding for the desmosomal components desmoplakin, plakoglobin, plakophilin, desmoglein and desmocollin have been identified, as well as non-desmosomal mutations in the ryanodine receptor (RyR2), transmembrane protein 43 (TMEM43) and transforming growth factor-|33 [TGF- p3).24'25
1.0.5.1 The Adhesion of Cardiomyocytes: The Intercalated Disk
In contrast to skeletal myocytes which are long and multinuclear, cardiac myocytes require mechanical and electrical interconnections at the level of the intercalated disk at the ends of the cell in order to transmit electrical activation and mechanical forces. The intercalated disk includes three different protein complexes
- adherens, desmosomal and gap junctions.23 The function of the intercalated disks are to not only ensure mechanical coupling between neighbouring cells, but also to allow for rapid propagation of electrical impulses from cell to cell, allowing for synchronous cardiac activation.23
Adherens junctions, composed of cadherins and catenins, are the main component of the intercalated disk, and they provide a strong mechanical connection between neighbouring cells sufficient to sustain the continuous stress which contraction imparts onto cells, thus keeping the cells tightly together. They achieve this strong connection via linkage to the thin actin filaments within the sarcomeres of neighbouring myocytes.16-26 The junctioiis also allow transmission of
8 the contractile force from one cell to the next, allowing for coordination of contraction.23
Gap junctions, composed of connexin proteins, provide the ability for neighbouring cells to communicate with one another, through the transmission'of various compounds such as metabolites, water and ions via passive diffusion. They directly connect the cytoplasm of one cell to that of the neighbouring cell, thus helping to ensure proper propagation of the electrical impulse through the intercellular transfer of current, which triggers the orderly contraction of cardiomyocytes.23 The gap junction channel between cells is composed of a total of
12 connexin proteins to form two interacting connexons (each is a multimeric structure of 6 connexins). The connexin of interest when discussing ARVC is connexin43 (Cx43), as it is the most important connexin present in the ventricular myocardium.23-27 It is of particular importance to note that given the nature of their primary role (ie: providing a low-resistance method for cells to electrically communicate), gap junctions would be at risk for shear stress damage as a result of cardiac contraction. Therefore, the gap junctions are surrounded by mechanical junctions (adherens and desmosomal junctions) at the level of the intercalated disk to protect them from shear stress related to cardiac contraction.16
The desmosome is a dense and robust structure that is found not only in cardiac tissue, but also epithelial tissue as it is also subject to strong contractile stress and abrasive forces. It is composed of an intercellular and intracellular component - the intercellular component is formed by cadherins (desmocollin and desmoglein), and the intracellular component is formed by plakoglobin, plakophilin
9 and desmoplakin.23 Besides providing a structural integrity role linking cardiomyocytes through a connection to the intermediate filament system (desmin), the desmosome may also act as a signaling centre, regulating the availability of signaling molecules and therefore participating in processes such as cell proliferation, differentiation and morphogenesis.28 The integrity of the desmosome is vital to the proper functioning of the gap junctions, given their role in "shielding" them from the shear forces at play in the contracting heart. This anatomic relationship between the gap junctions and the desmosome becomes of importance in the setting of failure of mechanical coupling due to alterations in the desmosome, which in turn affects the extent to which cardiomyocytes are electrically coupled via the stability of the gap junctions.16 Defective gap junction function can result in and contribute to slowed/blocked electrical conduction between cells, thus providing further mechanisms of arrhythmogenesis.23'26-29
1.0.5.2 The Cell Adhesion Theory for ARVC: A Disease of the Desmosome
The theory that ARVC represents a disease of the desmosome originated from identification of mutations in genes encoding for the protein constituents that make up the desmosome. The work that pioneered this discovery was the identification of the mutation responsible for Naxos syndrome, a rare form of cardiomyopathy that is also characterized by a cutaneous component, in the mid
1980's.30*31 This autosomal recessive disease is characterized by woolly hair, diffuse keratoderma over the palms of the hands and the soles of the feet, as well as the typical features described herein of ARVC.1 The name of the syndrome comes from
10 an island in Greece, where the prevalence of disease exceeds 1 in 1000 individuals.32
The natural history of the syndrome consists of expression of the cutaneous phenotype in infancy, with the cardiac phenotype developing from adolescence to the fourth decade of life, with sudden cardiac death being a common occurrence in younger individuals.1-10 Linkage mapping identified a two-base pair deletion in the plakoglobin gene responsible for the syndrome. Due to plakoglobin's key importance in the structural integrity of the desmosome, researchers began investigating the other constituents of the desmosome for additional mutations in the more typical form of ARVC. Using a candidate gene approach which was focused on the desmosome, causal mutations have since been identified in plakophilin-2, desmoglein-2, desmoplakin and desmocollin-2.1
The mutations are definitively linked to ARVC through segregational analysis.
The exact mechanism however may be debated. Two models have been proposed - the first model hypothesizes that the desmosomal mutation results in the compromise of cardiomyocyte cell to cell adhesion.12333'34 This defect in cell to cell adhesion therefore results in the inability of the myocytes to tolerate the shear stress from cardiac contraction, leading to cellular detachment and apoptosis with subsequent healing and replacement with fibrous and/or fatty tissue.1 This theory has left many to also speculate why the RV is preferentially affected, given the similar distribution of desmosomes in the left ventricle (LV). One proposal is that the RV, which had adapted to a wide variation in preload, is more likely to be affected by abnormal cell adhesion due to its inherent thinner walls and greater distensibility. This proposal would also seem in agreement given the prevalence of
11 the disease in competitive athletes, with whom due to their intense physical activity and wide variation in right heart preload and afterload, would be more likely to experience more shear mechanical stress on impaired desmosomes, accelerating cellular detachment leading to expression of the disease phenotype.12 In fact, it has been shown in the Veneto region of Italy that affected individuals involved in competitive sporting activity have a 5.4 times greater risk of sudden death than during sedentary activity.12
The second theory that attempts to connect desmosomal dysfunction to the
ARVC phenotype involves the desmosome's role as a signaling centre. Besides their role in maintaining cardiac tissue architecture, the desmosome plays a key role in cellular signaling, of which the Wnt/(3-catenin signaling pathway is best described.1
This pathway, which is evolutionary conserved, is involved in the regulation of cell fate, proliferation and apoptosis. Plakoglobin (also called gamma-catenin), a constituent of the desmosome, has structural and functional similarities to p- catenin. It therefore interacts and competes with (3-catenin at multiple cellular levels, leading to a net negative effect on the Wnt/(3-catenin signaling pathway. It has been shown experimentally in mice that cardiac specific suppression of desmoplakin results in the translocation of plakoglobin to the nucleus with resultant reduced Wnt/(3-catenin signaling. This results in an increased expression of adipogenic and fibrogenic genes in vitro, abnormal cardiac adipose tissue with fibrosis in vivo, and resultant ventricular arrhythmias similar to what is observed in
ARVC.35 Recently, it has been demonstrated via immunohistochemical analysis that there was a reduction in the immunoreactive signal for plakoglobin at the
12 intercalated disks in affected tissue from affected individuals compared to
controls.3637 This finding suggests and supports the finding in mice that a shift in
plakoglobin from junctional to the intracellular or intranuclear pool may play a
pivotal role in the pathogenesis of ARVC, possibly through altered Wnt/(3-catenin
signaling.36
1.0.6 The Genetics of ARVC
Evaluation of relatives of affected individuals with ARVC has revealed that up
to 50% of cases are familial, and the disease is most commonly inherited as an
autosomal dominant trait with variable penetrance.638 The variable penetrance,
which could be influenced by modifier genes, environmental influences and gender
effects39, makes the clinical diagnosis of familial ARVC a challenge due to a wide
clinical variability which is seen in affected individuals. In the remaining cases (the
"sporadic" form), ARVC is believed to be due to an acquired etiology or some other
unidentified mode of inheritance.22 The identification of compound heterozygosity
and digenic inheritance in some individuals has added to the complexity of ARVC
genetics, yet may partly explain the apparent incomplete penetrance of any single
disease gene.
To date, eight genes with 12 chromosomal loci have been identified as causal
mutations in the autosomal dominant form of ARVC, with the majority of the genes
encoding for desmosomal proteins. One of the more commonly implicated mutations is located on the gene encoding for plakophilin-2 (PKP2) protein - it has been found in up to 43% of ARVC patients, and affected families have the PKP2 gene
13 mutation in up to 70% of individuals.5 The other two more commonly implicated mutations involve the genes encoding for desmoplakin and desmoglein-2. Three extra-desmosomal gene mutations have also been identified. The first, TGF-|33 has been implicated in ARVC1, but it explains less than 1% of all ARVC presentations.25
The beta type transforming growth factors are polypeptides which act to control the differentiation and proliferation of multiple cell types, and has been shown in vitro to modulate the expression of desmosomal genes.40 A mutation in TMEM43 has recently been identified in a Newfoundland population. In this particular designation of ARVC (ARVC5], a high incidence of sudden cardiac death and heart failure has been observed.41 Little is known regarding the function of the TMEM43 gene, but it may be a part of an adipogenic pathway whose dysregulation may explain the fibrofatty replacement characteristic of ARVC.30 The final extra- desmosomal gene implicated to date is the gene encoding for the ryanodine receptor (RyR2) which is classified as ARVC2. RyR2 functions as a calcium induced- calcium release channel, and releases calcium from the sarcoplasmic reticulum in response to calcium influx through the L-type dihydropyridine receptor channel.42
Mutations of RyR2 are believed to increase RyR2 mediated calcium release into the cytoplasm and are associated with two clinically distinct forms of sudden cardiac death - ARVC2 and a stress-induced polymorphic ventricular tachycardia. Both forms are similar in terms of clinical characteristics (effort induced arrhythmia, sudden death and syncope), however ARVC2 differs with its progressive replacement of cardiomyocytes with fibrofatty replacement. However, RyR2 mutations represent a different SR-Ca++ -handling mechanism of arrhythmogenesis
14 rather than a defect of the intercalated disk, and as such likely represents a phenocopy of ARVC, partially as a result of overinterpretation of fatty infiltration in the original patients described with ARVC2.42
1.0.7 Clinical Signs of ARVC
One of the challenges of diagnosing ARVC within the human population is the wide clinical spectrum observed in the disease process. This heterogeneity in symptoms has been well documented in first degree relatives with inherited PKP2 mutations where symptoms varied from being completely asymptomatic and concealed in some affected relatives to sudden cardiac death and heart failure in others.39 In affected individuals there is typically a long pre-clinical phase where the disease is silent, with clinical signs only appearing in late adolescence and early adulthood in many.30 This long pre-clinical phase is referred to as the concealed phase of the disease where subtle RV structural changes are occurring, with clinical signs appearing when individuals progress to the next phase of overt RV electrical instability. However, it has been documented that sudden cardiac death can still occur in those individuals in the concealed phase of disease without overt structural cardiac changes, and in that regard, sudden cardiac death was the first clinical sign in 5 of 12 individuals in a case series of ARVC.943 In the next phase of overt RV electrical instability, clinical signs are attributable to the presence of ventricular arrhythmia (VPCs, VT, VF). In a review of 108 unrelated ARVC affected individuals, the most common clinical sign was palpitations and was present in 56% of patients, with dizziness (27%], syncope (21%) and chest pain (14%) being less common
15 clinical symptoms.7 During this stage more obvious structural changes typically occur within the RV, particularly involving the "triangle of dysplasia".15 As the disease progresses, there is continued loss of the RV myocardium which impairs mechanical and heart pump function, leading to the third phase of the disease, isolated right heart failure. The final phase of the disease is characterized by biventricular pump failure due to progressive LV involvement of the disease process, with clinical signs attributable to both left- and right-sided congestive heart failure.43
1.0.8 Etiology of Ventricular Arrhythmia in ARVC
The most obvious cause for the VA seen in patients with ARVC is a result of the anatomic changes that occur, namely the infiltration of the myocardium with fibrofatty tissue. This infiltration results in disruption of the normally homogenous depolarization of the myocardium, resulting in conduction block (secondary to presence of fibrosis) and slowed conduction (surviving myocytes embedded amongst the fibrofatty replacement tissue), setting the stage for the development of re-entry as a predominant mechanism for the VA in over 80% of cases.10*16 In order to explain why the risk for VA is higher during physical activity, it has been suggested that sympathetic nerve trunks are interrupted by the anatomic changes of
ARVC, resulting in a denervation supersensitivity to catecholamines. Therefore, a catecholamine surge in this instance could cause VA in the denervated supersensitive myofibers through re-entry, triggered activity, or a combination of both.16 It has been demonstrated that some patients with ARVC without overt RV
16 anatomic changes still have electrical instability and demonstrate VA.1 This has left investigators to postulate that abnormal gap junction function, as a result of impaired mechanical coupling due to desmosomal disease, alters electrical coupling between cells, thus slowing myocardial conduction and creating an arrhythmogenic substrate ("functional re-entry").1 Therefore, whether the VA seen with ARVC is related to anatomic changes or altered function of the gap junctions [or both), the basic arrhythmogenic mechanism appears to be re-entry.
1.0.9 Diagnostic Criteria for ARVC
ARVC often presents itself as a challenging diagnosis in human medicine as there is no single test or particular clinical finding that is definitively diagnostic.
Therefore, physicians rely on an integration of several complementary diagnostic methods to arrive at diagnosis of ARVC. In 1994, the European Society of Cardiology and the Scientific Council on Cardiomyopathies of the International Society and
Federation of Cardiology met and formed a Task Force to outline various major and minor diagnostic criteria required to reach a clinical diagnosis of ARVC.44 The Task
Force proposed several diagnostic categories including assessment of cardiac structure and function, tissue characterization, repolarization and depolarization abnormalities, arrhythmias and familial history. A diagnosis is achieved when two major, one major plus two minor, or four minor criteria are met. These criteria are considered highly specific and help to reduce diagnostic ambiguity, however, their sensitivity is lacking when applied to both early cases of ARVC as well as familial cases.45 They also lack specific quantitative values in the assessment of cardiac
17 dimensions and function (as assessed via angiography, echocardiography, computed tomography and cardiac magnetic resonance imaging).30 Therefore, modifications have been suggested and new guidelines for the diagnosis of ARVC, particularly in the diagnosis of familial ARVC, have been recently proposed in publication.46 These new modifications have incorporated new findings of ARVC in the past 16 years to improve the diagnostic sensitivity while maintaining specificity. New quantitative guidelines have been made regarding two-dimensional (2D) echocardiographic findings as well as magnetic resonance imaging (MRI) findings, which had not been previously included. Furthermore, additional quantitative measures of histological characterization have been made, and the electrocardiogram (ECG) and signal averaged electrocardiogram (SAECG) criteria have been expanded with additional cut-off values (see Table 1.1).
18 Original Task Force Criteria Revised Task Force Criteria I. Global or regipnal dysfunction and structural alterations Major Severe dilatation and reduction of RV By 2D Echo: ejection fraction with no (or only mild) Regional RV akinesis, dyskinesia or aneurysm LV impairment And 1 of the following (end diastole): Localized RV aneurysms (akinetic or o PLAX RVOT > 32 mm dyskinetic areas with diastolic bulging) o PSAX RVOT > 36 mm Severe segmental dilatation of the RV o Or fractional area change < 33%
By MRI: Regional RV akinesia or dyskinesia or dyssynchronous RV contraction And 1 of the following: o Ratio of RV end-diastolic volume to BSA > 110 mL/m2 (male) or > 100 mL/m2 (female) o Or RV ejection fraction < 40%
By RV Angiography: Regional RV akinesia, dyskinesia, or aneurysm Minor Mild global RV dilatation and/or ejection By 2D Echo: fraction reduction with normal LV Regional RV akinesia or dyskinesia Mild segmental dilatation of the RV And 1 of the following (end diastole): Regional RV hypokinesia o PLAX RVOT > 29 to < 32 mm o PSAX RVOT > 32 to < 36 mm o Or fractional area change > 33% to <40%
By MRI: Regional RV akinesia or dyskinesia or dyssynchronous RV contraction And 1 of the following: o Ratio of RVED volume to BSA > 100 to < 110 mL/m2 (male) or > 90 to < 100 mL/m2 (female) o Or EF > 40% to < 45% II. Tissue Characterization of Wall Major Fibrofatty replacement of myocardium Residual myocytes < 60% by morphometric analysis, with on endomyocardial biopsy fibrous replacement of RV free wall myocardium in > 1 sample, with or without fatty replacement of tissue on endomyocardial biopsy Minor Residual myocytes 60% to 75% by morphometric analysis, with fibrous replacement of the RV free wall myocardium in > 1 sample, with or without fatty replacement of tissue on endomyocardial biopsy III. Repolarization Abnormalities Major Inverted T waves in right precordial leads, or beyond in individuals > 14 years of age (in the absence of complete RBBB) Minor Inverted T waves in right precordial Inverted T waves in leads VI and V2 in individuals > 14 years leads (people > 12 years of age, in of age (with no complete RBBB) or in V4, V5 or V6 absence of RBBB) Inverted T waves in leads V1-V4 in individuals > 14 years of age in presence of complete RBBB. IV. Depolarization/Conduction Abnormalities Major Epsilon waves or localized prolongation Epsilon wave in V1-V3 of the QRS complex in V1-V3 Minor Late potentials (SAECG) Late potentials by SAECG in > 1 of 3 parameters in the absence
19 of a QRS duration of > 110 ms on standard ECG Filtered QRS duration > 114 ms Duration of terminal QRS < 40 uv> 38 ms RMS voltage of terminal 40 ms < 20 uv Terminal activation duration of QRS > 55 ms measured from the nadir of the S wave to the end of the QRS, including R' in VI, V2 or V3, in the absence of complete RBBB V. Arrhythmias Ma/or Nonsustained or sustained VT of LBBB morphology with superior axis (negative or indeterminate QRS in leads 11, 111 and aVF with positive in lead aVL) Minor LBBB-type VT (sustained and non- Nonsustained or sustained VT of RV outflow configuration, sustained) LBBB morphology with inferior axis (positive QRS in leads 11, Frequent ventricular extrasystoles (> HI and aVF and negative in lead aVL) or of unknown axis 1000 per 24 hours) > 500 ventricular extrasystoles per 24 hours (Holter) VI. Family History Major Familial disease confirmed at necropsy ARVC/D confirmed in 1st degree relative who meets current or surgery Task Force criteria ARVC/D confirmed pathologically at autopsy or surgery in a 1st degree relative Identification of a pathogenic mutation categorized as associated or probably associated with ARVC/D in the patient under evaluation Minor Family history of premature sudden History of ARVC/D in a 1st degree relative in whom it is not death ( < 35 years of age) due to possible or practical to determine whether the family member suspected ARVC/D meets Task Force criteria Familial history (clinical diagnosis based Premature sudden death (< 35 years of age) due to suspected on present criteria) ARVC/D in a 1st degree relative ARVC/D confirmed pathologically in 2nd degree relative Table 1.1: Comparison of Original & Revised Task Force Criteria Used with permission from: Marcus, F., et al., Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: Proposed Modifications of the Task Force Criteria. Circulation, 2010.121: p. 1533-41. In the new criteria, definite diagnosis is 2 major or 1 major and 2 minor criteria, or 4 minor from different categories; borderline: 1 major and 1 minor or 3 minor criteria from different categories; possible: 1 major or 2 minor criteria from different categories. PLAX - parasternal long-axis view; PSAX - parasternal short-axis view.
1.0.9.1 Physical Examination Findings
Physical examination in patients with ARVC is often normal, with abnormalities only being reported in about 50% of affected individuals.5 The abnormal findings are non-specific and none are pathognomonic for a diagnosis of
ARVC. Possible physical examination findings include a splitting of the second heart sound (S2) as a result of delayed closure of the pulmonic valve secondary to RV dysfunction. If RV dysfunction with significant myocardial disease is present, a third heart sound (S3 gallop) may also be appreciated. An arrhythmia may be detectable
20 with pulse deficits, and a murmur associated with tricuspid regurgitation may also be documented.5
1.0.9.2 Electrocardiographic Findings
Among patients with ARVC, ECG findings are typically the first appreciated abnormality, with ECG abnormalities reported in over 85% of cases.16 According to the 1994 Task Force criteria, both sustained and non-sustained ventricular tachycardia with a left bundle branch block morphology and frequent VPCs (greater than 1000 over 24 hours of ambulatory ECG monitoring) are minor criteria to satisfy the diagnosis of ARVC.44 The left bundle branch block morphology refers to
VPCs which appear to have an origin from the RV. (see Figure 1.1)
Sr^V
Figure 1.1: Lead II from a dog: Interpolated VPC of Left Bundle Branch Block Morphology
In terms of repolarization abnormalities appreciated on surface ECG, inverted T waves in the right precordial leads (V1,V2 and V3) in individuals older than 14 years of age are considered to be a major criterion in the new guidelines, in the absence of right bundle branch block (or a minor criterion if present only in VI
& V2).46 It is believed that this finding occurs as a result of ventricular dysfunction and RV dilation in patients with ARVC, but can also be seen in patients as a
21 manifestation of myocardial injury or myocardial replacement.47 Caution must be used however when applying this criterion to young patients, as it has been documented that T wave inversion can occur in up to 20% of normal adolescents without ARVC in the right precordial leads.16 Therefore, the Task Force suggests applying this criterion only to children over the age of 14 (2010 criteria); when applied to children under this age, T wave inversion only carries a specificity of
ARVC of 64%.10 In a recent review of 205 patients with ARVC by Steriotis et al., T wave inversion was only present in 49% of individuals,48 an incidence lower than previously reported by other investigators, where T wave inversion was appreciated in 85% of affected individuals.15 However, patients with more advanced
RV myocardial disease associated with ARVC are more likely to have T wave inversion as reported by previous investigators.49
Depolarization abnormalities on the surface ECG in patients with ARVC typically take the form of a prolonged QRS (greater than 110 ms) in the right precordial leads and the presence of epsilon waves. It is suspected that slowing of conduction through the abnormal fibrofatty tissue of the RV in affected patients causes these particular ECG findings.16 The Task Force criteria state that either of these criteria satisfy a major criteria for diagnosis of ARVC.44 QRS prolongation was noted in 29% of 205 patients in the study by Steriotis.48 Peters et al. however described a greater incidence of QRS prolongation in 343 affected individuals at
75%.47 These investigators also determined that the QRS duration in leads VI-V3 divided by the QRS duration in leads V4-V6 was greater than 1.2 in 98% of patients, and that this localized formula could also be used in patients with incomplete and
22 complete right bundle branch block (which has been found to be present in 15% of
ARVC patients).4750 However, this localized formula was not included in the recent modification of the Task Force criteria.46 Epsilon waves are low amplitude waves which extend just beyond the QRS complex prior to the onset of the T wave, and are best seen in the leads overlying the RV (the right precordial leads)(see Figure 1.2).51
It was first described in 1978 by Fontaine, and it represents regions of slowed conduction in the RV free wall as a result of fibrofatty infiltration or just gap junction dysfunction.52 He found that by altering the placement of the limb leads in a formation he described as the "Fontaine leads", where the left arm electrode is placed over the xyphoid process, the right arm lead placed over the manubrium sterni and the left leg lead over a rib roughly at the level of placement of V4, he could better recognize the presence of the epsilon wave.52 In the study by Steriotis et al.,
9% of affected patients had the presence of epsilon waves, while in the study by
Peters et al., 23% of affected patients had epsilon waves.47-48 A study by Wu et al. of
49 patients with ARVC found that 37% of affected patients had epsilon waves by using the standard 12-lead ECG, and 57% using the Fontaine leads. When patients were classified as having diffuse RV disease, as assessed with transthoracic echocardiography, 73% of patients had epsilon waves compared to only 23% with what was considered to be localized disease.52 This finding led these investigators to conclude that epsilon waves correlate with the progression of disease in ARVC, and could be used as a marker when following patients in the long-term.
To overcome the difficulties in the identification of epsilon waves, a study by
Peters et al. of 360 patients used the finding of QRS fragmentation to aid in the ECG
23 diagnosis of ARVC.53 QRS fragmentation is defined as deflections which occur on the surface ECG from either one right precordial lead or from more than one of the standard leads at the onset of the QRS, on top of the R wave, or the nadir of the S wave.53 This finding was present in 85% of the 360 patients and has clinical utility as lead amplification was not required, and the approach uses the standard 12 lead approach as opposed to using specialized ECG recording techniques, such as the
Fontaine lead system.53
Figure 1.2: ECG from patient with epsilon waves present (arrow). Source: Wu, S., et al., Epsilon wave in arrhythmogenic right ventricular dysplasia/cardiomyopathy. Pacing and clinical electrophysiology: PACE, 2009. 32(1): p. 59-63. Used with permission.
1.0.9.3 Signal-Averaged ECG and ARVC
SAECG is a signal processing technique whereby consecutive QRS complexes are compared and averaged, allowing for the removal of random signals (noise), and therefore enhancing signals located in the terminal portion of the QRS known as late potentials (LPs).54 LPs are analogous to the epsilon wave observed on the standard surface ECG, and represent regions of delayed electrical conduction, namely in the diseased RV myocardium, and are believed to represent a pathophysiologic marker
24 for re-entrant ventricular tachyarrhythmias.55 Initially its utility was a method of risk stratification for patients with myocardial infarction, and has since been applied to patients with ARVC for a similar purpose.9 SAECG in the time domain (temporal) is recorded using an X, Y and Z orthogonal lead system (such as the Frank lead system), which is used to collect a certain number of beats to be averaged in order to improve the signal to noise ratio. A seed beat is first selected by the software analysis system as the beat with the shortest QRS duration and is therefore considered to be the most normally conducted QRS. All subsequent collected QRS complexes must match the seed beat in order to be incorporated into the averaging process. These beats are then filtered using a high pass filter (25 or 40 Hz filtering), and combined into a vector magnitude that represents the square root of the sum of the squares of each lead.54-55 Time domain analysis is then applied to the vector magnitude to yield three parameters of interest: filtered QRS duration (fQRSd), duration of high frequency low amplitude (HFLA) signals under 40 \xV, and the root mean square (RMS) value of the terminal 40 ms. As late potentials occur at the terminal portion of the QRS, when they are present, they increase the filtered QRS duration, increase the duration of HFLA signals, and as LPs consist of low voltage signals, the RMS voltage is relatively low.55 (See Figure 1.3) SAECG is considered abnormal and LPs present when two of three of the parameters of interest are abnormal (1994 criteria), or when only one parameter is abnormal (2010 criteria).
Several investigators have used SAECG in the clinical work-up of patients with
ARVC, and it has been found to be abnormal in 50-81% of affected individuals7956, with a reported sensitivity and specificity of 57% and 95% respectively.5 The
25 reduced sensitivity reflects the inability for SAECG to detect patients with early localized disease of the RV myocardium.5 As the disease progresses in affected individuals, it has been demonstrated that the SAECG variables continue to become more abnormal, but not with a progressive linear increase.55
Besides analysis in the temporal or time domain, SAECG can also be performed in the spectral or frequency domain, as well as spatial averaging techniques. In frequency domain, fast Fourier transformation analysis is used to determine the amplitudes of the frequency components of the QRS complexes. This can therefore limit the impact of high-gain amplification and signal filtering which is used in time domain analysis to identify the onset and offset of the QRS.57 This technique however lacks reproducibility and provides no additional information when compared to the time domain analysis.58 In spatial averaging, LPs can be identified using one QRS (as opposed to hundreds with the other methods).
However, this entails the use of multiple electrodes on the patient, and the signal noise is reduced by comparing closely spaced electrodes, assuming the cardiac electrical activity between these electrodes is not random like noise.58
According to the Task Force criteria, the presence of LP by SAECG only fulfills a minor criteria for the diagnosis of ARVC (1994 criteria) with more quantitative guidelines regarding measures obtained from SAECG (including fQRSd, duration of the terminal QRS < 40 uV, and RMS voltage of the terminal 40 ms) being published in the 2010 criteria.44
26 |iV Normal 100- Ab ntorma l 100- 90^ ! 90- 1 80J f J 7t>l 70-
60J ! i 60- t ! •fH. *j 40- 40- / 30- 10- s / , |0J 20i \v*-h I i mm i Urf-AfiWfMKfW^ OJ VM VMp < T
200 nut's QRS 200 mir QRS Figure 1.3: A normal SAECG from a non-Boxer normal control dog on the left, and an abnormal SAECG on the right from a Boxer with > 1000 VPCs in 24 hours, presumably affected with ARVC. The arrow indicates the region where late potentials are identified.
1.0.9.4 Correlating SAECG to other diagnostic modalities and risk for arrhythmia in patients with ARVC
Given the ability of SAECG to aid in the diagnosis of ARVC through the identification of LPs, there have been several investigators who have also investigated how well it correlates with other modalities used in the diagnosis of
ARVC. In a 1999 study by Turnni et al., 38 patients with ARVC were enrolled to investigate the correlations of SAECG to tissue biopsy and angiography findings.
When compared to percentage of fibrosis, fatty tissue and surviving myocytes, only a weak correlation could be found between fQRSd, HFLA and RMS40 using a 25 Hz band-pass filter, and percentage of fibrous tissue.59 No correlation was found
27 between SAECG variables and angiographic hemodynamic variables of the left and right ventricle such as end-diastolic volume and ejection fraction; however, when the presence of LPs (either present or not present) was correlated to angiography, patients with LPs had significant increases in RV end-diastolic volumes (EDV), and significant reductions in RV ejection fractions (EF). When compared to the incidence of arrhythmia, 10 of 18 patients with LPs had sustained ventricular arrhythmia versus 5 of 20 patients who did not have the presence of LPs. Therefore, it was determined that a percentage of fibrous tissue of greater than 30% was a significant predictor of LPs, and RVEF less than 50% with the presence of LPs on
SAECG were associated with increased risk of development of VT or VF.59
A study by Folino et al. of 31 patients with ARVC investigated the correlation between SAECG and arrhythmic events and echocardiographic findings.55 They determined that RVEDV correlated moderately well with fQRSd and HFLA at all filter settings (25,40 and 80 Hz), and the RMS40 at the 25 and 40 Hz filter.
Interestingly, over the follow-up period of 8 years, only the SAECG parameters showed progression of late potentials, while the echocardiographic indices did not reveal significant changes. Therefore, they concluded that conduction disturbances, assessed via SAECG, seem to increase independently of anatomical abnormalities.
When considering the baseline SAECG values, those patients with sustained VT had more abnormal LPs characterized by longer fQRS and HFLA durations.55 A recent study by Park et al. also attempted to correlate SAECG and echocardiographic indices in 38 patients with ARVC. They found that the extent of SAECG abnormalities had no correlation with any of the 2D echocardiographic parameters
28 for either ventricle in any of the 38 patients.56 However, when only select patients with VT/VF were included, the fQRSd was significantly correlated to RV end- diastolic and systolic area. The investigators therefore concluded: "fragmented electrical activity may appear with no significant relation to the anatomical alterations in patients with ARVC".56
Finally, a study of SAECG and ventricular arrhythmias by Nava et al. of 138 patients with ARVC revealed that the highest percentage of abnormal SAECGs were found in patients with VF (71%) and sustained VT (72%); however, in patients with non-sustained VT and less severe ventricular arrhythmias, SAECG was less sensitive, detecting only 40.6% and 55.8% of patients, respectively.8 This lower sensitivity could be a result of SAECG's inability to detect patients with less severe disease (and therefore less severe ventricular arrhythmias), or that the ventricular arrhythmias in ARVC are multifactorial in nature and do not rely solely on the presence of anatomic re-entry (due to fibrofatty infiltration), but also depend on other modulating factors such as sympathetic nervous system stimuli that would not influence SAECG parameters.56
1.0.9.5 Echocardiography and ARVC
Echocardiography is widely used as one of the first diagnostic tools in evaluating patients suspected of having ARVC due to its low cost, availability and non-invasive qualities. The 1994 Task Force listed several major structural and functional criteria that could be evaluated with the use of echocardiography, although specific quantitative parameters were lacking. In the revised 2010
29 guidelines, specific measurements of the RVOT in both long and short-axis views have been proposed.46 Major criteria include regional RV akinesia, dyskinesia or aneurysm in the presence of an RVOT measurement at end-diastole of > 32 mm in the long-axis or > 36 mm in the short-axis view, or a fractional area change of =s
33%.46
One of the limitations of standard two-dimensional (2D) echocardiography is the RV itself. Due to its unique geometry and crescent shape, its position in the near field, the presence of prominent trabeculations and the limited number of imaging planes available for its evaluation, imaging of the RV is a challenge with subtle changes often missed by inexperienced observers who are not familiar with the normal variations of RV shape and motion.15 Furthermore, calculations of ventricular volume (which are used to calculate ejection fraction) are difficult when applied to the RV compared to the LV, due to its anatomy. The RV is anatomically divided into two functional parts - the inflow tract and the outflow tract, which are separated by a muscular ridge known as the crista supraventricularis.60 This division into two functional components makes the use of volumetric formulas in the calculation of RV volume and ejection fraction such as the Simpsons formula fraught with inaccuracies and therefore not commonly used in clinical practice.60
Therefore, assessments of RV function rely on the use of other imaging modalities, including tissue Doppler imaging (TDI). TDI applies colour Doppler to the myocardium (as opposed to the blood) and spectral pulsed Doppler is used to record the wall motion velocity at a particular point of interest. The myocardial motion is of lower velocity than the blood, and several investigators have validated
30 its use as a measure of both systolic and diastolic function in the RV compared to
more invasive, gold-standard measures including ejection fraction (via
angiography), ±dP/dT and Tau.60
Many investigators have reported the echocardiographic findings typically
found in patients with ARVC. A study by Lindstrom et al. reported findings in 15
patients with ARVC, compared to 25 normal subjects.61 Comparing the ARVC
affected patient group to the normal controls, the most common abnormality found was dilation of the RV, found in 73% of ARVC patients and not present in the normal
group. Other abnormal findings included dilation of the RVOT in 53% of patients,
dilation of the right ventricular inflow tract (RVIT) in 40% of patients, while 27% of
the ARVC affected individuals had normal cardiac dimensions.61 The investigators
also applied TDI to all subjects and found significant differences between the two
groups. The main findings included a decreased lateral tricuspid annular velocity in
early diastole (E'), as well as a decreased peak systolic annular velocity (S'). They also determined that TDI was abnormal at an earlier stage in the disease than when
using other traditional cross-sectional measures and subjective assessments.61 This finding highlights the utility of TDI in the evaluation of asymptomatic individuals suspected of being affected by ARVC in terms of being able to diagnose subtle functional abnormalities before they become overtly apparent on two-dimensional imaging.
A study by Yoerger et al. evaluated 29 subjects with ARVC compared to 29 normal subjects in a North American Registry echo study.62 They determined that at least one RV morphologic abnormality was present in 62% of individuals, with 38%
31 of individuals having two or more apparent abnormalities present. The most frequent morphologic abnormality was trabecular derangement noted in 54% of individuals. The RVOT and RVIT were also significantly increased compared to the control subjects, with the RVOT dimension being the most commonly enlarged dimension. Using an RVOT long axis dimension cutoff value of 30 mm, any measurement larger than 30 mm had a sensitivity of 89% and specificity of 86% in the diagnosis of ARVC. This cutoff would be useful in the screening of individuals suspected of having ARVC, as long as other causes of right heart enlargement such as congenital cardiac disease and pulmonary hypertension are ruled-out. RV function was also assessed using calculations of RV area as well as with TDI and global RV dysfunction was present in 62% of affected individuals, with 79% of affected individuals demonstrating at least regional abnormalities, most commonly of the anterior wall and apex.62
Recently, Kjaergaard et al. evaluated the use of advanced quantitative echocardiographic measures including three-dimensional echocardiography and
TDI in 20 patients with ARVC.63 Compared to 32 normal individuals, the patients with ARVC had a significant reduction in peak systolic annular motion measured both at the lateral tricuspid annulus and the lateral mitral annulus. RV ejection fraction, as measured with three-dimensional echocardiography, was also found to be significantly reduced compared to normals. Based on their findings, the authors concluded that both TDI and three-dimensional echocardiography were useful in identifying functional abnormalities in patients with ARVC, and may be useful in the identification of asymptomatic, pre-clinical individuals.63 Later, Prakasa et al found
32 similar findings in 30 patients with ARVC with the use of TDI. In fact, four patients with ARVC had reduced peak systolic tricuspid annular velocity yet a morphologically normal RV by conventional two-dimensional echocardiography, supporting a role for TDI in the detection of mild cases of ARVC.64
Teske et al. evaluated the use of TDI and tissue deformation (strain) in 34 patients with ARVC compared to 34 normal individuals.14 Morphologically, they found that the RVOT was significantly enlarged in 74% of patients and the RVIT enlarged in 68%. Using the current Task Force criteria, 82% of ARVC patients met major criteria, minor criteria in 15%, and 1 affected individual did not meet any of the structural criteria. There was a significant reduction in both the peak systolic annular velocity and the peak early diastolic annular velocity measured at the lateral tricuspid valve annulus in affected patients. Strain measures the rate of deformation of a particular segment of tissue and has been found to correlate well with gold standard invasive measures of cardiac function, both systolic and diastolic.65 In this study, strain as well as strain rate was found to be reduced in three myocardial segments analyzed, compared to the normals. The investigators concluded that both TDI and strain/strain rate were superior in the detection of cardiac functional abnormalities in patients with ARVC compared to conventional echocardiographic criteria such as measurement of fractional change of cardiac dimensions, isovolumic acceleration, and tricuspid annular plane systolic excursion as measured with M-mode, since all of the deformation parameters showed a higher accuracy to detect functional abnormalities.14
33 Over the past decade and a half since the Task Force released their recommendations for the diagnosis of ARVC, echocardiography has advanced to become a significant diagnostic tool in the identification of structural and functional abnormalities in patients with ARVC, particularly in those patients who would have not otherwise met the previous qualitative Task Force criteria. With the recent release of the 2010 Task Force criteria, quantitative measures of the right ventricular outflow tract (RVOT) diameter have become one of the major criteria used in the diagnosis of ARVC, highlighting the importance of this non-invasive diagnostic test.
1.0.9.6 Advanced Imaging in the diagnosis ARVC: Computed Tomography (CT) and Cardiac Magnetic Resonance Imaging (cMRI)
Advanced cardiac imaging provides the opportunity to non-invasively assess
RV structure and function, but with their multiplanar capabilities and special imaging techniques (such as dark blood imaging and bright blood cine acquisitions), the ability to provide evidence of fatty infiltration.66 Of the two imaging modalities, cMRI has been most investigated in the evaluation of patients with ARVC as it does not provide radiation exposure to the patient, and it provides superior temporal resolution.16 Several investigators have reported the CT findings in patients with
ARVC, often with the use of contrast enhanced volume mode scanning.6770 Reported morphological findings included the presence of intramyocardial fat deposition, scalloping of the RV free wall, low attenuated trabeculations, and RV enlargement.
Ventricular volumes can also be determined with CT using cine mode scanning and
34 when applied to patients with ARVC, reveal increased RV volumes and depressed global RV function.67
cMRI allows for excellent visualization of the RV compared to echocardiography and CT, and provides excellent spatial and temporal resolution of the myocardium.39 Therefore, it allows for both qualitative assessments of RV structure but also quantitative assessments of RV function. One of the challenges however with cMRI is that it requires extensive training and expertise in order to ensure accurate interpretation of the findings.18 Many researchers have investigated the ability of cMRI to detect one of the histopathological markers of ARVC, intramyocardial fat accumulation, given its excellent tissue characterization capabilities. However, even with its excellent tissue characterization capabilities, the specificity and sensitivity of cMRI detection of RV intramyocardial fat is tremendously variable, between 22-100%.39 The explanation for this variability is multifactorial. Firstly, it has been well demonstrated that even normal individuals will have a certain degree of intramyocardial fat deposition, particularly those patients who are obese or on chronic steroid therapy. Furthermore, the identification of intramyocardial fat is challenged by the normal presence of both epicardial and pericardial fat.18 Anatomically, some regions of the heart also contain a large amount of fat deposition, such as the atrioventricular sulcus, which can be difficult to differentiate from the subtricuspid region, a common region affected by
ARVC.71 The RV free wall, being inherently thinner than the LV free wall due to the reduced pulmonary artery resistance the RV pumps against, affects the spatial resolution ability of cMRI, making it difficult to make a reliable statement regarding
35 the presence of fat infiltration. Finally, it has been well shown that the interobserver variability can vary tremendously, particularly in those individuals with less experience in the interpretation of cMRI, leading to a high proportion of false positives. This high rate of false positive identification was noted by Bomma et al. who noted that 77% of patients who were initially noted to have abnormal findings of intramyocardial fat accumulation at initial assessment could not be confirmed at a re-evaluation.72
Other abnormal findings that can be noted on cMRI are divided into those which are functional and those which are structural. In terms of functional abnormalities, cMRI is able to demonstrate wall motion abnormalities such as hypokinesis and dyskinesis that could lead to RV aneurysm formation, and reduced ejection fraction. In fact, cMRI has been shown to identify some patients with systolic dysfunction (reduced ejection fraction) that were deemed to have a normal ejection fraction as measured with echocardiography; however, in the same report, it was demonstrated that cMRI was inferior to echocardiography in the identification of wall motion abnormalities.15 One group has reported a unique finding on cMRI in patients with ARVC termed the "Accordion Sign" - these investigators noted an abnormal contraction pattern of the RVOT and/or subtricuspid region that demonstrated a focal "crinkling" which became much more prominent during systole.71 Structural abnormalities which have been identified include RV dilation, prominent RV trabeculae, the presence of RV aneurysms, and
RV wall thinning.15-18-39 Due to the constellation of abnormalities which can be noted on cMRI, which on their own are not specific for ARVC, a diagnostic scheme
36 has been proposed using major and minor criteria findings. Using this scheme, there has been improvement in the sensitivity (82.3%) and specificity (88.8%) of cMRI, leading to fewer false positives and more consistency amongst observers.10
One of the limitations of cMRI, besides the expertise required for accurate interpretation, is the higher cost of use, particularly when considering the screening of relatives of affected family members. As well, patients with ARVC who have implantable cardioverter-defibrillators (ICDs) cannot be evaluated with cMRI, limiting its ability as a follow-up diagnostic tool in those patients already diagnosed.18
1.0.9.7 Angiography and ARVC
Despite the widespread use of advanced cardiac imaging, angiography still continues to be considered one of the gold-standard diagnostic tests for ARVC.16
Although considered to be invasive with an exposure to radiation for the patient and medical personnel, angiography can allow excellent delineation of the RV contour, allowing for the identification of subtle functional and structural abnormalities in patients with ARVC.73 In a study by Indik et al., they were able to quantify regions of abnormal wall motion in ARVC patients, and found that the area of greatest motion abnormality occurred in the tricuspid and inferior wall regions.73 This group analyzed angiographic contours of the RV in three different views, and calculated contour area movement during contraction. The development of such quantitative assessments addresses the purely qualitative statements set forth by the 1994 Task
Force criteria, and may also assist in the reduction of both inter and intra-observer
37 variability in the diagnosis of ARVC using angiography.73 The newest Task Force
criteria published in 2010 still rely on qualitative assessments of regional RV
akinesia, dyskinesia or aneurysm with no quantitative measures of RV function listed.46 Besides the functional abnormalities which can be noted with angiography, structural changes can also be observed with some findings being more specific to
ARVC. These include the presence of aneurysms in the RVOT, bulges in the subtricuspid region, and prominent trabeculations (thickness >4 mm) with deep fissures. As ARVC seems to spare the trabeculae, it has been speculated that they undergo a compensatory hypertrophy in patients with ARVC, leading to their
prominent and fissured appearance on the ventriculogram. This finding is
somewhat controversial, as it can be difficult to differentiate abnormal from normal
RV trabeculation.16 The combination of bulges in the subtricuspid region and prominent transverse trabeculations in the apical region has a reported sensitivity
of 87.5% and a 96% specificity for ARVC.73 Although invasive, angiography is considered to be a cornerstone for the initial diagnostic evaluation of a patients suspected of having ARVC, as it allows for not only functional and structural assessment of the right heart, but other assessments can be performed during the same procedure such as electrophysiological studies and endomyocardial biopsies.16
1.0.9.8 Endomyocardial Biopsy
Endomyocardial biopsy (EMB) provides the opportunity to definitively
characterize the histopathological changes which result in the clinical phenotype of
ARVC, and is therefore considered to be a potential hallmark diagnostic test for
38 disease, particularly when fibrofatty substitution is demonstrated with myocardial degenerative changes.16 The 1994 Task Force criteria determined that tissue characterization of fibrofatty replacement of the myocardium constituted a major finding for the diagnosis, however, no quantitative guidelines were provided in reference to the location and the extent of the RV tissue changes.19 Furthermore,
EMBs do not represent the one and only diagnostic test needed for ARVC, as many patients with the disease will have normal biopsy findings. This is often a reflection of not only the disease process itself, but also in sampling methods. It has been demonstrated that patients with early ARVC will have focal "hot spots" of disease, characterized by apoptosis and necrosis with inflammatory infiltration, and these areas may not be included in the EMB sample.19 Furthermore, these early changes often occur in the subepicardial and mid-mural layers, regions not routinely sampled through EMB. Due to the risk of perforation of the sometimes-thin right ventricular free wall (RVFW), EMB samples are often taken from the IVS, a region that has been shown to be rarely involved in ARVC.5 To further complicate the matter, relying on the presence of fibrofatty myocardial infiltrate alone may lead to a higher proportion of false positives, as not only can intramyocardial fat be present in normal individuals, but fibrosis can also be observed in many cardiomyopathies as well as non-cardiomyopathic conditions.19 A recent study by Basso et al. attempted to provide quantitative EMB findings to aid in the diagnosis of ARVC.
They simulated EMB by sampling tissue from explanted hearts which were both diffusely and segmentally affected by ARVC, as well as hearts from patients with
DCM and those from normal subjects.19 Based on their findings, they determined
39 that the antero-apical region was most commonly affected (90% of cases) and samples from this region were found to be most informative. They also confirmed that samples taken from the IVS were unlikely to be diagnostic. By comparing the
ARVC affected hearts to the DCM hearts, they determined a diagnostic cut-off value for percentage of residual myocardium of less than 59% would provide a sensitivity of 80% and a specificity of 95% in ARVC hearts. It was found that reliance on percentage of fatty tissue was a less reliable diagnostic marker, therefore these investigators emphasized that myocardial atrophy should be regarded as one of the most important morphological parameters for the diagnosis of ARVC by EMB.19
These investigators also sampled the LV from the ARVC hearts (due to the involvement of the LV in over 50% of ARVC cases), and found that LV EMB was not informative in terms of tissue characterization as the morphological changes were usually segmental and confined to the subepicardial and mid-mural layers, neither of which are usually accessible via EMB.
Electroanatomic mapping is an adjunctive diagnostic method which may enhance the diagnostic utility of EMB. Using a catheter-electrode system, multiple voltage points are sampled from within the RV along the endocardium, and a three- dimensional electroanatomic map is generated. This technique has been shown to demonstrate delayed RV activation in abnormal tissue due to delayed conduction as a result of fibrofatty infiltration in ARVC patients.16 Alternatively, delayed RV activation may occur as a result of abnormal anisotropic properties due to desmosomal changes in patients with ARVC.74 Regardless, identification of low
40 voltage areas on the electroanatomic map can guide EMB to enhance the diagnostic yield by increasing the probability of sampling from an ARVC affected region.75
1.0.9.9 Electrophysiologic Studies and Electroanatomic Mapping in Patients with ARVC
Electrophysiologic (EP) studies in patients with ARVC serve a multitude of purposes: they can assist in the diagnosis of ARVC, offer prognostic information, and provide options for therapy. In some centers, EP studies with programmed ventricular stimulation (PVS) are performed as the standard protocol for the evaluation of patients with ARVC, while in others, it is used in select patients only.
EP studies with PVS are able to assess a patient's arrhythmogenic potential through a ventricular pacing protocol, or through pharmacologic intervention, such as isoproterenol infusion.16 During these interventional procedures, physicians are also able to evaluate the hemodynamic alterations which occur as a result of the VT, as well as its propensity to progress to VF, an important method to assess a patient's risk for sudden death. They are also able to investigate the ability of the VA to be interrupted by anti-tachycardia stimulation, as would occur if the patient were to receive an ICD, as well as evaluate the efficacy of drug treatment on the inducibility of arrhythmias.5'16 EP studies with PVS have been cited as a method of risk stratification for patients with ARVC, however, an observational study by Corrado et al. of 132 patients with ARVC who received an ICD revealed that PVS was of limited value in terms of identifying those patients who were at risk of a tachyarrhythmia.76 Of the 98 patients who were identified as higher risk for a
41 tachyarrhythmia based on inducibility of VA with PVS, 50 did not experience ICD therapy during the follow-up period (mean follow-up of 39± 25 months], whereas 7 of 13 patients in whom VA could not be induced experienced appropriate ICD interventions. These findings yield a positive predictive value of only 49%, a negative predictive value of 54%, and test accuracy of 49% by using PVS.
Furthermore, the type of VA which was inducible at the time of the EP study did not predict the occurrence of VF during the follow-up period.76 Therefore, EP studies may have little value in the risk stratification of patients with ARVC, and their utility may be eclipsed with newer methods such as electroanatomic mapping.16 EP studies are also used to assist in the radiofrequency ablation of highly arrhythmic foci as a form of therapy for patients with ARVC. Finally, EP studies can be used to determine the mechanism of the arrhythmia in patients with ARVC, differentiating re-entry from an automatic or triggered focus.
Electroanatomic voltage mapping of the RV is being used more commonly in the diagnostic evaluation of those patients suspected of having ARVC, given its ability to assess the electrical consequences of ARVC and detect concealed ARVC lesions that may be missed with other imaging systems. The system commonly used is the CARTO system, which consists of an ultralow magnetic field generator which is placed beneath the fluoroscopic table, a specialized catheter which contains a magnetic sensor in its tip, a reference catheter, and the computer mapping system.16
The sensor mounted catheter is then moved sequentially through the RV to spatially record electrograms, with particular focus on the "triangle of dysplasia" - the subtricuspid region, RV apex and the RVOT. These measured signals are analyzed
42 using the computer system, and areas of low amplitude are noted which reflect RV fibrofatty myocardial atrophy.77 The areas of low amplitude correspond to what is known as an "electroanatomic scar", and are colour-coded on the three-dimensional reconstruction. This allows for differentiation between electrically normal tissue, the abnormal tissue, and the intermediate tissue in between, referred to as the
"border zone".16 It has been well documented that electroanatomic voltage mapping accurately identifies "dysplastic" areas in patients with ARVC, namely regions with wall motion abnormalities identified with cMRI or echocardiography, as well as regions with fibrofatty infiltration confirmed with EMB.10-30 Using this voltage mapping, investigators have been able to differentiate those patients truly affected with ARVC from those with diseases which fulfill some of the 1994 Task Force criteria and therefore "mimic" ARVC, such as inflammatory cardiomyopathies and idiopathic RVOT tachycardia.30'77 In these particular patients, despite meeting some of the criteria for a diagnosis of ARVC, they lacked abnormal voltage map findings which are present in patients affected with true ARVC. It can also help in the identification of patients with concealed ARVC who may not meet the Task Force criteria and are therefore misdiagnosed. Voltage mapping has also been shown to be very beneficial in the differentiation of EMB diagnosed primary myocarditis from
ARVC with superimposed inflammation. In these cases, without the combination of the voltage mapping with EMB, patients with ARVC and inflammation may be diagnosed as inflammatory cardiomyopathy.16 As previously noted, voltage mapping can be used to guide EMB to increase diagnostic sensitivity for ARVC, allowing sampling in the "border zone" to incorporate the transition from normal to
43 abnormal tissue. It can be used to assist radiofrequency ablation of arrhythmic foci, by allowing for the creation of linear ablation lesions which connect to or encircle the electroanatomic scar, therefore interrupting the re-entrant circuit.30 Finally, voltage mapping may represent a useful risk stratification technique for patients with ARVC, as patients with the abnormal voltage mapping were more likely to require an ICD due to serious VA, whereas those patients with normal myocardial voltage values were well controlled with oral anti-arrhythmic treatment.16 Given that not all patients with ARVC have abnormal electroanatomical mapping, it should not be considered an absolute diagnostic criteria.
1.0.10 New Options for the Diagnosis of ARVC
As knowledge of ARVC continues to evolve, it is expected that a more comprehensive understanding of the disease process itself will lead the way to new diagnostic tests which can be used to screen individuals for ARVC, as well as reduce the number of mildly affected individuals who are missed due to the limitations of the current guidelines for diagnosis. Recently, a new diagnostic test for ARVC has become available. This particular test is based on the finding that the immunoreactive signal level for plakoglobin is reduced at the intercalated disk in samples from patients with ARVC compared to normal individuals as well as those with HCM, DCM and ischemic heart disease.36-37 A reduced level of plakoglobin had a sensitivity of 95%, specificity of 90%, positive predictive value of 91% and a negative predictive value of 95% for the diagnosis of ARVC.36 A particularly noteworthy finding by the investigators was that signal levels were not only
44 reduced in regions which contained fibrofatty changes, but other areas which were grossly normal including the IVS and the LV. Therefore, this method may improve the utility of EMB taken from the IVS to diagnose ARVC, even in the absence of morphological changes in the EMB sample.36 The investigators determined that despite the reduced signal level, plakoglobin was not totally lacking, but present in lower concentrations, or bound to a different protein with reduced immunofluorescence. Although this test was performed on myocardial samples, it may be possible to use more accessible tissues that contain desmosomes, namely the skin, hair follicles and buccal mucosa. To date, the molecular mechanism explaining the reduction in plakoglobin signal levels is unknown, but it suggests a shift of plakoglobin from a junctional location to either intracellular or intranuclear pools, and this shift may play a key role in the pathogenesis of ARVC.36
1.0.11 Risk Stratification for Patients with ARVC
Due to the clinical nature of ARVC and the possibility of sudden death in affected patients, investigators have sought to determine which risk factors may increase the likelihood that an affected individual will experience an adverse outcome, in an effort to guide both the need for therapy as well as the most appropriate therapy. One of the fundamental challenges with risk stratification with
ARVC is that given that the disease is progressive, risk factors may change in an individual, prompting frequent routine follow-ups to re-evaluate the patient's risk as well as the success of therapy.78 Furthermore, given the infrequency of sudden death with ARVC, it is difficult to assess risk in predisposed patients. Frequently,
45 assessments of risk for VA and adverse outcomes are made based on diagnostic findings during patient evaluation. When evaluating SAECG and echocardiography, a group of investigators determined that the presence of LPs on SAECG and a RV EF
< 50% were associated with an increased risk for the development of VA.59
However, other investigators have failed to correlate SAECG findings and a risk for
VA. These investigators determined that SAECG is useful in ARVC patients to identify a marker for pathological substrate (LPs), but is not a tool to predict an individual's vulnerability to VA.9 Up to 92% of patients with ARVC have right ventricular enlargement, which can be documented on echocardiography, cMRI and angiography.9 These cardiac imaging modalities also allow for the assessment of RV structure and function, and also for determination of concomitant LV involvement in affected patients. Investigators have shown that severe RV dilation has been associated with a worse arrhythmic prognosis, and those patients with LV involvement are at higher risk for clinical arrhythmic events, severe cardiomegaly and heart failure.79 Investigators have also assessed 12-lead surface ECG findings and found that QRS dispersion greater than 40 ms was a strong independent predictor of sudden death with a sensitivity of 90% and a specificity of 77%.50 As previously discussed, the use of PVS during EP studies does not appear to be useful in the risk stratification of ARVC patients, however electroanatomic voltage mapping may prove to be more useful in the assessment of affected individuals.
Presenting complaints in patients have also been analyzed for risk factors, with a history of syncope being a strong independent predictor for sudden death with a sensitivity of 40% and a specificity of 90%. In fact, one group showed that syncope
46 was the only significant variable which separated 19 patients who died suddenly from a large group of affected individuals.9 Finally, familial history also plays an important role in risk stratification as a history of sudden cardiac death, particularly in a family member under the age of 35, has been shown to increase one's risk of
VA.80
Risk stratification has been a tremendous aid in reducing the incidence of sudden deaths in young athletes in Italy.12 During the past twenty years, systematic pre-participation screening consisting of a 12-lead ECG, thorough family history and physical examination has been employed in all athletes. This has led to a reduction in the rate of sudden death by approximately 90% through the identification of patients who had not only concealed ARVC (identification of T wave inversion in precordial leads and VA with left bundle branch block morphology), but also those individuals with asymptomatic HCM.12
1.0.12 Therapeutic Options for Patients with ARVC
Options for therapy in patients with ARVC often rely on assessments of risk, meaning that patients considered to be at high risk are treated more aggressively, such as with the placement of an ICD. Patients who are considered to be at lower risk are managed with anti-arrhythmic medications,, sometimes in combination with radiofrequency catheter ablation (RFA). However, there is limited information on the long-term outcomes in patients with ARVC managed with different therapeutics, therefore recommendations are largely based on small retrospective studies.16
One of the first recommendations in a patient with ARVC, particularly a
47 younger individual, is restriction from strenuous activity and participation in competitive sports, given the increased frequency of VA and sudden death during or after physical exercise.12 Anti-arrhythmic drugs are also typically recommended, even in a patient with ARVC who is asymptomatic. Unfortunately to date, most of the pharmacological recommendations have been empiric, as randomized, prospective studies are lacking.16 The goals of anti-arrhythmic therapy are the control of VA, particularly the occurrence of VT, reduction of symptoms in symptomatic patients, and potentially prevention or reduced risk of sudden death, although this benefit of therapy has yet to be proven.30 To date, one of the most commonly prescribed anti-arrhythmics is sotalol, an anti-arrhythmic that has both class II and class III properties. Although no large prospective studies have evaluated its use in ARVC, some investigators have studied its ability to control inducible VT during EP study in 68-83% of ARVC affected individuals at a dose of
320-640 mg/day.5'81 However, a recent report from the North American ARVC
Registry of 95 ARVC patients with ICDs who received anti-arrhythmic medications found that the 58 individuals who received sotalol did not appear to be protected from the risk of arrhythmia.81 It was the investigator's conclusions that sotalol could in fact be harmful to the patients who received it. This statement was based on the finding that despite adjustments for potential confounders, patients receiving sotalol were at higher risk for any clinically relevant VA or ICD intervention, and were at higher risk for the first clinically relevant arrhythmia, compared to those not on sotalol or those receiving no anti-arrhythmic medication.81 The investigators did note that the rate of the VT in patients receiving sotalol was significantly slower,
48 suggesting that the risk associated with sotalol was not due to pro-arrhythmic effects. They also concluded that this effect of slowed VT by sotalol would make
"arrhythmias more tolerable".81 Amiodarone was found to be protective in patients, regardless of confounders. However, only ten patients in the study received amiodarone, therefore these findings must be interpreted with caution in this observational study. Other investigators have discouraged the use of amiodarone as a first line of therapy in younger patients due to the high incidence of reported side effects, therefore this must also be taken into consideration when using amiodarone.16 Other reported anti-arrhythmic protocols consist of a combination of a p-blocker and amiodarone together, which controlled VA in six patients better than each drug individually.16 Other investigators have reported success with the use of class I drugs, such as mexiletine.1 Thus at the present time, there does not appear to be a generally accepted anti-arrhythmic treatment regimen for those patients with ARVC, but empirically speaking, the use of sotalol and fs-blockers appears to be most common.
For those patients where anti-arrhythmic therapy is not adequate in controlling the frequency or the symptoms association with VA, RFA can be a consideration. It can also be a consideration in those patients who have focal disease only. RFA has been shown to effective in treating VA by interrupting re entrant circuits involving diseased myocardium, as well as by ablating irritable foci which could result in VA through automaticity or triggered activity. RFA is typically guided through the use of electroanatomic voltage mapping, or through pace mapping, activation mapping and entrainment mapping. Ablation is considered to
49 be successful when the initially observed VT morphology cannot be induced using ventricular stimulation.82 A review of 24 ARVC patients who underwent RFA for control of VA revealed that ablation was successful in that VT could not be re- induced in 77% of patients. However, VT re-occurred in 85% of the patients, with a time to recurrence of approximately 8 months (range of 1 day to 44 months). This finding is not unexpected given the progressive nature of ARVC with continued morphological changes of the RV myocardium, leading to the development of new re-entrant circuits. Therefore, RFA in patients with ARVC is not curative, and should be considered as a palliative technique only in order to reduce the frequency of VA, especially in those patients whose VA is not adequately controlled with anti arrhythmics.82
In patients who have either failed anti-arrhythmic therapy and RFA, or those patients who are considered a high risk for sudden death through risk stratification, an ICD is a therapeutic option. A review of 321 patients who received an ICD, 73.5% received it for secondary prevention (previous arrest, history of syncope, episodes of sustained VT), while the remaining patients were treated for primary prevention
(history of first degree relative with sudden death).78 Many of the patients with ICDs received concurrent anti-arrhythmic therapy in an effort to reduce the number of required ICD interventions, making the device more tolerable to most.81 There have also been reports of patients with an ICD who not only receive oral anti arrhythmics, but also RFA to control frequent VT and ICD shocks.5 The ICDs function by sensing VT or VF, and respond with an appropriate intervention, either antitachycardia pacing or a defibrillation shock.16 In theory, they would seem to be
50 an ideal therapeutic option for a disease such as ARVC in which the predominant symptoms are related to VA. However, given the potential complications associated with an ICD as well as the emotional implications in young people with an ICD
(depression, anxiety and fear of discharge), patient selection is very important.78
Complications associated with ICDs have been reported to occur in approximately 6-
45% of patients, ranging from lead thrombosis, infection, fracture, and migration, to sensing problems which can result in inappropriate intervention or lack of appropriate intervention during VA.16 In ARVC, it can be a challenge to achieve adequate sensing threshold due to the progressive fibrofatty tissue infiltration, resulting in 13% of patients with ICDs experiencing undersensing.5 Furthermore, besides the sensing issues the fibrofatty infiltration may cause, it may also result in inappropriate defibrillation efficacy. As well, the progressive thinning of the RV wall which occurs in conjunction with the fibrofatty infiltration may increase the likelihood of lead perforation, with a reported incidence of between 0.6% to 5.2%.16
To date, there have been no prospective randomized trials to compare ICD to treatment regimens with oral anti-arrhythmics or RFA in patients with ARVC.
Patients who have been followed long term (1-7 years) have proven a benefit for
ICD, with a 3 year patient survival rate of 96% compared with a projected VF-free survival rate of 72% without the ICD.76 This study also identified that history of cardiac arrest, VT with hemodynamic compromise, young age and LV involvement were all independent predictors of potentially lethal VA, thus assisting in the risk stratification of patients with ARVC and allowing for proper therapeutic planning.76
These risk factors must be weighed when considering which patients should be
51 treated with an ICD, realizing that despite the complications which may occur, ICDs have been shown to be reliable in sensing and terminating sustained VA, therefore decreasing the risk of sudden death.16-83
In those patients who present with signs related to heart pump failure due to advanced ARVC, standard heart failure therapy is recommended. This includes the use of angiotensin-converting enzyme inhibitors (ACE-I), (3-blockers, diuretics and anti-coagulants.43 Patients who fail medical therapy for biventricular pump failure are candidates for heart transplantation, however the decision for transplantation is often delayed as long as possible as transplantation carries only a 50% survival at
12 years.16
1.1 Arrhythmogenic Right Ventricular Cardiomyopathy in Boxers
1.1.1 Identification and Natural History
In the 1980s, Dr. Neil Harpster identified an acquired, degenerative myocardial disease in the Boxer featuring myocyte atrophy and fatty tissue infiltration, preferentially affecting the myocardium of the RV.84-85 He observed these findings in a group of closely related Boxers in New England, with the affected dogs having evidence of VA of right ventricular origin, which was associated with syncopal episodes, and commonly occurred during exertion.8586 Since that time, a wealth of information has been published regarding the condition in the Boxer, and its surprising similarities to ARVC in people. Dogs afflicted with the disorder, like people with ARVC, had a wide range of clinical presentations from being completely
52 asymptomatic, to intermittent episodes of syncope, to rare cases of congestive heart failure. Sudden death was also observed intermittently amongst affected dogs, probably related to malignant VF. Based on these observed findings, Harpster described three types of "Boxer Cardiomyopathy": Type I dogs were asymptomatic but had evidence of VA; type II dogs were syncopal or experienced sudden death; type III dogs presented with signs attributable to congestive heart failure related to myocardial dysfunction, with or without evidence of VA.87 Due to the similarities in clinical qualities, natural history and RV histopathological changes to that observed in human ARVC, the disease was later appropriately classified as Boxer ARVC. This classification has lead to great interest in studying the Boxer as a spontaneous model for human disease, allowing for the development of new diagnostic assays, identification of new genetic mutations, and assessing new clinical therapies for human medicine.16
1.1.2 Prevalence of Boxer ARVC
It has been recognized for many years that Boxers are predisposed to VA with evidence of syncope, heart failure and sudden death.88 It has only been recently within the past two decades that the association between VA in Boxers and ARVC in the breed was made, meaning prior to this time affected dogs may have been misclassified as having a primary disease of myocardial dysfunction such as DCM.
Therefore, the exact prevalence of ARVC within the breed is unknown, however it is estimated to be relatively common.84 One of the difficulties in estimating the prevalence of the disease in the breed is related to the lack of a definitive gold
53 standard for ante-mortem diagnosis. As some dogs can be asymptomatic for the disease with the VA often found incidentally during a routine annual physical examination, many cases are likely missed or simply not reported to their veterinarian. With recognition of the disease entity as well as through the diligence of both veterinarians and Boxer breeders, an estimation of the prevalence of ARVC within the breed will be possible.
1.1.3 Pathological Changes of Boxer ARVC
Similar to human ARVC, the predominant pathological change with Boxer
ARVC is myocyte atrophy with fibrofatty and fatty infiltration within the RV myocardium. Grossly, affected hearts have been observed to be similar in weight and wall thickness when compared to non-affected controls, with approximately one third of the dogs having evidence of RV chamber dilation.89 RV aneurysms in the infundibular region may be observed, but they are relatively uncommon.16
Histologically the lesions of Boxer ARVC resemble those noted in people, however, it has been noted that the pure fatty form predominates in Boxers, while the fibrofatty form which is common in people, is less common in Boxers. A review of 23 hearts from affected dogs revealed that 65% had the fatty form, while 35% had the fibrofatty form. The fatty form was noted to be more diffusely distributed and multifocal, involving the RV wall and trabeculae with only mild interstitial fibrosis observed. The fibrofatty form was observed to be focal or diffuse in distribution with fatty replacement associated with marked replacement fibrosis and more extensive RV myocardial loss.89 Like in human ARVC, the histopathological
54 changes appeared to extend from the epicardium to the endocardium, with surviving myocytes located between islands of fat and fibrofatty tissue (see Figure
1.4).
Fatty infiltration can be found in normal canine hearts, however the percentage of fat in affected Boxer hearts was markedly increased compared to normal hearts (40.4% versus 13.8% in normal hearts), and more concentrated in the anterolateral and infundibular regions.89 Evidence of inflammation characterized by the infiltration of lymphocytes into the RV is present in about two-
55 thirds of affected dogs, with apoptosis present in just less than half. Closely mimicking what is observed in people, approximately 50% of affected Boxers with
ARVC also have LV involvement characterized by regions of focal fibrous tissue replacement with less fatty replacement, as well as inflammatory infiltration in 70% of these dogs. The left and right atrium can also be affected, with one third of the observed dogs having atrial myocyte loss with fatty or fibrofatty replacement.16-89
1.1.4 The Etiology of Boxer ARVC
To date, human ARVC is widely accepted as a disease of the desmosome.
Therefore, investigators have closely studied the composition of the intercalated disks of affected Boxer dogs in an effort to better understand the pathophysiology of the disease in the dog. Oxford et al. examined RV tissue samples from 12 dogs confirmed to have ARVC and compared them to samples from two control non-
Boxer dogs. Using immunolocalization for the proteins cadherin, plakophilin-2, desmoplakin, plakoglobin, connexin-43 and desmin, these investigators found the presence of cadherin (which makes up adherens junctions), plakophilin-2 and desmoplakin at the intercalated disks in affected dogs; however, plakoglobin and connexin43 immunofluorescence was not present in affected dogs, and in three of the four affected samples, a complete lack of desmin (a component of the intermediate filament) was observed at the intercalated disk.13 In order to determine whether the proteins which did not immunofluoresce at the intercalated disk were present at all in the tissue extracts from affected dogs, western blot analyses were performed. This revealed that loss of the detectable signal of some of
56 the proteins at the intercalated disk was not as a result of a complete absence of the proteins as all the proteins ran at the mobility predicted by their molecular weight, suggesting an inability for the proteins to localize at the intercalated disk and form a functional unit.13 These investigators concluded that Boxer ARVC affects the molecular integrity of the intercalated disk as a whole, leading to disarray of the intermediate filaments [as characterized by the lack of desmin at the intercalated disk] resulting in weak mechanical coupling between myocytes. This weak coupling could therefore result in the detachment of affected myocytes, leading to the clinical and pathologic characteristics of ARVC in Boxers. Furthermore, the loss of gap junction plaques (connexin43) may compromise electrical cell to cell interactions, and therefore promote the substrate for the malignant VA noted in affected
Boxers.13 To further support the concept of altered cellular mechanical and electrical coupling as the underlying substrate for Boxer ARVC, Oxford et al. recently demonstrated a significantly reduced average length of the desmosomes in affected
Boxers compared to non-Boxer controls in both the RV and LV. As well, a reduction in the number of gap junctions per examined field in ARVC samples compared to controls was also noted.90
One of the forms of dominant ARVC in people, ARVC2, has been linked with a mutation leading to RyR2 dysfunction. Given this finding in people, Meurs et al. investigated the ryanodine receptor as a possible etiology for Boxer ARVC. Through immunoblotting, they detected normal RyR2 expression in the control dogs, and decreased expression in the ARVC dogs. Furthermore, real time PCR detected differential expression of the RyR2 mRNA in normal dogs between the LV and RV,
57 and decreased message expression in ARVC affected dogs with a significantly lower quantity of RyR2 mRNA in all cardiac chambers.86 Given these findings, it is possible that Boxer ARVC, or at least some variants of Boxer ARVC, occur as a result of RyR2 dysfunction which predisposes affected individuals to myocardial electrical instability, contractile dysfunction with heart failure and possibly sudden death. An incidental finding in this particular study was the observation of differential expression of the RyR2 between the LV and the RV in normal dogs. It is hypothesized that this differential expression is a result of the differences in contraction and systolic wall tensions between the two ventricles, and given this differential, RyR2 dysfunction may preferentially affect the RV.86
An alternate hypothesis for the VA and heart disease in Boxers with ARVC was proposed by Oyama et al. These investigators analyzed tissue samples from affected Boxer dogs and compared them to samples from Doberman Pinscher dogs with DCM, Beagle dogs with experimental heart failure as a result of rapid ventricular pacing, and healthy non-Boxer control dogs. They determined that the transcriptional activity of calstabin2 was significantly lower in the Boxers and
Beagles compared to the Dobermans and the healthy controls. Calstabin2 is important in the modulation of the RyR2 receptor function, which serves to control calcium release from within the sarcoplasmic reticulum in myocytes.91 As well, immunoprecipitation of RyR2 revealed decreased calstabin2 levels in the RyR2 channel complex in the affected Boxer dogs compared to the control dogs. A decreased RyR2 concentration was not shown in this study, as previously identified in affected Boxers by Meurs et al.86 The investigators concluded that the observed
58 calstabin2 deficiency could potentially result in the abnormal function of the RyR2 channel in these dogs, resulting in intracellular calcium leak that could therefore promote VA. What is not apparent from this study is whether this deficiency is a primary or secondary abnormality, as it was also observed in the Beagles with pacing-induced heart failure. Therefore, this finding may represent a secondary change in response to the heart failure phenotype. Furthermore, no differences in
DNA sequencing in the regions of the calstabin gene were found between the affected Boxers and the control dogs. Regardless if it truly represents a primary or a secondary abnormality, as it may contribute to the VA noted with ARVC, it may represent a future therapeutic target in affected individuals.91
Much like human ARVC, the true etiology and pathogenesis of Boxer ARVC is still not clear. Like in human ARVC, there may be the possibility of variants in Boxer
ARVC, with some variants a result of RyR2 dysfunction [such as in ARVC2), whereas others may result because of abnormal mechanical-electrical cell coupling at the level of the intercalated disk.
1.1.5 The Genetics of Boxer ARVC
Given the marked similarity with human ARVC, there has been great interest in Boxer ARVC in an attempt to locate causative mutations, which could then be investigated and explored in people. The ability to closely follow affected families of
Boxers has proven that the disease is inherited as an autosomal dominant trait, as affected dogs occur in every generation with equal sex distribution, while affected parents have been noted to produce unaffected female dogs.87
59 In human ARVC, multiple genetic mutations involving genes that encode for proteins of the desmosome have been discovered. As the genetic code for the dog is known, and was actually sequenced from a Boxer, investigators have explored the same desmosomal genes in the Boxer. Meurs et al. evaluated 10 unrelated Boxer dogs with ARVC and 2 control unaffected dogs, and found no evidence of base-pair changes in the exonic and splice site regions of plakophilin-2, plakoglobin, desmoplakin and desmoglein-2 genes between the two groups. There were also no differences when comparing to the known Boxer genetic sequence.92 Corroborating these findings, Oxford et al. also sequenced the cDNA coding for junctional proteins in affected Boxers and compared them to a healthy non-Boxer control as well as the published Boxer sequence, and found no signs of early termination, frame-shifts or missense mutations in the sequences coding for desmoplakin, plakophilin-2, plakoglobin and connexin43.13 Furthermore, in another study Meurs failed to identify a genetic linkage of Boxer ARVC to the RyR2 gene through linkage analysis.86 The lack of identification of a genetic mutation in the proteins encoding for the desmosome in Boxers may simply reflect the heterogeneity which is observed in human ARVC, meaning that although the Boxers studied to date did not demonstrate desmosomal mutations, ARVC in some Boxers may truly be a result of desmosomal mutations.92
Recently an 8-base pair deletion within a non-coding conserved element in a regulatory region of a calcium modulating gene known as striatin was identified by
Meurs et al. in affected Boxers, but not observed in unaffected Boxers or in 31 dogs from other breeds tested.93-94 It has been observed that striatin co-localizes with
60 several desmosomal proteins including plakophilin-2, plakoglobin and desmoplakin and it also contains a calcium dependent calmodulin binding site, therefore the
mutation could affect the integrity of the desmosome and/or may influence calcium leak from the sarcoplasmic reticulum.93-94 Interestingly in this study, 11 of 35
Boxers who were classified as control dogs (under the age of six years, and had fewer than 100 VPCs in 24 hours) were heterozygous for the deletion. This suggests that either these dogs will develop disease at a later stage, or the degree of penetrance may vary among individuals due to different environmental factors or genetic modifiers.94
1.1.6 Other Species/Breeds Affected with ARVC
Besides being identified in the Boxer dog, ARVC has been reported in the domestic cat as well as isolated cases in several dog breeds including a Dachshund,
Bulldog, Bullmastiff, Labrador Retriever and Siberian Husky.9596
Fox was the first to report the findings of ARVC in 12 cats and determined that the pathophysiologic features were very similar to what is seen in both people and Boxers with ARVC.96 All affected cats had moderate to severe RV dilation, 7 of the 12 cats had right atrial (RA) enlargement, and 6 of the 12 cats had evidence of localized RV aneurysms in the apical, infundibular and subtricuspid regions
("triangle of dysplasia"), representing areas of akinetic or dyskinetic wall motion.
These features of right chamber enlargement and aneurysm formation are certainly more commonly observed in affected people with ARVC rather than affected Boxers with ARVC.16 On histopathology, there was marked myocardial atrophy in the RV
61 with 75% of affected cats having fibrofatty infiltration, and 25% of the cats having primarily fatty infiltration. This infiltration was noted to extend from the epicardium to the endocardium.96 This coincides with what is observed in people as well, with the majority of affected individuals having fibrofatty infiltration.16 All cats with the fibrofatty infiltration also had evidence of inflammatory infiltration, with only one of the cats with fatty infiltration showing evidence of myocarditis. LV involvement was noted in 10 of the 12 cats, characterized by fibrous infiltration within the LV free wall, and 9 of the 12 cats had evidence of apoptosis.96 A majority of the cats (8 of 12) died due to progressive right-sided congestive heart failure, and sudden death was not a feature of this feline model of ARVC.
Harvey et al. described two feline cases of ARVC and found similar features as previously described by Fox. Both cats presented with signs attributable to right heart failure (ascites, pleural effusion, hepatomegaly, jugular pulsation), both cats had third degree AV block, and one cat presented with a prior history of syncope.97
Both cats had marked right heart enlargement with wall motion abnormalities noted on echocardiography. Post mortem findings were only available on one of the cats, which revealed marked infiltration of the RV free wall with fibrofatty tissue and degeneration of the myocytes. This cat also had LV involvement characterized by inflammatory cell infiltration. The curious finding in these cats is presence of third degree AV block which has not been previously reported in any veterinary cases of ARVC, but has been reported intermittently in people with ARVC.97
Although the authors did not comment on any abnormal histopathological findings of the AV node, one would speculate that the fibrofatty infiltration may have been so
62 considerable in these cats that it may have affected the AV node function, such as the establishment of exit block surrounding the AV node, bundle of His, or both bundle branches.
Ciaramello et al. also recently described the findings in a cat in Italy affected by ARVC, with marked LV involvement.17 This cat presented with signs related to heart failure, with pleural effusion and tachypnea. A Holter monitor revealed the presence of multiple VPCs of left and right ventricular origin, predominantly from the RV. On echocardiography, there was marked RA and RV dilation, with a thin and hypokinetic RV wall. Aneurysms were present in the apical and subtricuspid region, coinciding to areas of the "triangle of dysplasia". There were no reports of historical syncope. Despite medical therapy for heart failure, this cat died 10 days after presentation and a post mortem examination was performed, revealing histopathological findings typical of ARVC affecting both the RV and the LV. The changes of the RVFW were so severe that it had the resemblance of parchment paper.17
One of the earlier reports of ARVC in a non-Boxer dog was by Simpson et al. who described the findings in a 3.5 year old Dachshund.88 This dog presented for signs of anorexia and lethargy, with signs of right heart failure (subcutaneous edema, ascites). Echocardiography revealed marked right heart dilation, with no evidence of left to right shunt, pulmonary hypertension, or tricuspid valve disease/dysplasia. Despite aggressive medical therapy, the dog was euthanized several days after presentation. Post mortem examination revealed marked thinning of the RV free wall, with widespread myocyte loss and fibrofatty
63 replacement noted on histopathology. Inflammatory cells were also present, with small mature lymphocytes and plasma cells noted.88
Santilli et al. recently described findings of ARVC confined to the RVOT in an
English Bulldog, the first case of segmental ARVC reported in the dog.95 This dog presented with a history of repeated syncopal episodes with profound fatigue.
Echocardiography revealed moderate right heart dilation, wall motion abnormalities and the presence of an aneurysm of the RVOT. LPs were present on
SAECG, and a standard surface ECG revealed sustained ventricular tachycardia of RV origin. The dog died of ventricular fibrillation after induction of anesthesia for planned RFA therapy. Histopathology revealed transmural fibrofatty replacement of the RVOT, with a normal appearing RV free wall, IVS and LV.95
The few reported cases of ARVC in veterinary medicine in non-Boxer patients underscores the presence of some differences between the findings reported in
Boxers, namely the presence of right heart failure. To date, heart failure is a rare outcome in the natural history of Boxer ARVC, with clinical signs attributable to the electrical component of ARVC, as opposed to overt myocardial dysfunctional disease leading to cardiac pump failure.84 Perhaps the difference lies in the fact that the majority of Boxers reported to date have predominant fatty infiltration, as opposed to fibrofatty infiltration. This could lead one to speculate that the fatty form of disease is less likely to result in functional disease, however, this theory has yet to be substantiated with clinical evidence. Interestingly in people, those with the fibrofatty variant are more likely to have thinner RV walls and typically have the appearance of RV aneurysms in the "triangle of dysplasia".16
64 1.1.7 Clinical Signs of Boxer ARVC
As outlined by Harpster, there are three clinical presentations of Boxers with
ARVC - the first are those dogs who have VA, but are completely asymptomatic for their arrhythmia with no reports of syncope, fatigue or exercise intolerance. The presence of VA is typically an incidental finding noted by a veterinarian during a routine physical examination. The second clinical presentation is Boxers with VA who are overt, meaning they are symptomatic for their arrhythmia. These dogs will have reports of syncope, both with and without activity, exercise intolerance and fatigue. Syncope is the most common finding in this group, being reported in over half of severely affected dogs. The final clinical presentation, representing the most uncommon form of disease, is Boxers who develop myocardial dysfunction and present with signs attributable to heart failure such as ascites, edema, syncope, fatigue, respiratory signs and depression. Sudden death is a possibility in any of the three stages or presentations of Boxer ARVC.16-84 However, it has been shown that
Boxers with the fibrofatty form and evidence of myocarditis are more likely to experience sudden death.89
As syncope is one of the most common presenting signs for Boxers with
ARVC, it is important to also point out that ARVC is not the only documented cause of syncope in the Boxer. Thomason et al. recently reported the findings of bradycardia-associated syncope in 7 Boxers who also had documented VA.98 In these cases, syncope was a result of documented SA arrest, typically using a Holter monitor. These findings are most consistent with neurally-mediated
65 syncope/bradycardia, an entity whose pathology is not completely understood. In affected individuals, episodes are typically triggered by situations which result in either a surge of sympathetic or parasympathetic nervous system dominance.98
This results in either a vagal-induced bradycardia or sympathetic nervous system withdrawal with profound vasodilation and hypotension. As VA co-existed with the neurally-mediated bradycardia in all dogs, the use of a beta blocker as an anti arrhythmic to control the VA could potentially worsen or precipitate the bradycardia related syncope, a consideration for Boxers whose syncope worsens despite medical management for VA.98 Anecdotally, these authors report that Boxers who have syncopal episodes related to VA typically will have numerous VPCs and paroxysmal VT on a Holter monitor performed within 1-2 days, a finding not seen in dogs with the bradycardia-associated syncope.
1.1.8 Diagnostic Criteria for Boxer ARVC
In human ARVC, diagnosis relies on meeting the criteria set forth by the Task
Force criteria. In veterinary medicine, no such diagnostic criteria exist for Boxer
ARVC. Although there is no gold standard antemortem diagnostic test, a Boxer is considered to be ARVC affected when demonstrating a certain frequency of ventricular ectopy on a 24-hour Holter monitor. There have been no studies that have investigated the definitive frequency of VA required, however having over
1000 VPCs in a 24-hour period is typically considered to identify a definitely affected individual.91 In reality, the actual VPC number may in fact be much lower, as it has been shown in a study of normal dogs that they rarely have VPCs, with no dog
66 having more than 24 VPCs in a 24 hour period." A study of asymptomatic Boxers revealed that a majority of the dogs had fewer than 75 VPCs in 24 hours, therefore it would appear that the Boxer is a breed which more commonly will have VA, even in normal individuals.84 As more diagnostic methods are investigated in dogs with
ARVC, definitive criteria for diagnosis may become available.
1.1.8.1 Physical Examination Findings in Boxer ARVC
The physical examination in the affected Boxer is often normal.84
Tachycardia or an irregular heart rhythm consistent with the presence of premature beats may be appreciated via auscultation or palpation of femoral arterial pulses.
The presence of a systolic heart murmur, particularly over the left heart base, is a common finding in many Boxers, including those who are both normal and those with ARVC. In fact, in a large study of 231 Boxers who were evaluated with phonocardiography, 77% of the dogs had the presence of a systolic heart murmur.100 Given the increased prevalence of congenital subaortic stenosis (SAS) among the Boxer breed, abnormal anatomy of the LVOT typically accounts for over a third of the Boxers with left basilar murmurs.101 In the remaining dogs, a left basilar murmur may be physiologic and related to an exaggerated response to sympathetic tone. Alternatively in France, it has been identified that the prevalence of atrial septal defects [ASDs] is high amongst Boxers (56.2%), and this could also result in a left basilar systolic murmur due to increased blood flow across the pulmonic valve.102
67 In those Boxers with myocardial dysfunction, a soft apical systolic murmur may be heard over the tricuspid or mitral valve. This murmur is considered to be functional, related to annular dilation secondary to eccentric hypertrophy, or due to altered papillary muscle geometry and function.84 An S3 gallop may also be appreciated in these dogs with myocardial dysfunction, along with signs typical of heart failure including tachypnea, jugular venous pulsation and distension, ascites, weak femoral arterial pulses, hepatomegaly, and respiratory crackles.16
1.1.8.2 Electrocardiographic Findings in Boxer ARVC
The classic ECG finding in Boxers with ARVC is the presence of VPCs with a left bundle branch block morphology where the QRS complexes in the ventrocaudal leads (II, III and aVF) have an upright positive morphology.103 (see Figure 1.5) This is typical of beats originating from the RV. In many affected dogs, a resting ECG performed in hospital may be normal due to the intermittent nature of the VA, therefore a normal ECG does not exclude the possibility of ARVC in a Boxer.104 The
VA can be of more complex morphology, with the presence of bigeminy and trigeminy, as well as couplets, triplets and runs of both non-sustained and sustained
VT. VPCs with a right bundle branch block morphology, those typically seen originating from the LV, can be seen in some affected Boxers, as well as supraventricular arrhythmias particularly in those with atrial enlargement and myocardial dysfunction. However, VPCs with a left bundle branch block morphology will predominate.16
68 40 i I liiS mn^» t' ' %Xt auWuV ' i' ' . !'. 1 l ' i Ut 15.!* 1 AVUMOIM . L!.L '' MAC6B!M>9A: ! i tfflL™vJ37 ' Figure 1.5: 9-lead ECG from an ARVC affected Boxer demonstrating frequent VPCs with a left bundle branch block configuration (upright in leads II, III, aVF]
In human medicine, there has been much analysis and investigation for surface ECG criteria that are consistent with ARVC, including the presence of epsilon waves, T wave inversion and QRS dispersion. To date, few if any of these criteria have been evaluated in Boxers with ARVC. A study by Spier et al. evaluated QT dispersion as an index to evaluate the severity of arrhythmia in Boxers with
ARVC.105 The QT interval on the surface ECG is a variable of ventricular repolarization, and the duration of the QT interval is not identical within all leads.
This is referred to as QT dispersion. Due to the ultrastructural changes of ARVC
(fibrofatty and fatty infiltration), there may be regional differences in ventricular repolarization which may manifest as an abnormal QT dispersion.105 In this study of
25 Boxers, there were no correlations between any QT variables and the total number of VPCs and the complexity of the arrhythmia. Furthermore, most of the
69 dogs had a QT dispersion of zero, indicating that substantial dispersion of repolarization was not found in Boxers with ARVC.105 This finding is in agreement with a human study by Peters et al. where patients with ARVC that had sustained VT did not have an increased QT dispersion.9
1.1.8.3 Ambulatory Electrocardiographic Findings in Boxer ARVC
The ambulatory ECG, or Holter monitor, is currently the diagnostic method of choice to aid in the diagnosis for Boxer ARVC and is used to assess the response to anti-arrhythmic therapy in dogs treated for ARVC. It is also useful in assessing syncopal Boxers to determine if their syncope is related to VA or to bradycardia.
However, it is recognized that it is not the ideal modality to aid in the diagnosis of
ARVC, given the substantial day-to-day variability of arrhythmia that occurs in
Boxers with ARVC. Additionally in human medicine, spontaneous VA variability of anywhere from 65-84% on consecutive Holters has been documented which has considerable implications when deciding if arrhythmia reduction is related to therapeutic intervention or just inherent variability.106 In a veterinary study by
Spier and Meurs, the spontaneous variability in the frequency of VA in Boxers with
ARVC was evaluated.107 They found that the variability of frequency of arrhythmia in dogs with greater than 500 VPCs in a 24-hour period did not exceed 80%.
Therefore, any changes in the frequency of VPCs less than 80% may fall within the limits of spontaneous day to day variability.107 This is important when evaluating the response to anti-arrhythmic therapy, but may also be a consideration when attempting to rule in or rule out ARVC in a Boxer. Therefore Boxers with fewer than
70 1000 VPCs per 24-hours, the arbitrary number above which dogs are considered affected, but more than 24 VPCs in 24 hours, a number considered to be higher than normal, should have a repeat Holter performed to re-evaluate the VA frequency. If the index of suspicion for ARVC is high and Holter results are inconclusive, an implantable loop recorder can be considered to identify paroxysms of VT with or without syncope since intermittent events can be identified over long periods of time.84 The investigators also compared the grade of the arrhythmia using a modified Lown grading scheme108, and found that in 50% of dogs, the VA did not differ by more than one grade, in 30% it did not change at all, and in the remaining
20%, a change of more than one grade was noted. Interestingly, it was noted that the dogs with the most frequent VPCs did not have any variability in the grade of arrhythmia, while the dogs with the least frequent arrhythmias had the greatest variability in grade. Therefore, when evaluating serial Holters in Boxers with ARVC it may be more useful to evaluate arrhythmia grade as opposed to evaluating frequency only to determine whether a real change has occurred whether due to treatment or disease progression.107
Holter monitors have also been used to evaluate heart rate variability (HRV) in Boxers with ARVC. HRV is a measure of the influence of the autonomic nervous system on the heart rate, with sympathetic nervous stimulation resulting in a decreased variability of R-R intervals, and parasympathetic modulation resulting in an increased variation of R-R intervals. In human medicine, it can be used to predict death as a result of congestive heart failure, as well as risk of sudden death in patients with VA.109 Spier and Meurs evaluated HRV in 24 Boxers and 10 normal
71 non-Boxer control dogs in order to evaluate the possible sympathetic nervous system involvement in the presence of VA in Boxers with ARVC. They found no differences in HRV between Boxers who were considered to be affected [via VA frequency) and those considered to be unaffected. Some of the evaluated Boxers had concurrent congestive heart failure, and a significant difference was found between this group of dogs and all other groups. This was not a surprising finding, given the high sympathetic nervous system tone present in those individuals in heart failure, yielding a low HRV.109 The lack of a significant difference between affected Boxers and non-affected Boxers and normal control dogs in terms of HRV does not rule out the involvement of the sympathetic nervous system in the modulation of VA in ARVC, as surges in sympathetic tone may be transient.109
The temporal variability of the VA in Boxers with ARVC has also been investigated with the use of Holter monitors.110 Scansen et al. evaluated 162 Boxers who had Holter monitors performed, and found that there appears to be a mild variation in the temporal frequency of VA in Boxers, with a nadir occurring between midnight and 0400, and a greater proportion of VA occurring between 0800-1200 and 1600-2000. These times typically correspond to times when dogs were awake and relatively active, and supports that in fact catecholamine release and the effects of the sympathetic nervous system may play a role in the expression of VA in Boxers with ARVC.110
Despite the potential limitations of Holter monitors, they will continue to be an important part of the diagnostic evaluation of Boxers with ARVC, as well as the evaluation of response to anti-arrhythmic therapy. To improve its sensitivity, future
72 studies investigating either the frequency or the complexity of VA and correlating it to severity of disease (such as evaluated by EMB, cMRI, echocardiography] may be useful.
1.1.8.4 Signal Averaged Electrocardiogram in Boxer ARVC
The use of SAECG is becoming more commonplace in veterinary medicine, with reports of using the technique in not only Boxers with ARVC, but also other breeds at high risk for VA including Doberman Pinschers. However, SAECG does pose some differences in application when compared to use in people. For example, as artifact can increase noise which negatively influences the signal to noise ratio,
LPs can be potentially missed as they become hidden in baseline noise. Veterinary patients who are restrained in lateral recumbency will often pant and have fine muscle tremoring due to anxiety, both of which can increase signal noise and therefore interfere with the averaging process. In some patients due to this limitation, an ideal noise level cannot be achieved and the diagnostic utility of the technique is impaired.111 Furthermore, it has been shown that the QRS duration in the dog is shorter than in humans, therefore when sampling the terminal 40 ms
(such as when calculating the RMS value), a greater proportion of the high voltage
QRS complex will be measured, skewing the RMS value. Therefore, it may be more ideal to sample the terminal 30 ms of the QRS in dogs as opposed to the standard 40 ms as performed in people.57-111112
Normal values for SAECG parameters have been previously published in healthy dogs by various investigators in both time and frequency domain.112 Calvert
73 et al. were some of the first veterinary investigators to apply SAECG to dogs with VA, and found possible LPs in four dogs that had documented episodes of VT.
Interestingly, three of the four dogs evaluated were Boxers with a history of syncope.113 More recently, Spier and Meurs performed SAECG in 93 Boxers (some normal, some ARVC affected) and compared results to 49 healthy non-Boxer dogs as controls using time domain analysis.58 They found that numerous false positives and negatives occurred, and that the proper identification of LPs is very dependent on the technique used and the filters applied. LPs, defined as having two abnormal
SAECG variables, correctly identified 14 of the 15 dogs that had a cardiac related outcome, be it sudden death or death due to congestive heart failure, yielding a sensitivity of 93%. It was observed that more severely affected dogs as assessed by
VPC frequency on a Holter monitor or clinical status, had significantly more abnormal SAECG findings. However, in 13 of the 79 clinically normal dogs LPs were also detected, yielding a specificity of 84%, with a positive predictive value of 52% and a negative predictive value of 98.5%.58 Therefore, although the application of the SAECG technique may pose more of a challenge in veterinary patients, it is still a useful modality in patients with cardiac disease associated with VA, and may be useful in the identification of those individuals at risk for a cardiac outcome with a fairly high sensitivity.
1.1.8.5 Echocardiography in Boxer ARVC
Despite the often advanced histological changes occurring in affected Boxers, it is frequently reported that transthoracic echocardiographic examination in these
74 patients is unremarkable, differing from the frequency in which abnormalities are detected in people with ARVC.16-114 Due to the difficulty in adequately imaging the
RV, and in many cases, a lack of experience in assessing the subtleties of the structure and function of the RV, it is possible that abnormalities do exist but subtle changes may be simply missed.84 Structural abnormalities which may be appreciated after careful examination may include RV dilation or possible thinning of the apical RV myocardium. Another possible limitation in the ability to detect structural and functional changes in affected dogs is that Boxers with ARVC often do not have follow-up echocardiographic examinations past their initial diagnostic work-up, as the severity of their disease is followed through Holter monitors.
Therefore, it is also possible that structural and functional changes occur over time in Boxers as they do in people, but the lack of frequent evaluations or follow-up precludes the ability to detect them. In those dogs who proceed to develop myocardial dysfunction [category III according to the Harpster classification), LV eccentric hypertrophy is observed with a reduced percentage of fractional shortening due to myocardial systolic dysfunction.115
Due to documented right ventricular dysfunction and sometimes subtle wall motion abnormalities in people with ARVC, veterinary investigators have started to use various noninvasive echocardiographic parameters to make statements about
RV function in an effort to find additional supportive diagnostic methods for Boxer
ARVC. To date, only the index of myocardial performance (IMP), or Tei index, has been assessed in affected dogs.116 The Tei index was proposed in human medicine well over a decade ago as a method of assessing both systolic and diastolic
75 performance of the heart, and when applied to the RV, it has been found to correlate with gold standard measures of function including RV peak ±dP/dT, Tau, and ejection fraction.117 Using pulsed Doppler echocardiography, the index is the sum of isovolumetric contraction and relaxation time, divided by the time for ejection.117-118
In the study of affected Boxers by Baumwart and Meurs, 12 affected Boxers were compared to 10 normal Boxers using the Tei index. They found no significant differences in the Tei index between the two groups, nor any other difference in cardiac structure (LV dimensions) or LV function (percentage of fractional shortening). Furthermore, the index was not found to correlate with the frequency of VPCs or the grade of the arrhythmia noted on a Holter monitor. Therefore, these investigators concluded that unlike their human counterparts, Boxers with ARVC do not have coexistent RV dysfunction with VA. Another possibility for these findings was the inclusion of Boxers with concealed disease in the control group, or the affected dogs had such mild disease that the IMP was not sensitive enough to detect any functional abnormality.116
In terms of other noninvasive echocardiographic measures such as TDI, which has proved to be very useful in human ARVC, these modalities have yet to be used in Boxers with ARVC.
1.1.8.6 Cardiac Magnetic Resonance Imaging in Boxer ARVC
To date, there have been only limited evaluations of affected Boxers using cMRI, and only one study used live patients whereas the other evaluation used explanted formalin-fixed hearts to assess the ability of cMRI to detect fatty and
76 fibrous tissue infiltration.6689 In the study of explanted hearts, 19 Boxer hearts were evaluated and compared to 7 controls. Of the 14 ARVC hearts with confirmed fatty replacement by histopathology, cMRI detected a high transmural signal intensity in the anterolateral and/or infundibular regions of the RV in all of these dogs.
Therefore, MRI was able to accurately detect intramyocardial fat in affected hearts, suggesting the utility of this test as an antemortem diagnostic aid in the work-up of suspected ARVC patients.89 A more recent study by Baumwart et al. used cMRI in 5 affected live Boxers compared to 5 unaffected control dogs. No intramyocardial fat was observed in either group of dogs, however, one of the affected Boxers did have evidence of an RV aneurysm, and the RV ejection fraction was significantly lower in the ARVC dogs compared to the normal dogs.66 These findings support the notion that in Boxer ARVC, myocardial dysfunction is indeed present in some Boxers and co-exists with VA, prior to the development of overt morphological changes. It also supports the investigation of other non-invasive tests to further assess myocardial function in these dogs, particularly using techniques that are more readily available such as echocardiography. Further studies with larger groups of affected dogs need to be evaluated, however, before specific cMRI criteria for Boxer ARVC can be made.
1.1.8.7 The Utility of Biomarkers for Boxer ARVC
In the past decade, the use of cardiac biomarkers in both human and veterinary medicine has increased substantially. These cardiac biomarkers allow for objective measurement of the markers of normal biologic processes as well as abnormal pathogenic processes, and can allow for an assessment of the response to
77 a particular therapy.119 Of particular interest in the veterinary field is the use of cardiac troponins as well as the natriuretic peptides, particularly brain natriuretic peptide or BNP.
Troponin is a regulatory protein which is made up of three isoforms which are components of the myofibrillar contractile apparatus responsible for the regulation of the interaction between actin and myosin filaments in cardiomyocytes.120 Troponin-T (cTnT) and troponin-I (cTnl) are the isoforms of interest as they have nearly absolute cardiac specificity based on their N-terminal extensions which are not present in the fast skeletal protein isoforms, while the troponin-C [cTnC], is present in both muscles and therefore not specific.120<121 In a normal individual, levels of cTnT and cTnl should not be detectable in the blood as they are structurally bound within cells. However after damage to the myocytes occurs, these proteins are released into circulation and can therefore be detected using immunoassays.120 These proteins can be detected within 4 hours and reach a peak concentration in circulation within 12-24 hours with their concentrations correlating to the severity of the inciting insult, then gradually decrease over a period of 5-20 days. In people, troponins have become the preferred method of diagnosis of acute myocardial infarction.122 The immunoassays used to detect troponin concentrations in people have been shown by numerous studies to be also usable in veterinary patients, as the troponin amino acid sequence is highly conserved between species, and the troponin antibodies used in the assay are not species-specific and are directed against a stable area of the molecule.122 To date, various cardiac disorders in veterinary patients are associated with elevated
78 troponin concentrations, including pulmonary hypertension, myxomatous mitral valve disease, HCM, subaortic stenosis, DCM, myocarditis and pericardial effusion.123
Non-cardiac causes can also result in troponin elevations, such as dogs with gastric- dilation volvulus (GDV), hyperthyroidism, renal failure, sepsis, Ehrlichia and
Babesiosis.124 Baumwart et al. have evaluated the concentration of cTnl in Boxers with ARVC and have found a significant correlation between the frequency of ventricular arrhythmia and the cTnl concentration exists. This finding is not surprising based on the morphological changes of myocyte atrophy, apoptosis and fibrofatty and fatty infiltration which occur with ARVC. This correlation also indicates that the cTnl concentration may indicate the severity of ARVC in an affected patient; however, overlap was noted in concentrations between non- affected normal Boxers and the Boxers with ARVC, therefore limiting its ability as a discriminating test.124
BNP is secreted predominantly from the ventricles in response to myocyte stretch in patients with cardiac disease, and has been shown to have natriuretic, diuretic and smooth muscle relaxant properties.125 BNP concentrations are reported to be increased in veterinary patients with myxomatous valvular degeneration, heart failure and those with muscular dystrophy cardiomyopathy.125 Baumwart and
Meurs evaluated plasma BNP concentrations in Boxers with ARVC compared to dogs with pacing induced heart failure, normal Boxers and non-Boxer control dogs. They identified no significant difference in BNP concentrations between ARVC affected and clinically normal Boxers, or between the non-Boxer control group and each of the Boxer groups.125 These findings are in contrast to a human study where human
79 patients with ARVC had significant increases in BNP concentrations compared to control healthy patients and those with RVOT tachycardia.126 These investigators hypothesized that the reason for the BNP increase in ARVC patients was due to local wall stress on the myocytes surrounding the atrophic area, as the fibrofatty region would be relatively resistant to contraction meaning the residual myocytes would experience more stretch as a result.126 A hypothesis for the difference in findings between Boxers with ARVC and humans with ARVC is that it has been well documented that the gross structural changes which occur in human ARVC (such as
RV dilation, wall motion abnormalities and aneurysmal formation) have not been noted to occur in the majority of Boxers evaluated to date for ARVC. Therefore at this time, BNP does not appear to be a useful diagnostic test in Boxers with ARVC.
1.1.8.8 Genetic Tests for Boxer ARVC
As previously discussed, a genetic test has recently become available to determine the status of ARVC affectedness in Boxers. The test is currently available through the Veterinary Cardiac Genetics Laboratory at the College of Veterinary
Medicine at Washington State University. Developed by Meurs et al., this test identifies a genetic deletion in the striatin gene. Dogs who were homozygous for the mutation have been found to have more severe disease based on Holter monitor findings such as VPC frequency.93-94 As with any genetic test which is used to identify genetic mutations, mere presence or inheritance of a mutation does not simply imply a diagnosis of ARVC, as some individuals with the mutation (genotype) may never express it clinically (phenotype). These tests are useful however to identify
80 those who are potentially at risk for the phenotype and are therefore followed more closely for clinical signs of the disease. These tests also assist in planned breeding programs. Due to the shear number of mutations identified to date in human ARVC, it is unlikely this mutation represents the only mutation present in Boxer ARVC, therefore continued genetic investigation will no doubt yield new information that will prove to be very useful in screening and breeding programs for Boxers.
1.1.9 Therapy for Boxer ARVC
Anti-arrhythmic treatment is the current cornerstone of therapy for affected
Boxers. A study by Meurs et al. investigated the use of four different anti arrhythmic protocols for Boxers with ARVC: atenolol, procainamide and sotalol monotherapy respectively, and mexiletine and atenolol combination therapy.127 In this study, 49 dogs were evaluated with all dogs receiving their respective medication protocol for 3-4 weeks. Dogs were evaluated for both anti-arrhythmic effect [defined as a greater than 85% reduction in the frequency of VPCs) and pro- arrhythmic effects (defined as a greater than 85% increase in the frequency of
VPCs). Dogs were excluded from the study during treatment if there was the development of syncope, worsening of syncope, depression or gastro-intestinal signs. They found that the best protocol was the sotalol monotherapy and the combination therapy of mexiletine and atenolol in terms of anti-arrhythmic effect, and fewest pro-arrhythmic and adverse side effects. In the 18 dogs who received sotalol, 9 had a greater than 85% reduction in VPC frequency with only one dog having a greater than 85% increase, and with only 2 dogs removed for syncope or
81 increases in syncope. In the 13 dogs who received the combination protocol, 8 had a greater than 85% reduction in VPC number, with only 2 having a greater than 85% increase and 2 dogs removed due to syncope.127 Procainamide demonstrated the worst pro-arrhythmic effect, with 5 of the 11 dogs having a greater than 85% increase in VPC number; however, there was no increase in syncope frequency in this group. Interestingly, none of the treatments reduced the incidence of syncope, but when all treatments were combined, there was a significant reduction in the incidence of syncope. Based on the results of this study, first-line anti-arrhythmic therapy in affected Boxers is often either a sotalol monotherapy or alternatively the combination therapy with mexiletine and a (3-blocker. To further investigate combination therapy, a study in sixteen Boxers revealed that dogs treated with a combination of mexiletine and sotalol had a better reduction in arrhythmia frequency (7 of 8 combination treated dogs had a greater than 85% reduction compared to only 2 of 7 dogs with monotherapy], a 100% reduction in VT or R on T
(monotherapy only had a 33% reduction), and significant reductions in maximum and mean heart rate compared to sotalol therapy alone.128 This study suggests that further investigation is required evaluating combination anti-arrhythmic therapy in more Boxers affected by ARVC.
A patient's response to therapy is best assessed via the use of follow-up
Holter monitors, evaluating for evidence of either anti or pro-arrhythmic effects.
Although therapy may not decrease the number of syncopal episodes and there is no evidence that they reduce the risk of sudden arrhythmic death, they improve the hemodynamic consequences of sustained VA in affected patients.84
82 A novel therapy which may be an additive consideration in patients already receiving anti-arrhythmics is omega-3 fatty acids.129 In people, it has been demonstrated that omega-3 fatty acids are cardioprotective, in that they have anti arrhythmic properties and potential anti-atherosclerotic effects. The mechanism of action is believed to be via the reduction in the excitability of cells by inhibiting voltage gated sodium and calcium channels.129 In a study of 24 Boxers with ARVC, it was determined that dogs who received fish oil (containing both eicosapentaenoic acid and docosahexaenoic acid) supplementation for 6 weeks had a reduction
(defined as a greater than 85% reduction) in the frequency of their arrhythmia.129
Further studies will be required in order to determine the ideal dosing recommendations for this therapy.
RFA is not currently routinely available in veterinary centers and therefore has yet to be used as a treatment adjunct for Boxers with ARVC. However, an ICD has been reported to have been used in one Boxer by Nelson et al.130 In this report, the ICD was used in a young Boxer who had sustained VT despite anti-arrhythmic monotherapy with first mexiletine and then sotalol, and then finally combination therapy with both atenolol and mexiletine. No complications occurred in the immediate post-operative period, however, when high energy defibrillation shocks were delivered, the dog became fearful and would flee and hide.130 Therefore, the high energy defibrillation was deactivated, relying on anti-tachycardia pacing and low-energy cardioversion as the primary interventional options for tachyarrhythmias.130 Following this adjustment, the dog did not have any syncopal episodes or any signs associated with a tachyarrhythmia. Unfortunately 10 months
83 after the initial implantation, the dog developed an infection of the ICD generator site and the entire system required removal. A new system was not replaced and the dog was maintained on oral anti-arrhythmics before the dog died of an unrelated event - intestinal perforation due to foreign body ingestion. This case provides support that ICD therapy is possible in Boxers with ARVC and may be useful particularly in those cases that have continued VA and/or clinical signs despite oral anti-arrhythmic therapy.
In the rare subset of Boxers who develop systolic dysfunction with biventricular heart failure, standard heart failure therapy consisting of diuretics,
ACE inhibitors and positive inotropes are recommended, in conjunction with an anti-arrhythmic if required.84 Carnitine therapy may also be warranted based on a report of improvement in myocardial function in a small number of Boxers following carnitine supplementation.131
1.2 Tissue Doppler Imaging Assessment of Systolic Function
Cardiologists are always attempting to find new methods to assess the systolic performance of the heart and how structural changes can impair it.
Traditionally, the gold standard methods to assess the systolic performance of the heart revolved around invasive measures such as cardiac catheterization with calculation of ejection fraction, dP/dT as well as via radionuclide ventriculography.
Although these measures give clinicians invaluable information regarding the systolic performance of the heart, they are costly, invasive, expose patients and personnel to radiation and contrast agents, and in many cases, are not readily
84 available, and with newer more advanced imaging techniques, are less than "gold" as a standard. With the introduction of echocardiography, many of these invasive measures are used less routinely in the clinical setting.132 Echocardiography allows for the determination of a variety of measures of surrogates for systolic function such as fractional shortening, velocity of circumferential fiber shortening, left ventricular internal dimension (systole) index, ejection fraction, index of myocardial performance, strain and strain rate, and TDI, all of which have been shown to correlate to the more invasive gold standard methods of measurement. Within the past decade, there has been great interest in the utility of TDI due to its ease of use, availability, and less dependence on preload and afterload than other echocardiographic derived measures of systolic function.133
In the past, Doppler echocardiography was used primarily for quantification of blood flow velocity by measuring the Doppler shifts from moving red blood cells.65 With TDI, Doppler is applied to the motion of cardiac tissue, and the resultant Doppler shifts are measured. These Doppler shifts are higher in amplitude than conventional Doppler signals from red blood cell motion, but are of lower velocity, therefore high-velocity filters are used to eliminate blood motion and track the motion of the tissue only.65132 There are two current applications of TDI - spectral Doppler and colour Doppler. With spectral TDI, a fixed sample volume is placed over a region of interest within the heart, typically the annulus of the atrioventricular valves for standardization, and the instantaneous velocities of wall motion at that point are measured. Velocities which are moving toward the transducer position are positive, while those moving away are negative, and a
85 velocity waveform is generated.65 (see Figure 1.6) The benefits of spectral TDI are that it is easy to acquire and can be performed "online", has been shown to be reproducible and has excellent temporal resolution, however, the spatial resolution has shown to be poor and it does not allow for separate analysis of myocardial layers (therefore represents a measure of "global" ventricular systolic performance).65 Angle dependence is very important with TDI, like in any other application of the Doppler principle, and suboptimal alignment with the region of interest will result in an underestimation of the tissue velocities.133 With colour TDI, the image is acquired and analysis is performed "offline". Here, the colour of the wall motion reflects its motion, and the brighter the shade of colour, the higher the velocity of wall motion. Wall segments encoded red are moving toward the transducer position, while those moving away are encoded blue.65*134 This modality allows for measurement of regional mean velocities, and has the advantage that several segments can be analyzed within one collected cardiac cycle, and unlike the spectral method, the sample area can be manually tracked along the moving tissue, ensuring that it does not move out of the myocardium. This method is particularly useful to compare the amount and timing of contraction and relaxation across multiple regions of the heart simultaneously. Although both modalities produce a similar waveform (Sm), it has been shown that spectral TDI produces higher velocity measurements.134 Furthermore, depending on the imaging window used, either longitudinal fiber motion (from the apical windows) or radial and circumferential fiber motion (from the parasternal windows) can be assessed with TDI.
86 Figure 1.6: TDI from a dog measured at the lateral mitral valve annulus with the upward peak corresponding to peak systolic wall motion (Sm), the downward peak corresponding to peak early diastolic wall motion (Em), and the downward peak following the P wave on the concurrent ECG corresponding to late diastolic wall motion (Am]
In people, TDI of the mitral annulus has been shown to correlate well with LV systolic function in normal patients, as well as patients with structural cardiac disease such as HCM, DCM, RCM, ARVC and ischemic heart disease.135 In veterinary medicine, it has been validated for use in dogs as well as those with structural cardiac disease such as mitral valve disease and DCM.135-137 In a study by Chetboul et al., TDI was used to quantify myocardial velocities in Golden Retrievers in the pre clinical phase of muscular dystrophy-associated cardiomyopathy.138 These investigators determined that there was a significant reduction in systolic myocardial velocities, both radial and longitudinal, in affected Golden Retrievers
87 compared to normal unaffected dogs, despite the fact that the majority of the affected dogs had no notable ventricular dilation or alteration in systolic function as assessed with conventional two-dimensional echocardiography. These results support the use of TDI to detect subtle myocardial dysfunction before overt structural and functional alterations occur. TDI has also proven to be very useful in assessing the function of the RV which is typically a challenge using conventional echocardiographic methods, due to its unique geometry and limited imaging windows available for assessment. TDI measured at the tricuspid annulus has been shown to correlate well with invasive measures of systolic function in both people and dogs.60-139 In fact, it has been shown that velocities measured at the lateral tricuspid annulus are typically higher than those measured at the lateral mitral annulus.65 When myocardial systolic function is depressed, reflected by a reduced ejection fraction, the Sm wave velocity of the TDI waveform is lower than normal.
Normal values for annular velocity have been reported by multiple investigators in both people and dogs and cats. Besides its ability to quantify systolic function, TDI can also identify systolic dysfunction in patients before overt signs of systolic dysfunction exist, such as the observation of increased end systolic dimensions. In human medicine, TDI has also been shown as a method of risk stratification in certain cardiac diseases, as low velocities have been shown to predict adverse cardiac outcomes.132'135
88 1.3 Tissue Doppler Imaging Assessment of Diastolic Function
TDI has also been shown to be remarkably useful in patients with diastolic dysfunction, particularly in those patients who may have been otherwise diagnosed with normal diastolic function based on the presence of a pseudonormal transmitral flow profile.140 It has been shown to correlate well with invasive measures of ventricular diastolic function, including Tau and - dP/dT in both dogs and people.136
With TDI, two distinct diastolic velocities are recognized; Em coincides with the early diastolic velocity relating to passive filling of the ventricle, while Amcoincides with the late diastolic velocity relating to the atrial systolic contribution to ventricular filling, (see Figure 1.5). Em has been shown to reflect myocardial relaxation, and is decreased in patients with impaired relaxation.140 As such, this value has been found to be one of the earliest echocardiographic markers for diastolic dysfunction. Prior to the common use of TDI, reliance on transmitral inflow patterns had the potential to miss patients with true diastolic dysfunction, namely those patients with a pseudonormal pattern. In these individuals, despite having impaired relaxation, the transmitral inflow pattern is "corrected" due to the presence of elevated filling pressures, yielding a mitral flow pattern resembling a normal filling pattern. This demonstrates the load dependent quality of transmitral flow assessment, making TDI superior for diastolic function assessment as it does not demonstrate as much load dependence.140-141 In the study of pre-clinical Golden
Retriever muscular dystrophy-associated cardiomyopathy by Chetboul et al., besides the significant reductions in systolic myocardial velocities in affected dogs, there was also a reduction in early diastolic myocardial velocities compared to
89 control dogs despite the absence of notable ventricular dilation, supporting TDI's ability to detect diastolic dysfunction before overt changes are noted on two- dimensional echocardiography.138 Doberman Pinschers with overt DCM have been shown to have diastolic dysfunction with the use of TDI, characterized by a significant reduction in both early and late diastolic annular velocities.142 The early diastolic annular velocities measured with TDI can also be used to estimate mean left atrial pressures (MLAP). It has been shown experimentally in animals and clinically in people that the ratio of Era to the transmitral inflow E correlates strongly with MLAP. In the dog, a ratio of E:Em > 9.1 has been shown to predict a MLAP of greater than 20 mmHg in a model of acutely induced mitral regurgitation.143 These indirect and noninvasive assessments of MLAP are important in the patient with diastolic dysfunction, as it is useful to predict which patients are at risk for the development of pulmonary edema. Furthermore, TDI of mitral annular motion has also allowed for the discrimination of constrictive pericarditis from restrictive cardiomyopathy, as both appear similar on transmitral inflow patterns. With constrictive pericarditis, early diastolic annular motion is rapid, while in restrictive cardiomyopathy, it is slower.141
Given its ability to detect both early systolic and diastolic dysfunction, TDI has become a useful application of echocardiography and has become a standard part of a routine echocardiographic examination. Furthermore, given the difficulty in adequately assessing RV function using traditional echocardiographic methods,
TDI has been shown to be a user-friendly method to quickly assess both RV systolic and diastolic function. Given its ability to diagnose subtle systolic and diastolic
90 dysfunction in affected people with ARVC who may not have gross structural changes, TDI has the potential to define myocardial function in Boxers with ARVC, which to this point in time, has been considered to be largely normal in the majority of affected dogs.
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84. Meurs KM. Boxer dog cardiomyopathy: an update. Vet Clin North Am Small Anim Pract 2004;34:1235-1244, viii.
85. Harpster N. Boxer cardiomyopathy. Vet Clin North Am Small Anim Pract 1991;21:989-1004.
86. Meurs KM, Lacombe VA, Dryburgh K, et al. Differential expression of the cardiac ryanodine receptor in normal and arrhythmogenic right ventricular cardiomyopathy canine hearts. Hum Genet 2006;120:111-118.
87. Meurs K, Spier A, Miller M, et al. Familial ventricular arrhythmias in Boxers. J Vet Intern Med 1999;13:437-439.
88. Simpson KW, Bonagura JD, Eaton KA. Right ventricular cardiomyopathy in a dog. J Vet Intern Med 1994;8:306-309.
89. Basso C, Fox P, Meurs K, et al. Arrhythmogenic right ventricular cardiomyopathy causing sudden cardiac death in boxer dogs a new animal model of human disease. Circulation 2004;109:1180-1185.
90. Oxford E, Maass K, Fox PR, et al. Phenotypic differences in the ultrastructure of cardiomyocytes from Boxer dogs afflicted with arrhythmogenic right ventricular cardiomyopathy (ARVC). In: ACVIM Forum, Montreal, Canada 2009.
91. Oyama MA, Reiken S, Lehnart SE, et al. Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency. J Vet Cardiol 2008;10:1-10.
100 92. Meurs KM, Ederer MM, Stern JA. Desmosomal gene evaluation in Boxers with arrhythmogenic right ventricular cardiomyopathy. Am} Vet Res 2007;68:1338- 1341.
93. Meurs KM, Mauceli E, Acland G, et al. Genome-wide association identifies a mutation for arrhythmogenic right ventricular cardiomyopathy in the Boxer dog. In: ACVIM Forum, Montreal, Canada 2009.
94. Meurs KM, Mauceli E, Lahmers S, et al. Genome-wide association identifies a deletion in the 3' untranslated region of Striatin in a canine model or arrhythmogenic right ventricular cardiomyopathy. Hum Genet 2010;128:315-324.
95. Santilli RA, Bontempi LV, Perego M, et al. Outflow tract segmental arrhythmogenic right ventricular cardiomyopathy in an English Bulldog. J Vet Cardiol 2009;11:47-51.
96. Fox P, Maron B, Basso C, et al. Spontaneously occurring arrhythmogenic right ventricular cardiomyopathy in the domestic cat: A new animal model similar to the human disease. Circulation 2000;102:1863-1870.
97. Harvey AM, Battersby IA, Faena M, et al. Arrhythmogenic right ventricular cardiomyopathy in two cats. J Small Anim Pract 2005;46:151-156.
98. Thomason JD, Kraus MS, Surdyk KK, et al. Bradycardia-associated syncope in 7 Boxers with ventricular tachycardia (2002-2005). J Vet Intern Med 2008;22:931- 936.
99. Meurs K, Spier A, Wright N, et al. Use of ambulatory electrocardiography for detection of ventricular premature complexes in healthy dogs. J Am Vet Med Assoc 2001;218:1291-1292.
100. Heine R, Indrebo A, Kvart C, et al. Prevalence of murmurs consistent with aortic stenosis among boxer dogs in Norway and Sweden. Vet Rec 2000;147:152- 156.
101. Koplitz S, Meurs KM, Spier AW, et al. Aortic ejection velocity in healthy Boxers with soft cardiac murmurs and Boxers without cardiac murmurs: 201 cases (1997- 2001). J Am Vet Med Assoc 2003;222:770-774.
101 102. Chetboul V, Trolle JM, Nicolle A, et al. Congenital heart diseases in the boxer dog: A retrospective study of 105 cases (1998-2005). J Vet Med A Physiol Pathol Clin Med 2006;53:346-351.
103. Kraus MS, Moi'se NS, Rishniw M, et al. Morphology of ventricular arrhythmias in the boxer as measured by 12-lead electrocardiography with pace-mapping comparison. J Vet Intern Med 2002;16:153-158.
104. Meurs K, Spier A, Wright N, et al. Comparison of in-hospital versus 24-hour ambulatory electrocardiography for detection of ventricular premature complexes in mature Boxers. J Am Vet Med Assoc 2001;218:222-224.
105. Spier A, Meurs K, Muir W, et al. Correlation of QT dispersion with indices used to evaluate the severity of familial ventricular arrhythmias in Boxers. Am J Vet Res 2001;62:1481-1485.
106. Toivonen L. Spontaneous variability in the frequency of ventricular premature complexes over prolonged intervals and implications for antiarrhythmic treatment. Am J Cardiol 1987;60:608-612.
107. Spier A, Meurs K. Evaluation of spontaneous variability in the frequency of ventricular arrhythmias in Boxers with arrhythmogenic right ventricular cardiomyopathy. J Am Vet Med Assoc 2004;224:538-541.
108. Lown B, Calvert A, Armington R, et al. Monitoring for serious arrhythmias and high risk of sudden death. Circulation 1975;52.
109. Spier A, Meurs K. Assessment of heart rate variability in Boxers with arrhythmogenic right ventricular cardiomyopathy. J Am Vet Med Assoc 2004;224:534-537.
110. Scansen BA, Meurs KM, Spier AW, et al. Temporal variability of ventricular arrhythmias in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy. J Vet Intern Med 2009;23:1020-1024.
111. Calvert CA, Jacobs GJ, Kraus M, et al. Signal-averaged electrocardiograms in normal Doberman pinschers. J Vet Intern Med 1998;12:355-364.
102 112. Bernadic M, Hubka P, Slavkovsky P, et al. High resolution electrocardiography in healthy dogs: time domain parameters and comparison of the non-stationary (Wigner distribution) versus standard stationary frequency domain analysis methods. Physiol Res 2005;54:477-484.
113. Calvert CA, Kraus M, Jacobs G, et al. Possible late potentials in 4 dogs with sustained ventricular tachycardia. J Vet Intern Med 1998;12:96-102.
114. Boujon CE, Amberger CN. Arrhythmogenic Right Ventricular Cardiomyopathy CARVC) in a Boxer. J Vet Cardiol 2003;5:35-41.
115. Baumwart RD, Meurs KM, Atkins CE, et al. Clinical, echocardiographic, and electrocardiographic abnormalities in Boxers with cardiomyopathy and left ventricular systolic dysfunction: 48 cases (1985-2003).} Am Vet Med Assoc 2005;226:1102-1104.
116. Baumwart RD, Meurs KM. An index of myocardial performance applied to the right ventricle of Boxers with arrhythmogenic right ventricular cardiomyopathy. Am J Vet Res 2008;69:1029-1033.
117. Teshima K, Asano K, Iwanaga K, et al. Evaluation of right ventricular Tei index (index of myocardial performance) in healthy dogs and dogs with tricuspid regurgitation. J Vet Med Sci 2006;68:1307.
118. Baumwart R, Meurs K, Bonagura J. Tei index of myocardial performance applied to the right ventricle in normal dogs. J Vet Intern Med 2005;19:828-832.
119. Boswood A. Biomarkers in cardiovascular disease: beyond natriuretic peptides. J Vet Cardiol 2009;11 Suppl l:S23-32.
120. Gupta S, de Lemos J. Use and misuse of cardiac troponins in clinical practice. Prog Cardiovasc Dis 2007;50:151-165.
121. Parmacek MS, Solaro RJ. Biology of the troponin complex in cardiac myocytes. Prog Cardiovasc Dis 2004;47:159-176.
122. Spratt DP, Mellanby RJ, Drury N, et al. Cardiac troponin I: evaluation I of a biomarker for the diagnosis of heart disease in the dog. J Small Anim Pract 2005;46:139-145.
103 123. Ljungvall I, Hoglund K, Tidholm A, et al. Cardiac troponin I is associated with severity of myxomatous mitral valve disease, age, and C-reactive protein in dogs. J Vet Intern Med 2009.
124. Baumwart RD, Orvalho J, Meurs KM. Evaluation of serum cardiac troponin I concentration in Boxers with arrhythmogenic right ventricular cardiomyopathy. Am J Vet Res 2007;68:524-528.
125. Baumwart RD, Meurs KM. Assessment of plasma brain natriuretic peptide concentration in Boxers with arrhythmogenic right ventricular cardiomyopathy. Am J Vet Res 2005;66:2086-2089.
126. Matsuo K, Nishikimi T, Yutani C, et al. Diagnostic value of plasma levels of brain natriuretic peptide in arrhythmogenic right ventricular dysplasia. Circulation 1998;98:2433-2440.
127. Meurs KM, Spier AW, Wright NA, et al. Comparison of the effects of four antiarrhythmic treatments for familial ventricular arrhythmias in Boxers. J Am Vet Med Assoc 2002;221:522-527.
128. Prosek R, Estrada A, Adin D. Comparison of sotalol and mexiletine versus stand alone sotalol in treatment of Boxer dogs with ventricular arrhythmias. In: 24th Annual American College of Veterinary Internal Medicine Forum, Louisville, KY 2006.
129. Smith CE, Freeman LM, Rush JE, et al. Omega-3 fatty acids in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy. J Vet Intern Med 2007;21:265- 273.
130. Nelson OL, Lahmers S, Schneider T, et al. The use of an implantable cardioverter defibrillator in a Boxer dog to control clinical signs of arrhythmogenic right ventricular cardiomyopathy. J Vet Intern Med 2006;20:1232-1237.
131. Keene BW, Panciera D, Atkins CE, et al. Myocardial L-carnitine deficiency in a family of dogs with dilated cardiomyopathy. J Am Vet Med Assoc 1991;198:647-650.
132. Dittoe N, Stultz D, Schwartz BP, et al. Quantitative left ventricular systolic function: from chamber to myocardium. Crit Care Med 2007;35:S330-339.
104 133. Oh J, Seward J, Tajik A. Assessment of Systolic Function and Quantification of Cardiac Chambers. In: The Echo Manual, 3rd ed. Philadelphia: Lippincott Williams &Wilkins; 2007:109-153.
134. Wess G, Killich M, Hartmann K. Comparison of pulsed wave and color Doppler myocardial velocity imaging in healthy dogs.} Vet Intern Med 2010.
135. Teshima K, Asano K, Sasaki Y, et al. Assessment of left ventricular function using pulsed tissue Doppler imaging in healthy dogs and dogs with spontaneous mitral regurgitation. J Vet Med Sci 2005;67:1207-1215.
136. Hori Y, Sato S, Hoshi F, et al. Assessment of longitudinal tissue Doppler imaging of the left ventricular septum and free wall as an indicator of left ventricular systolic function in dogs. Am J Vet Res 2007;68:1051-1057.
137. Chetboul V, Gouni V, Sampedrano CC, et al. Assessment of regional systolic and diastolic myocardial function using tissue Doppler and strain imaging in dogs with dilated cardiomyopathy. J Vet Intern Med 2008;21:719-730.
138. Chetboul V, al. e. Tissue Doppler assessment of diastolic and systolic alterations of radial and longitudinal left ventricular motions in Golden Retrievers during the preclinical phase of cardiomyopathy associated with muscular dystrophy. Am J Vet Res 2004;65:1335-1341.
139. Hori Y, Kano T, Hoshi F, et al. Relationship between tissue Doppler-derived RV systolic function and invasive hemodynamic measurements. Am J Physiol Heart Circ Physiol 2007;293:H120-125.
140. Oh J, Seward J, Tajik A. Assessment of Diastolic Function and Diastolic Heart Failure. In: The Echo Manual, 3rd ed. Philadelphia: Lippincott Williams & Wilkins; 2007:120-142.
141. Hoit BD. Left ventricular diastolic function. Crit Care Med 2007;35:S340-347.
142. O'Sullivan M, O'Grady M, Minors S. Assessment of diastolic function by Doppler echocardiography in normal Doberman Pinschers and Doberman Pinschers with dilated cardiomyopathy. J Vet Intern Med 2007;21:81-91.
105 143. Oyama M, Sisson D, Bulmer B, et al. Echocardiographic estimation of mean left atrial pressure in a canine model of acute mitral valve insufficiency. J Vet Intern Med 2004;18:667-672.
106 Chapter 2: Assessment of Systolic and Diastolic Function via Tissue Doppler Echocardiography in Boxers Affected with ARVC Compared to Normal Boxers and Non-Boxer Control Dogs
2.1 Introduction:
Arrhythmogenic right ventricular cardiomyopathy, or ARVC, is a common cause of cardiac disease and sudden death in the Boxer dog. It is a disorder characterized primarily by electrical instability of the right ventricular myocardium, manifested in the form of tachyarrhythmias such as premature ventricular contractions [VPCs], sustained and paroxysmal ventricular tachycardia (VT) and ventricular fibrillation (VF). It is these tachyarrhythmias which result in the clinical presentations of syncope, collapse, weakness and sudden death observed in affected dogs.1 The clinical presentation can vary from an incidental arrhythmia in an otherwise normal dog, to syncope, or to sudden death as the first clinical sign. A rare subset of dogs may also develop congestive heart failure related to ARVC.2 The abnormal electrophysiological properties of the right ventricle in affected dogs occur as a result of histopathological changes of the cardiac myocytes. In most affected dogs, the condition is characterized by a progressive atrophy of the cardiac myocytes leading to fibro-fatty or fatty tissue replacement.3 The current thought is that mutations within the genes which encode for the cardiac intercalated disks and gap junctions (collectively referred to as the desmosome), result in alterations of mechanical and electrical cell to cell interactions, resulting in myocyte death and infiltration with fibrous and fatty tissue.3 Initially these pathological changes occur in the right ventricle and then progress to affect the left ventricle late in the disease process. These alterations in the right ventricular myocardium create areas of
107 differing excitability and disparity in conduction, creating the substrate for reentrant circuits which provides the predominant mechanism for the tachyarrhythmias observed.4 Despite these changes affecting the RV, on echocardiographic examination the hearts of affected dogs often appear morphologically normal.
Besides the Boxer, ARVC has been noted in other canine breeds as well as cats and humans.1 As the disease in the Boxer closely mirrors what is observed in people, parallels between the two species can be drawn, thus leading to an interest in studying the Boxer as a model for human disease. The prevalence in people is about 1 in 5000 individuals, and is responsible for as many as 20% of the sudden deaths which occur in young people.5 In both the Boxer and in people, ARVC has been shown to be a familial disease. In people, it is typically inherited as an autosomal dominant trait with incomplete penetrance and variable expression, though an autosomal recessive form has been described.5 Mutations at a dozen chromosomal loci encoding a dozen different genes have been discovered to date in people.4 As previously noted, a majority of these genes encode for components of the desmosome. In Boxers, an autosomal dominant form of inheritance is suspected, and 4 desmosomal genes have been investigated to date with no mutations observed in those particular genes encoding for the desmosome.3'6
Recently, a deletion in the 3' untranslated region of the striatin gene has been identified in Boxers affected by ARVC. Dogs homozygous for the deletion were more severely ARVC affected, as assessed with ambulatory ECG. Striatin appears to colocalize with several desmosomal proteins, including desmoplakin, plakophilin-2
108 and plakoglobin, thus potentially explaining how a mutation in this particular gene could result in the ARVC phenotype in the Boxer.7
The diagnosis of ARVC in people relies on an algorithm which incorporates familial history, arrhythmia and electrical abnormalities (as assessed with ECG,
Holter monitors and signal-averaged ECG [SAECG]), cardiac structural and functional abnormalities (echocardiography, angiography and cardiac MRI) and myocardial tissue characterization (via endomyocardial biopsy or post-mortem examination).5 SAECG is a diagnostic tool which is frequently employed when evaluating people affected with ARVC, and its use has been reported in affected
Boxers.8-9 It is a signal processing technique which identifies the presence of late potentials, which are low voltage signals that occur at the end of the QRS complex and are often hidden in baseline noise. These late potentials represent regions of delayed conduction and have been shown to be an indicator for the increased risk of development of arrhythmias, increased severity of disease, and an increased risk of sudden death. Therefore in people, it is often used as a method of risk stratification in terms of identifying those individuals who are at higher risk for an adverse outcome such as sudden death and congestive heart failure.8 Additionally, the identification of structural abnormalities or systolic/diastolic dysfunction plays an adjunctive role in the diagnosis in people. Abnormalities may include RV dilation, focal RV aneurysms, hypokinesis or dyskinesis of RV wall segments and global systolic and diastolic dysfunction.15 These abnormalities are often identified on cardiac MRI as well as standard two-dimensional echocardiography. A sensitive method to identify subtle systolic and diastolic dysfunction with echocardiography
109 is through the use of tissue Doppler imaging (TDI). TDI is a Doppler technique which allows quantification of the speed of wall motion, and has been shown to be an effective method to evaluate regional or global systolic and diastolic ventricular function.10 In people with ARVC, both reduced RV and LV systolic and diastolic myocardial velocities have been observed compared to normal controls.11 These changes have even been observed in those individuals without obvious RV or LV dilation or systolic dysfunction as assessed via two-dimensional echocardiographic measures, therefore supporting that TDI may identify cardiac dysfunction before overt structural changes are noted.1112
In the Boxer dog, diagnosis of the condition relies almost exclusively on the presence of historical syncopal events as well as ECG and ambulatory Holter findings. However, other diagnostic modalities such as SAECG and two-dimensional echocardiography have been reported in a few case series to date.1315 Compared to the diagnosis in people, there are no clear diagnostic criteria. It is generally accepted that Boxers with greater than 1000 right-sided VPCs in a 24-hour period are affected, while non-affected Boxers should have fewer than 24 VPCs in a 24-hour period.16 The challenge of Holter interpretation lies with VPC frequency occurring between these ends of the spectrum, which is compounded by the fact that day-to day variability in VPC frequency of up to 80% has been observed when evaluating consecutive 24-hour Holter analyses.1718 Additional clinical challenges include the assessment of disease severity and risk of sudden death or congestive heart failure in affected dogs. A recent veterinary study using SAECG in the Boxer correctly identified 14 of 15 dogs (based on the presence of late potentials) who went on to
110 have a cardiac related death [either sudden death or death due to congestive heart failure). It was also found that Boxers with higher frequencies of VPCs and with LV systolic dysfunction were found to have more abnormal SAECG findings, supporting that late potentials paralleled other indices of disease severity. However, the same study identified 13 of 79 clinically normal dogs as also having late potentials, thus affecting the specificity of this non-invasive diagnostic test.919
In contrast to its diagnostic utility in people, echocardiography has been of limited value to date in the diagnosis of disease in the Boxer as myocardial function appears to be typically preserved in affected individuals.20 This is likely related to the fact that the majority of Boxers with ARVC reported have subjectively normal heart structure, which may perpetuate the sense that they are functionally normal as well. However, no quantitative assessments and subsequent guidelines of RV systolic and diastolic function have been reported in affected Boxers through the use of echocardiography. This may be related to the difficulty in adequately assessing RV size and function with two-dimensional echocardiography due to its unique geometry (crescent shape with an irregular endocardial surface), as well as the limited number of imaging planes which are available for its evaluation.21
Tissue Doppler imaging (TDI) is a fairly recent development in echocardiography which has demonstrated utility in detecting and quantifying myocardial function.22 This modality evaluates myocardial motion using pulsed- wave Doppler, providing a means to quantify global as well as regional ventricular function. Assessment of global ventricular function is achieved through measurement of both mitral and tricuspid annular velocities. In people, it has been
111 shown that there is a positive correlation between left ventricular ejection fraction and peak systolic mitral annular velocity.10 Tricuspid annular velocities have also shown good correlation with systolic annular velocity and right ventricular ejection fraction, as assessed by radionuclide ventriculography.10'2324 In veterinary medicine, only one study has investigated TDI of the tricuspid annulus - this study was undertaken by Chetboul in 2005 in normal awake patients.25 Various studies have looked at mitral annular velocities in the setting of both normal and diseased canine hearts, and have revealed good correlation with other indices of systolic and diastolic function.2627 There have been no studies evaluating tricuspid annular velocities in the diseased right heart, such as in ARVC, in veterinary patients.
Whereas in the human literature, there have been a variety of studies which have evaluated the use of TDI to assess cardiac function in those patients diagnosed with
ARVC. These studies revealed a reduction in both systolic and diastolic function when evaluating both the tricuspid and mitral annular velocities.11
Given the histological changes of ARVC in Boxers, as well as the understanding of the disease as it occurs in people, subclinical alterations in systolic and diastolic function may be present in affected dogs, just as they are in people.
Furthermore, some Boxers do develop RV or LV enlargement and congestive heart failure.2 It is suggested that those with the fibrofatty form are prone to the development of RV or LV wall thinning, chamber enlargement and systolic dysfunction1, however to date there is no known easy clinical means to identify dogs at future risk of structural cardiac changes and congestive heart failure. Therefore, given that TDI is a non-invasive and reliable method to quantify both RV and LV
112 systolic and diastolic function in dogs, it may be of use in Boxers with ARVC. To date, no studies have reported TDI findings in Boxers afflicted with ARVC.
113 2.2: Statement of Objectives and Hypotheses
Objectives:
1. Determine whether systolic tricuspid or mitral valve annular velocities by TDI are reduced in Boxers with ARVC, suggesting global RV or LV systolic dysfunction respectively, compared to normal non-affected Boxers and normal non-Boxer dogs.
2. Determine whether diastolic tricuspid or mitral valve annular velocities by TDI are reduced in Boxers with ARVC, suggesting global RV or LV diastolic dysfunction respectively, compared to normal non-affected Boxers and normal non-Boxer dogs.
3. Determine whether reductions in systolic and diastolic tricuspid and mitral annular velocities are detected in Boxers with lesser degrees of ventricular arrhythmia (a degree that is suspicious for but not definitely diagnostic for ARVC), compared to normal non-affected Boxers and normal non-Boxer dogs.
4. Determine whether ARVC-affected Boxers have abnormal SAECG findings compared to normal Boxer dog and normal non-Boxer dogs.
5. Determine whether tricuspid and mitral annular velocities correlate with the degree of ventricular arrhythmia as assessed by ambulatory ECG monitoring
(Holter) or the potential for ventricular arrhythmia as assessed by signal averaged electrocardiogram (SAECG) in Boxers.
114 Hypotheses:
1. Boxer dogs affected with ARVC will have abnormal cardiac function (systolic, diastolic or both) as assessed with TDI compared to non-affected normal Boxer dogs and non-Boxer control dogs.
2. More severely ARVC affected Boxer dogs will have greater reductions in cardiac function (systolic, diastolic, or both) as assessed with TDI as well as more abnormal
SAECG as compared to non-affected normal Boxer dogs and non-Boxer control dogs.
3. Boxer dogs affected with ARVC will have the presence of late potentials on SAECG compared to non-affected normal Boxer dogs and non-Boxer control dogs.
4. There will be correlations between the degree of arrhythmia (the frequency of ventricular premature contractions, the presence of late potentials) and TDI variables.
115 2.3: Materials and Methods
This prospective study was approved by the Animal Care Committee of the
University of Guelph with an intended study population of 75 dogs, and informed consent was obtained before enrolling dogs into the study from all participating owners.
2.3.1 Study Design
In this prospective study, four groups of Boxers and one group of healthy non-Boxer dogs were studied. The Boxers were classified into groups based on the number of ventricular premature contractions (VPCs) present during a twenty-four hour ambulatory Holter analysis: 15 dogs having greater than 1000 VPCs in 24 hours (group 1), 10 dogshaving between 200-999 VPCs in 24 hours [group 2], 15 dogshaving between 25-199 VPCs in 24 hours (group 3) and 15 dogs having fewer than 24 VPCs in 24 hours (group 4). In the non-Boxer group, 15 healthy dogs of various breeds were age matched to the Boxer groups and had fewer than 24 VPCs in 24 hours (group 5). Dogs were age matched by grouping the Boxers into four age groups (aged 1-3, 3-5, 5-7 and 7-10 years), and using the percentage of dogs in each age group to determine how many non-Boxer normal dogs should fill each age category. All dogs underwent the same evaluation: cardiovascular physical examination with history, transthoracic echocardiogram to evaluate cardiac structure and function, blood pressure, an electrocardiogram (ECG), signal averaged electrocardiogram (SAECG), a 24 hour Holter recording, and blood sampling for measurement of packed cell volume, total solids, a complete biochemical profile and analysis of serum troponin (CTnl). Initially at the onset of the study, all Boxers were
116 evaluated first with physical examination, echocardiogram, ECG, SAECG, blood pressure, and bloodwork. The 24-hour Holter was then applied at the time of discharge from the hospital and once analyzed, the Boxer dogs were classified into one of the four groups. This process was continued until one of the Boxer groups had fifteen participants. When this occurred, eligible Boxers were then pre- screened with the Holter recording to determine their candidacy [based on degree of ventricular arrhythmia) for enrolment in the remaining groups. Qualifying
Boxers were then evaluated in hospital as described above within five weeks from the date of performing the Holter examination. Once data collection was complete on the four Boxer groups, age matching was performed to select fifteen healthy non-
Boxer dogs to act as controls. Eligible non-Boxer dogs were pre-screened with the
Holter recording to ensure they had fewer than 24 VPCs in 24 hours, and once qualified, they then were assessed in the same fashion as the Boxers.
2.3.2 Enrollment Criteria
Enrollment to the four Boxer groups was restricted to purebred Boxer dogs, whereas enrollment to the non-Boxer group was restricted to dogs of other breeds who were similar in size to Boxers. All dogs were required to be older than one year of age and not older than ten years of age. Dogs with a history of hypothyroidism were allowed to participate with evidence of well-controlled disease
[documentation of a normal T4 level).
117 2.3.3 Exclusion Criteria
Cardiac criteria precluding study enrollment included concomitant congenital cardiac disease such as subaortic stenosis (evidence of an aortic velocity of > 2.4 m/s with continuous wave Doppler from the left parasternal apical window or evidence of a moderate to severe left basilar systolic murmur [grade 4/6 or greater]], or evidence of acquired cardiac disease such as degenerative valvular disease. Dogs currently receiving any anti-arrhythmic medication or oral steroid therapy were also excluded from participation. Additionally, dogs suffering from concurrent renal disease (as defined by a serum creatinine concentration of > 150 umol/L and/or urea > 9.0 mmol/L), hepatic disease (as defined by a serum alanine transferase concentration > 200 U/L and/or alkaline phosphatase concentration >
300 U/L], pancreatic disease (as defined by a serum amylase concentration > 2000
U/L and/or serum lipase concentration > 2000 U/L), anemia (packed cell volume <
37%), systemic hypertension (average systolic blood pressure > 160 mmHg) or known endocrinopathies such as diabetes mellitus or hyperadrenocorticism were excluded from study participation. Dogs of breeds known to be at high risk for cardiomyopathy such as Doberman Pinschers or mixed breed dogs that were believed to include Boxer or another breed at high risk for cardiomyopathy were excluded from participation as a non-Boxer control dog.
2.3.4 Patients
All dogs evaluated were client-owned and were older than one year of age and younger than 10 years of age. The dogs were evaluated between August 2008
118 and December 2009. The Boxer dogs were recruited through the assistance of
Boxer associations, Boxer breeders, local veterinary hospitals and word of mouth.
The non-Boxer dogs were recruited from the staff and students of the Ontario
Veterinary College.
Interested Boxer owners were initially interviewed by telephone and the eligibility of the dogs for participation was assessed through a questionnaire (see
Appendix 3.1), assessing for any exclusion factors. Boxers were excluded on the basis of the following: younger than one year of age, older than 10 years of age, dogs receiving any concurrent anti-arrhythmic medication or oral steroid therapy, dogs currently being treated for any systemic illness (such as liver, renal or pancreatic disease for example), or dogs with moderate to severe heart murmurs
(grade 4/6 or greater) as reported by their primary care veterinarian. Dogs with moderate to severe murmurs were excluded on the basis of presumed moderate to severe subaortic stenosis, a congenital cardiac disorder common in the Boxer breed, or presumed presence of other congenital or acquired cardiac disease other than
ARVC. If a Boxer qualified on the basis of the telephone questionnaire, they were either evaluated at the hospital with a Holter recording at discharge (as performed until one of the Boxer groups had 15 participants) or they were pre-screened with the Holter monitor to determine if they had sufficient ventricular arrhythmia to meet candidacy for enrolment in one of the remaining open groups.
119 2.3.5 Data Acquisition
The evaluation of eligible study participants at the Ontario Veterinary College
consisted of a pertinent history and a complete physical examination including body
weight. A transthoracic echocardiogram was performed by one operator (JO) using
either a 3 MHz or 5 MHz probe (depending on size of the patient) and the Vivid 7
Dimension echocardiographic system (General Electric, Vingmed Ultrasound,
Horten, Norway). For this echocardiogram, dogs were conscious, unsedated, and
manually restrained in both right and left lateral recumbency. Owners were
encouraged to provide restraint for the duration of the examination, and when
unavailable, technical assistance was provided. All standard imaging planes were
performed as recommended by the Echocardiography Committee of the Specialty of
Cardiology, American College of Veterinary Internal Medicine.28 The ECG leads of
the ultrasound system were attached for continuous recording of heart rate and
rhythm. Initially the dogs were evaluated in left lateral recumbency using the left
parasternal caudal apical window, with colour Doppler applied to blood flow across
each valve assessing for valvular insufficiencies. Valvular insufficiency, if present, was subjectively graded as mild, moderate or severe in order to assess if
degenerative valvular disease (an exclusion factor) was present. Dogs with
moderate to severe regurgitation were considered to be suspicious for valvular
disease and were to be excluded from the study based on this finding. Aortic velocity was recorded at a sweep speed of 100 mm/s using continuous wave
Doppler from this window (with optimization of the left ventricular outflow tract).
Left ventricular wall motion at the mitral valve annulus from both the medial
120 (interventricular septum] and lateral (free wall) walls was measured using pulsed wave TDI. The sample volume and cursor were placed at the margin of the mitral annulus (medial and lateral) in an effort to be aligned as parallel as possible to the longitudinal axis of LV wall motion, with the sample volume set at 4.9 mm. Three cine-loops with re-acquisition of the image for each and each containing at least eight cardiac cycles were obtained for each measurement, with a maximal sweep speed of 200 mm/s. At the time of measurement, Doppler gain was minimized to facilitate identification of peak systolic myocardial velocity (Sm), peak early diastolic myocardial velocity (Era) and peak late diastolic myocardial velocity (Am) (see figure
4.1). The ratio of peak early to late diastolic velocity (Em/Am) was calculated. The average of three measures, one from each of the three cine-loops stored, was used for each variable in the analysis. Right ventricular wall motion at the lateral tricuspid valve annulus was measured (Sm, Em, Am, Em/Am) using pulsed wave TDI as described above for left ventricular wall motion. The average of three measures, one from each of the three cine-loops stored, was used for each variable in the analysis. The dogs were then positioned in right lateral recumbency and a complete two-dimensional examination was performed with colour Doppler applied to each valve. Valvular insufficiencies, if present, were reported. Measurement of pulmonic velocity was obtained from the right parasternal short axis view using continuous wave Doppler at a sweep speed of 100 mm/s. A two-dimensional guided M-mode from the right parasternal long axis view (with left ventricular inflow and outflow tracts optimized) was then recorded at a sweep speed of 100-200 mm/s with at least eight cardiac cycles. Three such M-modes were obtained with re-acquisition of
121 the image for each. The collected M-modes were used to measure left ventricular internal dimension in diastole (LVIDd), and left ventricular internal dimension in systole (LVIDs). Fractional shortening percentage (FS%) was calculated as [(LVIDd-
LVIDs)/LVIDd] x 100%. The average of three measures from the three M-modes was used for each parameter.
Figure 4.1: Pulsed-wave tissue Doppler Recording from a dog measured at the lateral mitral annulus with the systolic (Sm), early diastolic (Em) and late diastolic (Am) velocities labeled with a simultaneous electrocardiogram below.
All echocardiographic measurements were performed on stored images using either the echocardiographic machine or an offline computer station (EchoPac
PC, Vivid 7, GE Vingmed Ultrasound, Horten, Norway). One ideal cardiac cycle was selected from each cine-loop stored and used to measure the TDI (LV and RV) and
M-mode variables.
122 A nine-lead ECG (leads I, II, III, aVF, aVL, aVR, VI, V2 and V3) was performed with the dogs manually restrained in right lateral recumbency. Electrode plates moistened with alcohol and fastened by rubber straps were used to facilitate good skin-plate contact. Following the routine ECG, a SAECG (General Electric MAC 5500,
Marquette 12SL Hi-Res ECG analysis, Milwaukee, Wisconsin) was performed with the patient in continued right lateral recumbency. Additional electrodes were placed per the Frank orthogonal lead system as recommended by the ECG manufacturer (see figure 4.2). This lead system requires seven electrodes with five electrodes placed in the horizontal (transverse) plane around the thorax at the approximate level of the fifth intercostal space: one in the right mid-axilla (I), one along the sternum (E), one in the left mid-axilla (C), one at the level of dorsal midline
(M), and one halfway between dorsal midline and left mid-axilla (A).
Figure 4.2 : Placement of electrodes on the Boxer per the Frank orthogonal lead system.
123 Figure 4.3: Photograph of participating Boxer in right lateral recumbency with ECG electrodes placed for the acquisition of the SAECG.
These electrodes were attached to plates that were attached to a one-inch wide rubber band that wrapped around the thorax at the approximate level of the fifth intercostal space. The remaining two electrodes were placed on the left hindlimb and on the side of the neck (H, ground electrode), respectively (see figure 4.2,4.3).
The GE MAC5500 was equipped with SAECG software to detect ventricular late potentials. This was achieved by aligning approximately 250 similarly shaped sinus beats in the signal averaging process. The first step in the signal averaging process was the collection of approximately 8 seconds of X,Y, and Z lead data by the ECG system. The beat within this collected data that appeared to be the most normally conducted (assessed as having the shortest QRS duration) was marked as the seed
124 beat. All subsequent beats within the 8-second strip which were shaped like the seed beat were averaged to form the template beat (see figure 4.4). The template report was reviewed by the operator (JO) to ensure a sinus beat was selected as the seed beat as opposed to a VPC prior to beginning the averaging process. The averaging process was then started by acquiring approximately 250 beats that matched the template beat (within 3%). In this mode (known as Hi-Res), data is sampled from X, Y and Z leads at 1000 samples per second. Identification of the QRS and fast Fourier transform (FFT) filtering was applied to the template beat as well as all incoming beats prior to their insertion within the average. FFT allows conversion of the QRS information from the time domain to the frequency domain.
This process allows for proper alignment of each beat prior to insertion into the average, as well as the accentuation of late potential information present in the terminal QRS with the use of band-pass filtering which recovers the high frequency low amplitude information present in the terminal QRS. The final report generated by the Hi-Res software through time domain analysis highlighted and quantified late potential activity, if present, on a vector magnitude signal. The vector magnitude
(VM) plot contains information from all three leads as follows: VM = V(X2 + Y2 +
Z2).29 This yielded the QRS duration both filtered and unfiltered, high frequency low amplitude (HFLA) duration (the duration of time the voltage of the terminal QRS remains below 40 uV prior to its termination), and the root-mean-square (RMS) voltage of the terminal 40 ms of the QRS. A noise level was also reported, which represented the average baseline noise measured in a short segment of the ST region. Attempts were made to ensure that the noise level remained below the
125 desired level of 1 uV which was achievable in most patients. In some patients however, noise levels were greater than 1 uV and despite repeating the averaging process while attempting to optimize skin-electrode plate contact and patient motion, noise levels in some patients remained greater than 1 uV.
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127 Following the acquisition of the SAECG data, patients were left with their owners in order to reduce anxiety. Once patients were calm, systolic blood pressure was measured in right lateral recumbency from the right forelimb using an ultrasonic Doppler flow detector [Parks Medical Electronics, Aloha, OR). The palmar aspect of the paw was clipped of hair and ultrasound gel applied to facilitate good contact between the 8 MHz transducer and the artery. An appropriate cuff was selected based on the size of the forelimb of the patient, and three consecutive systolic pressures measured with a sphygmomanometer were obtained.
Once systolic blood pressure was obtained, the dogs were kept in right lateral recumbency and jugular venipuncture was performed using a Vacutainer™ system [Becton-Dickinson, Franklin Lakes, NJ) to obtain approximately 20 cc of venous blood. Blood was collected into 2 x 6 cc purple top K2 EDTA [10.8 mg)
Vacutainer tubes [Becton-Dickinson, Franklin Lakes, NJ). These tubes were inverted gently to mix the samples, and they were promptly placed into cold refrigeration at
4°C until the time of transportation to laboratory at the Hospital for Sick Children in
Toronto for a separate genetic analyses study, the results of which are not reported here. Blood was also collected in a 4 cc red top serum tube [no additives), a 4 cc green top tube [containing sodium heparin, 68 USP units), and two heparinized microHCT capillary tubes for packed cell volume [PCV) and total solid (TS) analyses.
The capillary tubes were centrifuged for 3 minutes [Readacrit, Becton Dickinson,
Sparks, MD) at 7200 RPM, and the PCV determined from a micro-hematocrit capillary tube reader. The separated serum was then placed on a refractometer to determine total solid concentration (g/dL). The red top tube was allowed to clot
128 (approximately ten minutes) before centrifugation (Horizon Easy-Spin 12, Drucker
Co., Philipsburg, PA) at 3150 RPM for five minutes. The separated serum was collected and submitted to the Animal Health Laboratory (AHL) at the University of
Guelph for biochemical profile analysis (albumin, alkaline phosphatase [ALP], alanine aminotransferase [ALT], aspartate aminotransferase [AST], amylase, conjugated bilirubin, total bilirubin, calcium, cholesterol, creatine kinase [CK], creatinine, globulin, glutamyltransferase [GGT], glucose, lipase, sodium, potassium, chloride, magnesium, total protein and urea). The green top tube was immediately centrifuged following collection (Horizon Easy-Spin 12, Drucker Co., Philipsburg,
PA) at 3150 RPM for five minutes. The separated plasma was collected and placed in 3 cc cryovials (Cryovials, Canemco Inc., Canton de Gore, QC) and immediately placed into a -70°C freezer for storage until transportation to an off-site laboratory for analysis of cardiac troponin-I (cTnl). Once all dogs had been collected, the stored samples were sent to Guelph General Hospital for batch analysis of cTnl concentrations using the AccuTnl assay (Beckman Coulter, Fullerton, CA). Fifteen months was the longest period a sample was stored, and all samples were thawed only once at the time of analysis.
In the initial stages of the project prior to filling one of the Boxer groups with
15 participants, a 3 channel ambulatory ECG Holter monitor (Rozinn Electronics,
Genesis Medical Corporation, Surrey, BC) was placed following the in-hospital evaluation and the dog was sent home in order to collect data from the dog in its normal environment. Owners were provided with a Holter activity log to record any cardiac events, exercise or any other activity of concern. Owners were encouraged
129 to maintain a normal routine for each dog while wearing the Holter. Once the Holter was worn for a full 24 hours, owners removed the Holter and promptly returned the unit for analysis at the Ontario Veterinary College. The tapes were reviewed and analysed by one of two trained operators (CW, JO) using the Pathfinder Digital software (version 8.701, Delmar Reynolds Medical Ltd., Edinburgh, UK). All final reports were reviewed by JO. Any recordings that did not have at least 16 hours of readable data were excluded. The total number of VPCs, the approximate percentage of right ventricular origin VPCs (needed a minimum of 80% of the VPCs to be of right ventricular origin) and the number of VPCs per hour taking into account total artifact seconds were tabulated. Based on the total number of VPCs, dogs were then placed into one of the four Boxer groups. If a dog failed to have 16 hours of readable Holter data, the Holter was repeated as soon as possible such that all Holter exams occurred within the five-week window from the time of evaluation.
Once one Boxer group had fifteen participants (the first group to be filled), the
Holter monitors were sent to interested participants for pre-screening to determine if their dog qualified for enrolment in one of the remaining three groups. If a dog qualified, they were evaluated (as described above) at the Ontario Veterinary
College within five weeks of the Holter exam.
2.3.6 Statistical Analysis
Descriptive statistics were performed on the demographic characteristics of each group and on the variables measured and calculated for each group. Each of the variables obtained was assessed for a significant difference between groups. To
130 assess the ANOVA assumptions, residual analyses were performed. For continuous data, an analysis of residuals was performed for each response variable amongst all groups to assess normality using a Shapiro-Wilk test. If the p-value was >0.05, we concluded that the data was normally distributed. For data that was not normally distributed, a log transformation was performed and a repeat Shapiro-Wilk test was used to determine whether the data was now normally distributed. If the p-value for the Shapiro-Wilk test remained < 0.05, a square root transformation was applied and analysis of the residuals was repeated with the Shapiro-Wilk test. If at this time the data remained non-normal, no additional transformations were attempted. For data that was normal or normal after a transformation, a one-way analysis of variance (ANOVA) was used to assess significant difference between groups.
Descriptive statistics and p-values are reported for these analyses. For variables that yielded a significant result (p-value < 0.05), the means for the groups were then compared using a Tukey-Kramer HSD (honestly significant difference) to determine where the significant difference occurred (between which groups). For data that was not normally distributed or for ordinal and nominal data, the nonparametric
Wilcoxon/Kruskal-Wallis test was used to check for differences across groups. For categorical data, a chi-squared test or Fisher's exact test was used to check for differences in variables across groups. For data that was not normally distributed, a post-hoc nonparametric Mann-Whitney-Wilcoxon test was used to assess significant pairwise differences in variables between groups. Descriptive analyses included determination of the mean, standard deviation, minimum and maximum for each
131 normally distributed variable. For non-normal data, including categorical data, the median, quartile, minimum and maximum are reported for each variable.
In addition more complex analysis was performed to examine for significant difference between groups while adjusting for potential covariates. These covariates included age, weight, gender, and left ventricular outflow tract velocity
(LVOT); interaction terms were also included in this model (age * gender, age * weight, gender * weight, age * gender * weight). A model was fitted using the least squares method with group as a fixed effect and included the interaction terms noted above. We restricted the number of terms in the model to no more than ten at any one time. All models were simplified by first removing the most non-significant term then re-running the model. This process continued until all non-significant terms were removed. Non-significant main effect terms were left in the model if these terms were present in a significant interaction term. Group was left as a fixed effect in the model in order to determine whether the variable of interest was different between groups. The outcome variables of interest included all of the TDI variables measured, as well as other echocardiographic variables of interest (LVID- s, FS%), the SAECG variables (filtered QRS duration, duration of HFLA < 40 uV, and
RMS 40 ms) and troponin I concentration. Each model was refined in a backward stepwise fashion by removing from the model one at a time the most non-significant variable (defined as p-value > 0.05). The significant variables are reported with their associated p-value.
To determine whether a relationship existed between the variables of interest and the frequency of VPCs (VPCs/hr), correlations were performed while
132 attempting to adjust for potential covariates. The correlation analysis was performed using a fitted model using the least squares method with VPCs/hr as an explanatory variable and including the same covariates and terms as used in the above model including the new covariate Boxer yes/no (in an attempt to determine if being a Boxer had an effect on our variables of interest). The response variables of interest included relevant echocardiographic variables (TDI variables, LVID-s, FS%),
SAECG variables and cTnl level. As described above, non-significant covariates were removed from the model one at a time in a backward stepwise fashion, starting with the least significant interactions. A linear fit was applied and the R2 values and the p-value of the analysis of variance are reported.
In an identical fashion, potential correlations between the TDI and SAECG data were investigated. This was performed using the same least squares fit model using the same covariates as noted above. For each of the SAECG explanatory variables (duration of filtered QRS, RMS voltage of the terminal 40 ms, and HFLA <
40 uV] each of the TDI variables was investigated as a response variable, with the
SAECG variable of interest left in the model as a fixed effect. The significant response variables are reported with their associated p-value, as well as the model
R2 value.
All statistical analyses were performed using a statistical analysis software for Macintosh (JMP 8.0.2, SAS Institute Inc., Cary, NC).
133 2.4 Results
Seventy dogs out of the desired 75 met the enrollment criteria for the study and were evaluated, while an additional 103 Boxers were screened via Holter recording but did not qualify. Of the 70 dogs, 55 were purebred Boxers with the following numbers per Boxer group: group 1 (> 1000 VPCs in 24 hrs) had 15 dogs, group 2 (200-999 VPCs in 24 hrs) had 10 dogs, group 3 (25-199 VPCs in 24 hrs) had
15 dogs, and group 4 (< 24 VPCs in 24 hrs) had 15 dogs. The Boxer group with fewer than 24 VPCs in 24 hours met the 15 participant goal first. The Boxer group with between 200 and 999 VPCs in 24 hours only had ten of the fifteen desired dogs due to an inability to find Boxers with the desired degree of arrhythmia with pre- screen Holter recordings within the available time frame. The remaining 15 dogs
(group 5) were non-Boxer breeds including the following: three Golden Retrievers, two Labrador Retrievers, one Standard Poodle, one Coonhound, one Brittany
Spaniel, two Springer Spaniels, one Nova Scotia Duck Tolling Retriever, one
Australian Shepherd, and three mixed breed dogs (two Border Collie crosses and one Labrador Retriever cross). None of the mixed breed dogs were apparently
Boxer cross breeds. Of the 70 dogs, there were 43 female dogs (28 intact), and 27 male dogs (15 intact). Of the 55 evaluated Boxers, only four dogs were receiving concurrent medications: one dog was receiving levothyroxine (Thyrotabs,
Novopharm Animal Health, Toronto, Ontario) for controlled hypothyroidism
(normal serum T4 level), one dog was receiving diethylstilbestrol (Stilbestrol,
Orman Veterinary Supply, Ancaster, Ontario) for urinary incontinence, one dog was receiving meloxicam (Metacam, Boehringer-Ingelheim, Burlington, Ontario) for the
134 treatment of osteo-arthritis, and one dog was receiving both levothyroxine and meloxicam for controlled hypothyroidism (normal serum T4 level) and osteo arthritis respectively. None of the non-Boxer dogs were receiving medications.
Eleven Boxers reported a history of syncope, with five of the dogs having more than one syncopal event. Only five of the Boxers had previous Holter recordings performed. Thirty of the enrolled Boxers had a systolic heart murmur, with no dogs having a murmur grade greater than 3/6. None of the non-Boxer dogs had a history of a heart murmur and none of these dogs had a history of syncope or exercise intolerance. In the non-Boxer group, none of the dogs had more than 4 VPCs present in a 24-hour period (three dogs had one VPC, one dog had two VPCs, and one dog had four VPCs in 24 hours, while the remaining dogs had none).
The sample characteristics and descriptive statistics of the five groups of dogs are presented in Table 4.1. The mean age of the 70 dogs was 4.54 years (range
1.07-9.96 years), and mean weight was 27.16 kg (range 14.9-40.5 kg). When comparing the variables of interest among all five groups of dogs, there was a significant difference in body weight between group 2 and group 5, with the mean body weight in group 2 being higher (30.04 +/- 5.23 kg) than in group 5 (mean weight of 24.24 +/- 7.06 kg). When comparing only Boxers, dogs in group 2 had a higher mean body weight than dogs in group 4 who had a mean weight of 26.01 +/-
2.03 kg. There was no significant difference between groups of dogs in terms of age or average systolic blood pressure.
As expected, there were significant differences between total number of VPCs between the groups since the groups were selected to be different based on number
135 of VPCs. Despite performing both a log transformation and a square root transformation, the residual of the total number of VPCs was not normally distributed. There was a significant difference between all possible pairwise comparisons of groups, both when all groups were examined and when Boxer-only groups were examined. These same differences existed between the groups when analyzing the number of VPCs/hour (Table 4.1).
Troponin I concentrations were available in 68 of 70 dogs. In 2 of the dogs, the troponin assay did not yield a result. A log transformation was required in order to make the distribution of the residuals for the troponin I concentration normal.
When comparing all groups of dogs, there was a significant difference between each of the four groups of Boxers (groups 1-4) and the non-Boxer control group (group
5), with tne Boxer dogs having a higher mean troponin I concentration than the non-
Boxer cohort. When comparing only the Boxer dogs, troponin I concentration differed significantly only between groups 1 and 4, and groups 2 and 4 (Table 4.1).
Therefore, Boxers with more frequent arrhythmia who are presumably more severely affected with ARVC had a higher mean troponin I concentration.
In terms of the signal averaged ECG variables, the ECG analysis software performed the averaging process in 67 of the 70 dogs. In 3 of the dogs (all Boxers), signal averaging was performing on the T wave and not the QRS, and post-hoc analysis was not possible in these dogs. This error occurred early in the data collection portion of the study and may be attributable to the learning curve associated with the operation of the signal averaging ECG software package. There were no significant differences in the three signal averaged variables (filtered QRS
136 duration in ms, duration of high frequency low amplitude signals < 40 uV, and root mean square value in the terminal 40 ms) in the four group and five group analyses
(Table 4.1).
Echocardiographic variables (two-dimensional, Doppler and tissue Doppler
[TDI]) were available for all 70 dogs evaluated. There were no significant differences in the M-mode data (such as LVID in diastole and systole and fractional shortening percentage) between any of the five groups (Table 4.1). The right ventricular outflow tract (RVOT) velocity did not differ significantly between any of the groups; however, the left ventricular outflow tract (LVOT) velocity differed significantly between group 5 and each of the Boxer groups (1-4), evidence that
Boxers have a higher mean LVOT velocity than non-Boxer dogs. In the four group analysis, there was a significant difference in LVOT velocity between groups 1 and 3
(Table 4.1).
In the analysis of the TDI variables, there were no significant differences between groups for any of the variables measured at the lateral or medial wall of the mitral valve annulus (Sm, Em, Am, Em/Am). Finally, there were no significant differences between groups (both four group and five group analyses) for the variables measured at the lateral tricuspid valve annulus (Table 4.1).
In the analyses of the difference between groups for the variables of interest when modeled with several covariates and their interaction terms (gender, age, weight, gender x age, weight x age, weight x gender, age x weight x gender, and
LVOT velocity) (all groups in Table 4.2, and only Boxers in Table 4.3), a significant difference was observed between the groups (between groups 2 and 3, 2 and 4 and
137 1 and 3) for the variable early diastolic wall motion velocity (Em) measured at the medial mitral annulus, with age being a significant covariate within the model
(Table 4.2). However, the model R2 was only 0.3298, meaning only a small portion of the variance between the groups could be accounted for by the model.
Additionally a significant difference (between groups 2 and 5, 2 and 3 and 2 and 4) was observed for the variable the ratio of Em/Am measured at the medial mitral annulus with age again being a significant covariate, with a model R2 value of 0.3589
(Table 4.2). In the four-group analysis (Table 4.3), a significant difference (between groups 2 and 4, 2 and 3,1 and 4, and 1 and 3) was observed for the variable Em measured at the medial mitral annulus, with both age and weight remaining significant covariates. The variable the ratio of Em/Am measured at the medial mitral annulus was also significant between groups 2 and 3, and 2 and 4 with age a significant covariate (Table 4.3). Finally in Table 4.3, the variable the ratio of Era/Am measured at the lateral tricuspid annulus was significantly different between groups
1 and 3, with age remaining a significant covariate in the model. Troponin I concentration also differed significantly in the five group analysis, with differences observed between all Boxer groups (1-4) and the non-Boxer control group (5).
Gender was found to be a significant covariate in the model, as well as the interaction term of weight x age, yielding a model R2 value of 0.7304. In the four group analysis (Table 4.3), troponin I concentration also differed significantly between groups 1-3 and 4. No significant covariates were observed in this model, and the model R2 value was lower (0.2885).
138 In Table 4.4, results of the correlation analyses between the explanatory variables of interest (in all dogs] and the response variable VPCs/hr using a least squares fit model are reported. A correlation was found between VPCs/hr and late diastolic wall motion velocity (Am) measured at the lateral mitral annulus, however the model R2 value indicates that only 7.79% of the variability in VPCs/hr could be accounted for by the variable Am at the lateral mitral annulus. A correlation was also observed for the variable Em measured at the medial mitral annulus, with age, gender and LVOT velocity remaining significant covariates in the model, and with an
R2 value of 0.2723. A weak correlation (R2 value of 0.0599) was observed with the
2 medial mitral annulus Am variable. Finally, a similarly weak correlation (R value of
0.0853) was observed with the HFLA < 40 uV variable.
Tables 4.5-7 present the results of the correlation analyses (in all dogs) between the explanatory variables of interest (the TDI and select echocardiographic
[FS %, LVID-d and LVID-s] variables) and the response variables the SAECG parameters using a least squares fit model. No significant correlations were observed between the filtered QRS duration variable and the variables of interest
(Table 4.5). Two explanatory variables demonstrated a significant correlation with the RMS voltage in the terminal 40 ms variable (Table 4.6). The first explanatory variable was the lateral mitral annulus Sm variable with the age x weight interaction term remaining a significant covariate. The model R2 value for this particular model was low (0.2437). The lateral mitral annulus Em variable was the second explanatory variable significantly correlated with the RMS voltage in the terminal
40 ms variable after accounting for age as a significant covariate, with a model R2
139 value of 0.3253. Finally several explanatory variables demonstrated a significant
(albeit weak) correlation with the response variable HFLA < 40 uV (Table 4.7). The explanatory variable the lateral mitral wall Sm was significant after accounting for age as a significant covariate, but the correlation was weak (R2 = 0.1709). The medial mitral wall Sm variable also demonstrated a significant but weak correlation
2 (R = 0.0818). Finally, the medial mitral wall Em variable was observed to correlate with the HFLA < 40 uV variable (R2 = 0.1839), while accounting for age as a significant covariate. Although these explanatory variables in Tables 4.5-7 were found to be significant, they are minimally useful to account for the changes in the
SAECG variables.
140 Table 4.1: Sample Characteristics and Descriptive Statistics
Parameter Group 1 Group 2 Group 3 Group 4 Group 5 P value (all P value (n=15) (n=10) (n=15) (n=15) (n=15) groups) (Boxers only) Gender 6FI 2FI 9FI 10 FI 1FI 0.0005 0.0847 3FS IFS 3FS OFS 8FS 4 MI 4 MI 3 MI 3 MI IMI 2MC 3MC OMC 2MC 5MC Age* (years) 5.16+/-3.10 5.82 +/- 3.43 4.27+/- 2.33 3.57+/- 2.18 4.29 +/- 2.61 0.5839 0.4398 (1.15-9.9) (1.12-9.96) (1.07-9.48) (1.07-8.9) (1.12-9.46) Weight* (kg) 28.67+/- 30.04 +/- 27.81 +/- 26.01 +/- 24.24 +/- 0.0198 (2 and 0.0321 (2 and 3.96(21.2- 5.23 (23.4- 2.61 (23.9- 2.03 (21- 7.06 (14.9- 5) 4) 34.7) 40.5) 33.5) 29.3) 40.2) Average 133.93 +/- 126.97 +/- 135.53 +/- 133.87+/- 134.53 +/- 0.4803 0.3429 Systolic 8.29 (120- 8.57(115- 14.50 13.81 12.39 Blood 147.33) 141.33) (110.67- (113.33-156) (105.33-156) Pressure* 158.33) (mmHg) Total VPCSA 2701, (1617, 393, (264.75, 70, 2, (1,8),(0- 0, (0,1), (0-4) < 0.0001(1 < 0.0001(1 7502),(1309- 523.5), (206- (25.6,116), 17) and 5) and 4) 74129) 648) (25-189) < 0.0001(1 < 0.0001 (2 and 4) and 4) < 0.0001(1 < 0.0001(1 and 3) and 3) < 0.0001 (1 < 0.0001 (3 and 2) and 4) < 0.0001 (2 < 0.0001 (1 and 3) and 2) < 0.0001 (2 < 0.0001 (2
141 Parameter Group 1 Group 2 Group 3 Group 4 Group 5 P value (all P value (n=15) (n=10) (n=15) (n=15) (n=15) groups) (Boxers only) and 4) and 3) <0.0001 (2 and 5) <0.0001 (3 and 5) < 0.0001 (3 and 4) 0.0016 (4 and 5) VPCS/HYA 203, (66.71, 16.39,(11.75, 3.5, (1.49, 0.09, (0.04, 0, (0, 0.04), < 0.0001(1 < 0.0001(1 430.41), 24.24), (9- 5.07), (1.01- 0.35), (0- (0-0.17) and 2) and 4) (56.79- 32.4) 8.61) 0.72) < 0.0001(1 < 0.0001 (2 3416.08) and 3) and 4) < 0.0001(1 < 0.0001 (1 and 4) and 3) < 0.0001(1 < 0.0001 (3 and 5) and 4) < 0.0001 (2 < 0.0001(1 and 5) and 2) < 0.0001 (2 < 0.0001 (2 and 4) and 3) <0.0001 (2 and 3) <0.0001 (3 and 4) < 0.0001 (3 and 5)
142 Parameter Group 1 Group 2 Group 3 Group 4 Group 5 P value (all P value (n=15) (n=10) (n=15) (n=15) (n=15) groups) (Boxers only) 0.0015 (4 and 5) Troponin 0.123, (0.073, 0.0915, 0.0865, 0.044, 0.001, (0- < 0.0001(1 < 0.0001(1 +A(ng/mL) 0.189), (0.0675- (0.0595- (0.033- 0.0235), (0- and 5) and 4) (0.051-0.215) 0.15125), 0.198), 0.1028), 0.086) < 0.0001 (2 0.0052 (2 and (0.051-0.203) (0.017-0.238) (0.024-0.137) and 5) 4) < 0.0001 (3 and 5) < 0.0001 (4 and 5) Filtered QRS 87.4 +/- 9.75 83.30 +/- 82.29 +/- 80.69 +/" 84.93 +/- 0.3864 0.3097 Duration* (73 -106) 10.29 (69 - 10.03(70- 9.08 (68 - 8.06 (73 - (ms) 104) 95) 102) 100) HFLA < 40 24.80 +/- 24.90 +/- 21.21 +/- 20.31 +/" 22.73 +/" 0.469 0.3243 uV* (ms) 8.62 (11-44) 7.37 (14-36) 8.03 (10-33) 7.03 (9-36) 7.66 (10-33) RMS in term. 137, (74, 156.5, (101.5, 142, (89.25, 1000, (113, 151, (67, 0.5536 0.5401 40 msA 1000), (8- 1000), (36- 1000), (74- 1000), (69- 1000), (21- 1000) 1000) 1000) 1000) 1000) LVID-d* 38.53 +/- 38.49 +/- 37.65 +/- 36.49 +/- 37.50+/- 0.6362 0.4666 (mm) 4.65 (28.22- 3.51 (32.88- 3.34 (30.97- 3.63 (27.83- 4.08 (30.14- 46.76) 43.48) 43.19) 41.74) 45.11) LVID-s* 29.38 +/- 29.40 +/- 27.43 +/- 27.46 +/" 27.27 +/- 0.3738 0.3365 (mm) 4.80 (21.06- 4.79 (21.85- 3.31 (20.33- 2.32 (24.59- 3.91 (21.28- 37.99) 37.32) 35.24) 33.24) 36.31) FS %* 23.07+/- 24.03 +/" 27.08 +/' 26.03 +/- 27.47 +/- 0.1259 0.223 5.97 (9.17- 6.62 (13.70- 6.14(18.38- 3.91 (19.72- 3.58 (19.46- 33.54) 33.54) 38.50) 32.06) 32.11)
143 Parameter Group 1 Group 2 Group 3 Group 4 Group 5 P value (all P value (n=15) (n=10) (n=15) (n=15) (n=15) groups) (Boxers only) Lat Wall Sm* 15.90 +/- 15.20 +/- 15.80 +/- 15.14+/- 14.70 +/- 0.7476 0.8217 (cm/s) 2.75 (9.11- 2.19(11.06- 2.46 (12.42- 3.04(10.15- 3.13 (9.17- 20.44) 19.07) 19.82) 21.14) 18) Lat Wall Em* 10.47 +/- 9.27+/-1.95 10.74 +/- 11.03 +/- 10.80 +/- 0.355 0.2065 (cm/s) 2.34(6.72- (5.33-12.47) 2.24 (6.88- 1.66 (7.91- 2.58 (6.39- 15.12) 13.98) 13.77) 15.12) Lat Wall Am*+ 2.05 +/- 0.25 1.87+/-0.30 2.01+/-0.27 2.01 +/- 0.23 2.05+/-0.21 0.4116 0.3933 (cm/s) (1.64-2.53) (1.47-2.46) (1.36-2.42) (1.64-2.49) (1.68-2.57) Lat Wall 1.42 +/- 0.46 1.57+/-0.71 1.58 +/- 0.64 1.55+/-0.36 1.42 +/- 0.38 0.8445 0.8489 km/Am (0.72-2.10) (0.46-3.05) (0.83-3.21) (0.96-2.16) (0.73-1.98)
Med Wall Sm* 13.64 +/- 13.02 +/- 12.63 +/- 12.07+/- 12.43 +/- 0.5308 0.3817 (cm/s) 2.47 (9.22- 3.35 (7.92- 2.29 (8.25- 2.03 (8.76- 2.88 (7.38- 17.3) 18.81) 15.95) 16.01) 16.21)
Med Wall Em* 9.36 +/- 2.99 9.64 +/- 3.09 7.50 +/-1.94 7.95 +/-1.73 7.98 +/-1.73 0.0791 0.0763 (cm/s) (4.59-13.61) (5.15-14.69) (4.33-11.99) (5.36-11.65) (5.47-10.63)
Med Wall Am* 7.09 +/-1.34 5.99 +/-1.71 6.48 +/-1.56 6.21 +/-1.40 6.80 +/-1.53 0.3485 0.2687 (cm/s) (5.15-9.75) (3.46-9.75) (4.06-9.65) (4.49-8.97) (4.39-9.38) Med Wall 1.38 +/- 0.45 1.73 +/- 0.74 1.26+/-0.47 1.33+/-0.28 1.21+/-0.20 0.0582 0.1186 Cm/Am (0.72-2.03) (0.93-3.68) (0.71-2.26) (0.80-1.72) (0.94-1.58)
TV Wall Sm* 13.91 +/- 13.37 +/- 13.01 +/- 14.69 +/- 13.40 +/- 0.6436 0.5313 (cm/s) 2.87(8.51- 1.97 (9.37- 3.34 (7.75- 4.06 (8.82- 2.84 (9.22- 17.45) 15.89) 19.43) 24.51) 17.73)
TV Wall Em* 8.36+/-2.85 7.80 +/-1.87 7.14+/-1.98 8.36+/-2.82 8.77 +/-1.08 0.3376 0.4877 (cm/s) (3.44-14.77) (4.23-10.46) (4.12-10.31) (4.29-14.52) (7.05-10.76) TV Wall Am** 2.02 +/- 0.20 2.08 +/- 0.22 2.16+/-0.28 2.08 +/- 0.20 2.13+/-0.26 0.5479 0.4182 (cm/s) (1.67-2.47) (1.64-2.44) (1.70-2.80) (1.78-2.43) (1.78-2.50)
144 Parameter Group 1 Group 2 Group 3 Group 4 Group 5 P value (all P value (n=15) (n=10) (n=15) (n=15) (n=15) groups) (Boxers only) TV wall 0.064 +/- -0.035 +/- -0.219+/- 0.0017 +/- 0.047 +/- 0.2468 0.2714 Em/An,** 0.45 (-0.92 - 0.38 (-0.58- 0.37 (-0.79- 0.41 (-0.72 - 0.20 (-0.34- 0.71) 0.75) 0.44) 0.71) 0.38) LVOT 1.64+/-0.31 1.74+/-0.31 1.98+/-0.31 1.84 +/- 0.29 1.34+/-0.16 < 0.001 (3 0.0194 (3 and Velocity* (0.88-2.12) (1.24-2.18) (1.32-2.4) (1.27-2.37) (1.01-1.58) and 5) 1) (m/s) < 0.001 (4 and 5) 0.0078 (2 and 5) 0.0132 (3 and 1] 0.0432 (1 and 5) RVOT 1.21 +/- 0.28 1.23+/-0.17 1.27+/-0.25 1.34+/-0.15 1.12+/-0.16 0.154 0.5459 Velocity* (0.52-1.72) (0.94-1.41) (0.74-1.71) (1.13-1.57) (0.87-1.38) (m/s)
Notes: t- Log Transform performed. * for normally distributed data the mean +/- standard deviation, (range) are reported. A for non-normally distributed data the median, (25, 75% quartile), (range) are reported.
Abbreviations: FI - Female Intact MI - Male Intact FS - Female Spayed MC - Male Castrated
145 VPCs - Ventricular Premature Contractions HFLA - High Frequency Low Amplitude RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract RVOT - Right Ventricular Outflow Tract mmHg - Millimeters of Mercury Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity Group 1 - Boxers with greater than 1000 VPCs in 24 hours Group 2 - Boxers with between 200-999 VPCs in 24 hours Group 3 - Boxers with between 24-199 VPCs in 24 hours Group 4 - Boxers with less than 24 VPCs in 24 hours Group 5 - Non-Boxer control dogs with less than 24 VPCs in 24 hours
146 Table 4.2: Complex Analysis with adjustment for covahates - Least Squares Fit Model - All group Analysis (5 groups)
Parameter Model R2 Value Group P Covanates P value Difference Between Groups value Troponin 0.7304 < 0.0001 Gender 0.0386 < 0.0001 (1 and 5] Age 0.1336 < 0.0001 (2 and 5) Weight 0.1715 < 0.0001 (3 and 5) Weight x Age 0.0495 < 0.0001 (4 and 5) Filtered QRS 0.0637 0.3864 Duration HFLA < 40 0.1133 0.1612 LVOT Vel 0.0494 uV RMS term 40 0.0605 0.4155 ms BP 0.0514 0.4803 Lat Wall Sm 0.1741 0.5669 Age 0.0210 LVOT Vel 0.0443 LatWall Em 0.3149 0.7771 Age < 0.0001 Lat Wall Am 0.1538 0.2975 Age 0.0051 LatWall 0.3158 0.5072 Age < 0.0001 tra/Am Med Wall Sm 0.2549 0.1234 Age 0.0328 Gender 0.0402 LVOT Vel 0.0311
Med Wall Em 0.3298 0.0020 Age < 0.0001 0.0128 (2 and 3) 0.0254 (2 and 4) 0.0310 [land 3) Med Wall Am 0.0652 0.3485 Med Wall 0.3589 0.0011 Age < 0.0001 0.0011 (2 and 5]
147 Parameter Model R2 Value Group P Covariates P value Difference Between Groups value Em/Am 0.0029 [2 and 3) 0.0048 {2 and 4)
TV Wall Sm 0.1020 0.3720 LVOTVel 0.0354
TV Wall Em 0.1617 0.2778 Age 0.0089 TV Wall Am 0.1493 0.2476 Age 0.0091 TV Wall 0.2821 0.0733 Age < 0.0001 0.0395 [1 and 3) Em/Am FS% 0.1033 0.1259 LVID-d 0.3291 0.6202 Gender 0.1106 Weight 0.1421 Gender x Weight 0.0035 LVID-s 0.2532 0.5189 Gender 0.2967 Weight 0.3274 Gender x Weight 0.0443
Abbreviations: HFLA - High Frequency Low Amplitude RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity
148 Group 1 - Boxers with greater than 1000 VPCs in 24 hours Group 2 - Boxers with between 200-999 VPCs in 24 hours Group 3 - Boxers with between 24-199 VPCs in 24 hours Group 4 - Boxers with less than 24 VPCs in 24 hours Group 5 - Non-Boxer control dogs with less than 24 VPCs in 24 hours
149 Table 4.3: Complex Analysis with adjustment for covariates - Least Squares Fit Model - Boxer group Analysis (4 groups)
Parameter Model R^ Value Group P Covariates P value Difference Between Groups value Troponin 0.2885 0.0002 0.0001 (land 4) 0.0076 (2 and 4) 0.0433 (3 and 4) Filtered QRS 0.0712 0.3097 Duration HFLA < 40 0.0691 0.3243 uV RMS term 40 0.0530 0.450 ms BP 0.0626 0.3429
LatWallSm 0.0176 0.8217 LatWall Em 0.2542 0.5430 Age 0.0015 Lat Wall Am 0.2135 0.1881 Age 0.0019 LatWall 0.3059 0.5751 Age < 0.0001 Eim/Am Med Wall Sm 0.0577 0.3817 Med Wall Em 0.3988 0.0005 Age 0.0031 0.0049 (2 and 4) Weight 0.0475 0.0044 (2 and 3) 0.0157 (land4) 0.0177 (land 3)
Med Wall Am 0.0735 0.2687 Med Wall 0.3656 0.0034 Age < 0.0001 0.0041 (2 and 3) fcim/Am 0.0056 (2 and 4)
TV Wall Sm 0.1140 0.3004 LVOT Vel 0.0490 (cm/s)
150 Parameter Model R2 Value Group P Covariates P value Difference Between Groups value
TV Wall Em 0.1455 0.3778 Age 0.0196 TV Wall Am 0.2295 0.1137 Age 0.0018 TV Wall 0.3225 0.0477 Age < 0.0001 0.0360 [1 and 3) tm/Am FS% 0.0815 0.2230 LVID-d 0.1422 0.1745 LVOTVel 0.0233 LVID-s 0.0635 0.3365
Abbreviations: HFLA - High Frequency Low Amplitude RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity Group 1 - Boxers with greater than 1000 VPCs in 24 hours Group 2 - Boxers with between 200-999 VPCs in 24 hours Group 3 - Boxers with between 24-199 VPCs in 24 hours Group 4 - Boxers with less than 24 VPCs in 24 hours Group 5 - Non-Boxer control dogs with less than 24 VPCs in 24 hours
151 Table 4.4: Analysis of Variables Correlated with VPCs/hr: Least Squares Fit Model - All Dogs (Boxer Yes/No)
Parameter Model R2 Value VPCs/hr P value Covariates P value Troponin 0.6602 0.3508 Boxer yes/no < 0.0001 Weight 0.0027 LatWallSm 0.1809 0.0590 Age 0.0089 LVOT Vel 0.0234
Lat Wall Em 0.3187 0.1418 Age < 0.0001 Lat Wall Am 0.0779 0.0192 Lat Wall Em/Am 0.2803 0.8819 Age < 0.0001 Med Wall Sm 0.2723 0.0028 Age 0.0081 LVOT Vel 0.0308 Gender 0.015 Med Wall Em 0.1868 0.0341 Age 0.0003 Med Wall Am 0.0599 0.0411 Med Wall 0.1549 0.5804 Age 0.0009 tm/Am
TV Wall Sm 0.0091 0.4304 TV Wall Em 0.0994 0.5038 Age 0.0085 TV Wall Am 0.1370 0.9312 Age 0.0034 Weight 0.0390 TVWallEm/Am 0.1855 0.5530 Age 0.0003 Filtered QRS 0.0416 0.0975 Duration HFLA < 40 uV 0.0853 0.0164 RMS term. 40 0.0202 0.2508 ms FS% 0.1464 0.0983 LVOT Vel 0.0074
152 Parameter Model R2 Value VPCs/hr P value Covariates P value Boxer Yes/No 0.0043 LVID-d 0.3292 0.0995 Weight 0.0792 Gender 0.0263 Gender x Weight 0.0021 LVID-s 0.0323 0.1361
Abbreviations: VPCs - Ventricular Premature Contractions HFLA - High Frequency Low Amplitude RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity
153 Table 4.5: Analysis ofCovariance Results - Least Squares Fit Model - Filtered QRS Duration - All Dogs (Boxer Yes/No)
Parameter Model R2 Value Filtered QRS Covariates P value Duration P value Troponin 0.6391 0.7060 Weight 0.0034 Boxer Yes/No < 0.0001 LatWallSm 0.1254 0.0629 LVOT Vel 0.0288
LatWallEm 0.3035 0.1137 Age < 0.0001 LatWallAm 0.1010 0.6479 Age 0.0103 LatWallEm/Am 0.2797 0.3821 Age < 0.0001 Med Wall Sm 0.2034 0.1577 Age 0.799 Weight 0.893 Gender 0.0288 Age x Weight 0.0181
Med Wall Em 0.1381 0.2438 Age 0.0041 Med Wall Am 0.000051 0.9542 Med Wall 0.1758 0.1294 Age 0.0013 Em/Am TV Wall Sm 0.0022 0.7039 TV Wall Em 0.0954 0.2776 Age 0.0215 TV Wall Am 0.1290 0.9177 Age 0.0042 Weight 0.0327 TV Wall Em/Am 0.1874 0.2084 Age 0.0006 FS% 0.1186 0.3785 Boxer Yes/No 0.0120 LVOT Vel 0.0170 LVID-d 0.3243 0.1138 Gender 0.2179 Weight 0.1232 Gender x Weight 0.0088
154 Parameter Model R2 Value Filtered QRS Duration P value LVID-s 0.0530 0.0608
Abbreviations: HFLA - High Frequency Low Amplitude RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity
155 Table 4.6: Analysis of Covariance Results - Least Squares Fit Model - RMS Terminal 40 ms - All Dogs (Boxer Yes/No)
Parameter Model R2 Value RMS Terminal 40 Covariates P value ms P value Troponin 0.6588 0.4279 Boxer Yes/No < 0.0001 Weight 0.0030 LatWallSm 0.2437 0.0032 Age 0.1664 Weight 0.6293 Age x Weight 0.0247 LatWallEm 0.3253 0.0335 Age < 0,0001 LatWallAm 0.1105 0.3466 Age' 0.0131 Lat Wall Em/Am 0.2711 0.9319 Age < 0.0001 Med Wall Sm 0.2140 0.0954 Age 0.5902 Weight 0.9768 Gender 0.0325 Age x Weight 0.0239
Med Wall Em 0.1601 0.0831 Age 0.0023 Med Wall Am 0.0170 0.2926 Med Wall 0.2104 0.302 Age 0.0001 Em/Am Weight 0.0443
TV Wall Sm 0.000596 0.8445 TV Wall Em 0.0863 0.4622 Age 0.0271 TV Wall Am 0.1349 0.5069 Age 0.005 Weight 0.0333
TV Wall Em/Am 0.1802 0.3121 Age 0.0009 FS% 0.1170 0.4156 Boxer Yes/No 0.0112 LVOT Vel 0.0163
156 Parameter Model R2 Value RMS Terminal 40 Covariates P value ms P value LVID-d 0.3184 0.1571 Gender 0.0829 Weight 0.1110 Gender x Weight 0.0042 LVID-s 0.2208 0.4177 Gender 0.3059 Weight 0.2523 Gender x Weight 0.0447
Abbreviations: HFLA - High Frequency Low Amplitude RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity
157 Table 4.7: Analysis ofCovariance Results - Least Squares Fit Model - HFLA <40uV-All Dogs (Boxer Yes/No)
Parameter Model R2 Value HFLA < 40 uV - P Covariates P value value Troponin 0.6553 0.9091 Boxer Yes/No < 0.0001 Weight 0.0038 LatWallSm 0.1709 0.0043 Age 0.0275 LatWallEm 0.3154 0.0581 Age < 0.0001 Lat Wall Am 0.1008 0.6554 Age 0.0111 LatWallEm/Am 0.2874 0.2302 Age < 0.0001 Med Wall Sm 0.0818 0.0189 Med Wall Em 0.1839 0.028 Age 0.0023 Med Wall Am 0.0115 0.3871 Med Wall 0.2424 0.056 Age 0.0001 km/Am Weight 0.0466
TV Wall Sm 0.0157 0.3121 TV Wall Em 0.0856 0.4518 Age 0.0204 TV Wall Am 0.1354 0.4906 Age 0.0043 Weight 0.0309 TVWallEm/Am 0.1707 0.592 Age 0.0006 FS% 0.1079 0.8866 LVOT Vel 0.0244 Boxer Yes/No 0.0164 LVID-d 0.3138 0.2033 Gender 0.1645 Weight 0.1167 Gender x Weight 0.0057 LVID-s 0.0095 0.4309
Abbreviations: HFLA - High Frequency Low Amplitude
158 RMS - Root Mean Square LVID-d - Left Ventricle Internal Diameter - diastole LVID-s - Left Ventricle Internal Diameter - systole FS % - Fractional Shortening Percentage Lat - Lateral Mitral Valve Annulus Med - Medial Mitral Valve Annulus TV - Lateral Tricuspid Valve Annulus LVOT - Left Ventricular Outflow Tract Sm - Peak Systolic Myocardial Velocity Em - Peak Early Diastolic Myocardial Velocity Am - Peak Late Diastolic Myocardial Velocity
159 2.5 Discussion
In the present study, Boxers presumed to be clinically affected with ARVC based on the presence of ventricular arrhythmia of predominantly right ventricular origin did not have systolic or diastolic dysfunction as assessed by TDI, compared to clinically normal Boxers and non-Boxer control dogs. Additionally, SAECG failed to identify dogs that were more severely affected with ARVC (those dogs having more
VPCs).
To date, many similarities have been observed between ARVC in people and in the Boxer dog.1 In both species, affected individuals may be symptomatic for ventricular arrhythmia of right ventricular origin. Symptoms may include lethargy, syncope, exercise intolerance and sudden cardiac death.2-30 Histologically, fatty or fibro-fatty infiltration of the right ventricular myocardium is considered to be hallmark finding in both species. Currently, there has been a vast amount of published data as well as diagnostic recommendations for the disease in humans.
However, the scope of the information available as pertaining to the Boxer is much more limited. Therefore, it may be commonplace to extrapolate data from the human form of ARVC and apply it to the Boxer in terms of diagnostic tests (such as echocardiography and cardiac MRI) to reach a diagnosis.
One of the largest challenges in people with ARVC is the diagnosis of the disease, particularly in its early stages. Many diagnostic criteria have been suggested, including a recommended set of guidelines that consist of both major and minor criteria, which has been very recently revised to incorporate new knowledge of the disease and improve the diagnostic sensitivity of the guidelines.31-32 One of
160 the major criteria listed pertains to global and/or regional dysfunction and structural alterations, as assessed by echocardiography, angiography, MRI or radionuclide scintigraphy.3236 Other criteria include tissue characterization of the ventricular myocardium, ECG abnormalities (such as the presence of epsilon waves), and the familial presence of the disease.31 In terms of applying these criteria to the
Boxer, we are somewhat limited as to what is available and what is practical in clinical veterinary medicine. Furthermore, many of these diagnostic criteria have yet to be validated or assessed in the Boxer with ARVC. Currently, the mainstay or
"gold standard" of diagnosis is based on establishment of an ARVC phenotype - namely, the presence of ventricular arrhythmia of right ventricular origin as assessed by ambulatory ECG (Holter monitoring).2 Admittedly, this approach is not ideal given the known day-to-day variability which exists in consecutive Holter recordings, as well as the intermittent nature of the ventricular arrhythmia in some affected individuals.16*17'37 Relying on this diagnostic test has the potential to misclassify some individuals who are truly affected as unaffected and vice versa.
Tissue characterization of the walls, which is often performed via endomyocardial biopsy in people, is not a practical diagnostic test in the Boxer dog due to its invasive nature, need for general anesthesia and specialized equipment, as well as the cost burden to the owner. Other minimally invasive techniques are presently available in some specialized institutions such as cardiac MRI (cMRI), scintigraphy and angiography, but these tests are again prohibitive due to the experience necessary for their interpretation as well as the cost associated with these specialized tests.
To date, there has only been one small study evaluating the use of cMRI in Boxer
161 dogs with ARVC.38 In this 2009 study by Baumwart and Meurs, five affected Boxers were evaluated with cMRI and were found to have reduced right ventricular ejection fraction compared to five healthy non-Boxer dogs. Morphologically, one dog was observed to have an RV aneurysm, a common morphological finding in people with
ARVC.39-40 However, myocardial fatty changes were not observed in affected dogs, as evaluated using dark blood imaging for fat visualization.38 The authors concluded that it was therefore possible that ventricular arrhythmias and myocardial dysfunction occur prior to overt morphological changes (such as fibrofatty infiltration) in Boxers with ARVC.38 Given the need for specialized equipment, expertise in interpretation of acquired images, as well as cost and need for general anesthesia, there is certainly a need for more non-invasive and readily available diagnostic tests that can aid the veterinary practitioner in the diagnosis of ARVC.
The most ideal diagnostic test for evaluating patients suspected of being affected by ARVC is one that assesses their genotype, namely a genetic test which evaluates for the presence of a known mutation. To date in human medicine, over a dozen mutations have been recognized in patients with ARVC.30'41 In the Boxer dog, several of the candidate genes recognized in people have been evaluated (namely those encoding for desmosomal proteins), with no mutations being recognized to date in these particular genes.1-3'42'43 Recently, an 8-base pair deletion in the regulatory region of a calcium modulating gene called striatin has been identified in affected Boxer dogs, but not in unaffected Boxers or in 31 dogs from other breeds tested.7 Striatin appears to co-localize with several desmosomal proteins (such as desmoplakin) and it also contains a calcium dependent calmodulin binding site.
162 Therefore, a mutation in striatin could affected the structural integrity of the desmosome and/or may influence calcium leak from the sarcoplasmic reticulum.7
Affected dogs who were homozygous for the deletion were also observed to be more severely affected, while some of the control dogs (who had fewer than 100 VPCs in
24 hours) were heterozygous for the deletion. This finding in the control dogs suggests that these particular dogs may develop disease at a later stage, or the degree of penetrance may vary among individual dogs due to different environmental factors or genetic modifiers.7 A genetic test is now available for the mutation, however results must be interpreted with caution as a negative finding may not represent a truly unaffected dog, as four dogs in the referenced study who were phenotyped as having ARVC tested negative for the mutation.7 This finding implies that the Boxer form of ARVC is a result of more than just one mutation potentially affecting more than just one gene.
Due to the possible limitations of genetic testing in the Boxer dog (such as more than just the striatin mutation), as well as many of the other non-invasive or minimally invasive procedures used to aid in the diagnosis in people, there is a need for better (readily available and less expensive) tests for Boxers with ARVC. Thus, we were interested in evaluating a specialized application of echocardiography and
Doppler, namely TDI, in Boxers with ARVC. Although 2D echocardiography is typically the first test of choice in evaluating people suspected of having ARVC in order to assess for morphological and functional RV changes, it is often limited by the unique geometry of the RV.40 More specifically, the anatomy of the RV makes it difficult to impossible to accurately calculate RV volumes and ejection fraction,
163 methods which are routinely applied to the left ventricle when assessing its function. The availability of 3D echocardiographic volume reconstruction in patients with ARVC may prove to be useful in the near future, addressing the previous limitations of 2D echocardiography, however, the new ARVC task force criteria does not address this new application of echocardiography.44 It is for this reason that TDI is routine in the evaluation and work-up of ARVC-suspected human patients. TDI allows for determination of myocardial wall motion velocity at a particular point of interest, and this myocardial motion has been found to correlate well to invasive measures of cardiac function of both the left and right ventricle.44
Many investigators have reported the TDI findings in affected individuals, including a decrease in the lateral tricuspid annular velocity in early diastole (Em) as well as a decreased peak systolic annular velocity (Sm) when compared to unaffected individuals.39 In fact, some investigators have reported abnormal TDI findings prior to the presence of morphological changes of the RV as assessed by conventional two-dimensional echocardiography, thus supporting a unique role for TDI in the detection of "mild or early" cases of ARVC.12 It has been reported that over 75% of people with ARVC will have abnormalities of RV structure and function, as assessed with non-invasive tests such as echocardiography.38 Some investigators have also proposed that TDI may be useful in detecting and monitoring the progression of both RV and LV dysfunction over time in ARVC patients. A recent study identified significant reductions in the systolic wall motion velocity measured at the lateral tricuspid annulus in affected patients between their initial and last investigation, demonstrating the progressive nature of the disease.45 Thus, given the useful
164 diagnostic utility of TDI in people, as well as its non-invasive nature and availability, as well as the similarities of ARVC between people and Boxers, we were optimistic that TDI may indeed detect abnormal RV (and/or LV) function and establish a place as an adjunctive test in the diagnosis of Boxer ARVC.
Based on our results, Boxer dogs that appeared to have the ARVC phenotype, as assessed with ambulatory ECG, did not have TDI abnormalities compared to normal non-Boxer age-matched control dogs, as well as normal Boxer dogs.
Furthermore, dogs that had more frequent VPCs, again assessed with ambulatory
ECG, did not have TDI abnormalities compared to both normal Boxers and Boxers with lesser degrees of arrhythmia. No significant correlations could be made between the TDI parameters and Holter and SAECG findings. Similarly in people, it has been shown that no correlations exist between SAECG parameters and 2D echocardiographic parameters (measurements of RV inflow tract, outflow tract, and cavity) in human patients with ARVC.46 On subjective assessment of RV structure, none of the dogs had evidence of RV enlargement, or aneurysm formation.
Furthermore, there were no significant differences in LV parameters (2D echocardiography) between the groups of dogs. These findings are in contrast to what has been reported to date in the human literature regarding ARVC and right ventricular dysfunction.30'40 To date, there have been limited evaluations of cardiac function in affected Boxers, and this study represents the largest study to date evaluating both RV and LV function in affected dogs using TDI. Therefore, it appears based on our findings that many affected Boxers maintain normal cardiac function, despite being affected with ARVC. Alternatively, TDI may not be a sensitive enough
165 to detect cardiac dysfunction. The finding of apparently normal cardiac function does agree with a previous veterinary study that reported the use of the Tei or myocardial performance index, a pulsed Doppler echocardiographic method which assesses both systolic and diastolic performance of the heart by measuring the isovolumic indices.20-47'48 In this study performed by Baumwart and Meurs, 12 affected Boxers were compared to 10 normal Boxers using the Tei index. They found no significant differences between the two groups, and none of the affected dogs evaluated had evidence of abnormal cardiac structure [such as chamber enlargement) or abnormal LV function, as assessed with fractional shortening percentage. Furthermore, no correlations could be found between the measured index and arrhythmia findings on Holter recordings.20 Our findings do appear to contradict the cMRI study performed by the same authors, where affected Boxers were shown to have reduced RV function as assessed by RV ejection fraction.38
However, this particular study was limited by the number of affected dogs evaluated
(only 5), and by the fact that dogs treated with anti-arrhythmics such as beta blockers, which can affect cardiac function, were included.38
When Harpster first described the Boxer ARVC phenotype nearly two decades ago, he reported that there was a spectrum of disease in affected individuals - some dogs were asymptomatic, others clinical for their arrhythmia, and a small subset of dogs progressed to congestive heart failure.49 There has certainly been much speculation about the natural progression of the disease in the
Boxer, and whether or not congestive heart failure is a possible outcome in all affected dogs if they do not first succumb to their arrhythmia. Based on the
166 literature and case reports to date2, congestive heart failure is a rare outcome in the
Boxer, and our results corroborate those findings as none of our dogs evaluated had cardiac dysfunction, a necessary finding which would have placed them at risk for the development of congestive heart failure. This finding is in contrast to ARVC in people, where congestive heart failure is a relatively common outcome and the cause of death in as many as 59% of human patients.50 The difference between the two species may lie in the histological differences which exist between them. While human patients typically have the fibrofatty form of the disease, Boxers are more often afflicted with the fatty form only.4 Furthermore, it has been suggested that those with the fibrofatty form of disease are more prone to the development of RV or LV wall thinning, chamber enlargement and both systolic and diastolic dysfunction.1 When considering other veterinary species afflicted with ARVC, such as the cat, cardiac dysfunction is very common with most to all cats having evidence of severe RV dilation and evidence of right sided congestive heart failure.51-52 In
Fox's report of ARVC in the cat, 75% of affected cats had the fibrofatty form of disease, similar to people, and therefore may be the explanation for the apparent presence of cardiac dysfunction and heart failure in affected felines.52 In other veterinary case reports in the canine population, those dogs who presented with signs of right-sided congestive heart failure also had the fibrofatty variant of the disease on histopathological analysis.53-54 This study did not perform histological characterization in affected dogs, and therefore cannot comment on the histological types present in our study population. This may be considered a limitation of the study, as status of disease affectedness was based on a qualitative phenotypic test
167 (Holter monitor) and not a quantitative assessment such as histological characterization. Regardless, our findings suggest a clear morphologic and functional difference in ARVC in Boxers when compared to people as well as other veterinary species, which may be in part due to the apparent genetic differences when compared to people (such as the lack of mutations of desmosomal proteins in the Boxer to date).3-7 The other possibility to explain our findings is that the most severely affected dogs (group 1 with over 1000 VPCs within a 24 hour period) were early in the progression of their disease, and were evaluated prior to the development of cardiac dysfunction and wall motion abnormalities. This consideration is somewhat unlikely given the number of dogs in our most severe group, and the range of ages in this group, meaning it would have been more likely a consideration if our population had been comprised of mostly younger dogs.
In an attempt to further characterize ARVC affectedness, SAECG was evaluated in all dogs in the present study. Previous studies have evaluated SAECG in
Boxers with ARVC, with the largest evaluating 93 affected Boxers by Spier and
Meurs.9 In that study, numerous false positive and negatives occurred, and the investigators concluded that the proper identification of late potentials was very dependent on the technique used and filters applied. SAECG correctly identified 14 of 15 dogs who had an adverse cardiac related outcome (sudden death or death due to congestive heart failure). However, 13 of the 79 clinically normal dogs were incorrectly identified as having late potentials, yielding a positive predictive value of
52%.9 Based on the results of this study, and other veterinary studies evaluating the use of SAECG5557, three parameters were used to assess the presence of late
168 potentials: the root mean square [RMS) voltage value of the terminal 40 ms of the
QRS complex, duration of high frequency low amplitude (HFLA) signals less than 40 uV, and the filtered QRS duration. In the current study, there were no significant differences in these three parameters between the five groups of dogs. In fact in the present study, 11 of the normal dogs (both non-Boxer and Boxer dogs) were positive for one late potential parameter, and 4 were positive for two late potential parameters. These findings are consistent with the previous Spier and Meurs study, and are likely attributable to technical artifacts or spurious signals which were consistently present during the averaging process. In the human literature, as many as 6% of healthy individuals will have the presence of late potentials as evaluated with SAECG.9 Therefore, based on the results of our study, SAECG is a technique whose results are highly dependent on the equipment and technique used. In our study, it was sometimes not possible to reduce the noise level adequately in all dogs in order to identify late potentials, as panting and trembling seemed to increase the noise level in anxious dogs, despite trying to increase the number of beats averaged to decrease the noise level. In these cases, sedation may be beneficial but was not part of our study protocol. Furthermore, the use of metal contact plates to facilitate electrode contact, as used in this study, may not be ideal as poor skin-electrode plate contact may also further increase the noise level, interfering with the ability for the
SAECG software to correctly identify the presence of late potentials. Finally, given that the QRS duration in the dog is shorter than in people, considering the RMS value of the terminal 30 ms instead of the terminal 40 ms (as is standard in people) may have been ideal.9 The present study evaluated the terminal 40 ms and may
169 therefore have allowed inclusion of a greater portion of the QRS, skewing our results and interfering with our ability to correctly identify late potentials in affected individuals. Thus, based on the technical limitations of this diagnostic modality, it may not be appropriate as a first line test when evaluating dogs suspected of having
ARVC. The utility of this test may lie in its ability to identify those dogs at risk for an adverse cardiac outcome, and it would be ideal to follow the dogs in the present study who were positive for late potentials to determine their risk for such an outcome. This is the primary use of SAECG in the human population, where the presence of late potentials is an indicator of increased risk of development of ventricular tachyarrhythmias, increased severity of disease, and increased risk of sudden death.8-19
Serum troponin I concentration was assessed between the five groups of dogs in the present study. As expected, there were significant differences between serum concentrations when comparing the most severely affected group of Boxers
(group 1] and the normal non-Boxer dogs (group 5) and the normal Boxer dogs
(group 4, those having fewer than 24 VPCs in a 24-hour period). Furthermore, when looking at Boxers only, there was a significant difference in troponin I concentrations between the normal Boxers (group 4) and Boxers with lesser degrees of arrhythmia (group 2). This finding may suggest that troponin I concentrations may be useful in terms of determining the severity of ARVC affectedness in Boxers. The elevations in troponin I are expected given the histological changes that occur in ARVC, namely fatty or fibrofatty infiltration with cell death and inflammatory infiltration, all risk factors for leakage of the cardiac
170 troponins into the circulation. An ideal comparison therefore would have compared the degree of myocardial changes assessed on myocardial tissue samples with the serum troponin I levels, but this was not assessed in the scope of the current study.
The findings herein are consistent with a previous study which evaluated serum troponin I concentrations in 10 Boxers with ARVC compared to 10 non-Boxer control dogs.15 In that study, the mean troponin concentration in affected Boxers was 0.142 +/- 0.05 ng/ml, and 0.023 +/- 0.01 ng/mL for control non-Boxer dogs. In the present study, the mean concentration in the most severely affected group was
0.129 +/- 0.05 ng/mL, and 0.017 +/- 0.03 ng/mL in the non-Boxer control group, and 0.053 +/- 0.03 ng/mL in the normal Boxer control group. Interestingly, the troponin I concentration was lower in the non-Boxer normal control group when compared to the "normal" Boxer group. This was also noted in the other veterinary study by Baumwart et al.15 Given that it would be unusual for the Boxer as a breed to have higher than normal troponin I concentrations, it is more likely that our normal Boxer group may have included dogs who were truly affected by ARVC but who did not fit the accepted ARVC phenotype as assessed with Holter monitoring.
An interesting application of serum troponin I concentration would be trending the concentration in affected dogs who are treated with anti-arrhythmics, to determine if there is any correlation between troponin concentration and the subjective assessment of arrhythmia control or to survival or the development of congestive heart failure. If correlations could be determined, then troponin I could be used as an adjunctive test when evaluating the progression of affected dogs who are treated.
Regardless, correlations between severity of disease (as assessed by histological
171 analysis or other non-invasive means of quantifying fatty infiltration such as cMRI) and troponin concentrations are needed in order to better determine how measurement of troponin concentrations might be useful in the assessment of ARVC affected dogs.
172 2.6 Conclusions
The present study failed to identify any left or right ventricular dysfunction
(either systolic or diastolic) in Boxers with ARVC as assessed with noninvasive tissue Doppler echocardiography, compared to both normal Boxers and normal non-
Boxer dogs. This finding suggests that ventricular dysfunction may be uncommon in affected dogs, or that ventricular function is maintained until the later stages of the disease. These findings concur with other studies designed to assess ventricular function in affected Boxers, and contrast to what is found in people with the disease where ventricular dysfunction develops in the majority of affected patients.
Furthermore, significant correlations between tissue Doppler and signal averaged
ECG variables were not found, as also reported in people with ARVC. Future studies evaluating ventricular function in Boxers with ARVC, particularly studies evaluating ventricular function over time, should consider different criteria for disease affectedness, namely the degree of fibrofatty/fatty infiltration, Which is currently difficult to perform reliably and non-invasively. The future availability of genetic testing may also be helpful in the classification of a Boxers ARVC status.
In the context of the present study, signal averaged ECG did not provide any additional information regarding a dog's severity of disease, however, it may be more useful to identify those dogs at risk for sudden death or symptomatic ventricular arrhythmia (syncope). Future long-term studies following Boxers with
ARVC and correlating their outcomes with signal averaged ECG data would be useful.
173 Finally, the present study found a significant difference in troponin I concentrations between groups of dogs (namely Boxers and the non-Boxer control dogs). However, a correlation between VPCs/hr and troponin I concentration was not found. Therefore, additional studies correlating the severity of disease based on histopathological myocardial changes as well as patient outcomes may allow troponin I concentrations to have prognostic value in dogs with ARVC, and may allow for monitoring the progression in affected dogs.
174 2.7 Limitations
There are several limitations that must be considered in the present study.
The largest limitation is how dogs were classified as being affected with ARVC or being normal through the use of arrhythmia frequency (i.e.: total VPCs in 24 hours).
To date, there is no data determining the degree of ventricular arrhythmia that is diagnostic for Boxer ARVC, and the classification of the frequency of VPCs for each of the four Boxer groups was arbitrary. However, most investigators would agree that as the frequency of VPCs increases, the likelihood of being affected with ARVC increases.1-2 Furthermore, the "normal Boxer group" arrhythmia frequency of less than 24 VPCs in a 24-hour period was also an arbitrary cutoff due to a lack of evidence of the definitive arrhythmia frequency in normal Boxers. A recent veterinary study reported that clinically normal Boxers generally have fewer than
91 VPCs in a 24-hour period, however that study like the present study, is limited by an inability to definitively define which Boxers are truly normal and not ARVC affected.58 Therefore, there is the potential that affected Boxer dogs were included in the normal group, thus potentially affecting our results. It has been shown that there is more Holter variability in Boxers, since arrhythmia frequency has been shown to change by as much as 80% from one Holter exam to a subsequent Holter exam.17 This variability may have lead to misclassification of some dogs and it may have been more ideal to perform several consecutive 24-hour analyses with an averaging of the results in order to better classify our dogs. We also used the frequency of arrhythmia as a measure of severity of disease (potential surrogate for degree of fibrofatty or fatty infiltration) with dogs having more severe arrhythmia
175 assumed to have more severe myocardial changes. To date, no studies have reported a correlation in Boxers between VPC frequency and the degree of fibrofatty or fatty infiltration. In human studies, it has been shown that fibrous substitution was a significant predictor for the presence of late potentials on SAECG and of reduced right ventricular ejection fraction via angiography in patients with ARVC.19
Ideally, dogs would have been classified by degree of right ventricular myocardial changes, either invasively through endocardial biopsies, or non-invasively via cMRI.
We had hypothesized that dogs with more severe disease would have more functional impairment based on the presumption that dysfunction would ensue when myocardial cells were lost and replaced. However, without a true measure of disease severity such as the degree of myocardial loss and fibrofatty/fatty replacement, it may be difficult to make accurate correlations between echocardiographic functional parameters (tissue Doppler) and severity of disease.
Furthermore, the dogs we considered to be most severely affected may have been early in their disease progression, and therefore may not have developed functional impairment that was detectable via tissue Doppler. This is a limitation of a one-time examination versus a longitudinal study, where serial examinations may have allowed for the detection of the development of ventricular dysfunction over time.
An additional limitation in the present study is the lack of investigator blinding once dogs were pre-screened with a Holter recording. Initially all dogs who were willing to participate were included and the echocardiogram and SAECG were performed prior to the ambulatory ECG being applied. Therefore, the echocardiographic measures were performed when the status of the dog's
176 affectedness was unknown. However, once the first group of Boxers was filled, all dogs required pre-screening with Holters prior to inclusion into the remaining groups. Therefore, the primary investigator was aware of their Holter results at the time that the echocardiogram was performed. This may have allowed for operator bias when selecting tissue Doppler cycles to measure or reject. Ideally, the primary investigator would not have been aware of the Holter results prior to performing the echocardiogram.
In an attempt to further examine for a relationship between the degree of
ARVC affectedness and assessments of functional impairment, SAECG was included in conjunction with the ambulatory ECG. Although we were able to collect SAECG data in all but three dogs, several of the dogs had high levels of signal noise presumably as a result of panting, shivering and trembling. The high level of noise will interfere with the ability for the SAECG software analysis program to properly identify the presence of late potentials. Thus, there was the possibility for both false positives and false negatives in the current study. The SAECG software did not permit analysis of the RMS voltage on the terminal 30 ms, but rather only on the terminal 40 ms which is the standard in people. In the Boxer, the implication of this software limitation is that more of the QRS is sampled (as the QRS in the dog is shorter than in people). Therefore, late potentials may have been missed, increasing the probability of false negatives results.
Every effort was made to standardize each echocardiographic examination so that all data was collected in the same order in each and every dog. This allowed for standardization of sweep speed, number of cycles included, and the Doppler
177 sample volume size and position of measurement of annular velocities. However, factors such as operator experience and image quality varied from examination to examination. Initially in the early stages of the study, the primary investigator's experience level in tissue Doppler was lower than at the end of data collection (one year later). Therefore, given the angle dependency of TDI analysis, there may have been more alignment issues early in data collection which would have underestimated annular velocity measurements, potentially skewing results. As well, the conformation of some dogs precluded the ability to attain good quality images, which may have impaired the ability to detect structural changes, as well as measurement of annular velocities. Although tissue Doppler is felt to be relatively load independent, loading conditions would have varied between dogs and were not accounted for, which may have influenced the echocardiographic results as well.
Finally, like in many veterinary studies, we may have been limited by the relatively small sample sizes present. However, if no significant differences are present with relatively small sample numbers, then a biologically significant difference may not be necessarily present.
178 2.8 References
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3. Meurs KM, Ederer MM, Stern JA. Desmosomal gene evaluation in Boxers with arrhythmogenic right ventricular cardiomyopathy. Am J Vet Res 2007;68:1338- 1341.
4. Oxford E, Everitt M, Coombs W, et al. Molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia/cardiomyopathy. Heart Rhythm 2007;4:1196-1205.
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7. Meurs KM, Mauceli E, Lahmers S, et al. Genome-wide association identifies a deletion in the 3' untranslated region of Striatin in a canine model or arrhythmogenic right ventricular cardiomyopathy. Hum Genet 2010;128:315-324.
8. Nava A, Folino A, Bauce B, et al. Signal-averaged electrocardiogram in patients with arrhythmogenic right ventricular cardiomyopathy and ventricular arrhythmias. Eur Heart J 2000;21:58-65.
9. Spier A, Meurs K. Use of signal-averaged electrocardiography in the evaluation of arrhythmogenic right ventricular cardiomyopathy in Boxers.J Am Vet Med Assoc 2004;225:1050-1055.
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179 11. Kjaergaard J, Hastrup Svendsen J, Sogaard P, et al. Advanced quantitative echocardiography in arrhythmogenic right ventricular cardiomyopathy. J Am Soc Echocardiogr 2007;20:27-35.
12. Prakasa K, Wang J, Tandri H, et al. Utility of tissue Doppler and strain echocardiography in arrhythmogenic right ventricular dysplasia/cardiomyopathy. Am J Cardiol 2007;100:507-512.
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17. Spier A, Meurs K. Evaluation of spontaneous variability in the frequency of ventricular arrhythmias in Boxers with arrhythmogenic right ventricular cardiomyopathy.! Am Vet Med Assoc 2004;224:538-541.
18. Spier A, Meurs K. Assessment of heart rate variability in Boxers with arrhythmogenic right ventricular cardiomyopathy.} Am Vet Med Assoc 2004;224:534-537.
19. Turrini P, Angelini A, Thiene G, et al. Late potentials and ventricular arrhythmias in arrhythmogenic right ventricular cardiomyopathy. Am J Cardiol 1999;83:1214.
20. Baumwart RD, Meurs KM. An index of myocardial performance applied to the right ventricle of Boxers with arrhythmogenic right ventricular cardiomyopathy. Am J Vet Res 2008;69:1029-1033.
180 21. Feigenbaum H, Armstrong W. Feigenbaum's Echocardiography, 6th ed. Philadelphia: Lippicot Williams & Wilkins; 2004.
22. Chetboul V, al. e. Tissue Doppler assessment of diastolic and systolic alterations of radial and longitudinal left ventricular motions in Golden Retrievers during the preclinical phase of cardiomyopathy associated with muscular dystrophy. Am J Vet Res 2004;65:1335-1341.
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24. Meluzin J, Spinarova L, Bakala J, et al. Pulsed Doppler tissue imaging of the velocity of tricuspid annular systolic motion; a new, rapid, and non-invasive method of evaluating right ventricular systolic function. Eur Heart J 2001;22:340-348.
25. Chetboul V, Sampedrano CC, Gouni V, et al. Quantitative assessment of regional right ventricular myocardial velocities in awake dogs by Doppler tissue imaging: repeatability, reproducibility, effect of body weight and breed, and comparison with left ventricular myocardial velocities. J Vet Intern Med 2005;19:837-844.
26. Wess G, Killich M, Hartmann K. Comparison of pulsed wave and color Doppler myocardial velocity imaging in healthy dogs. J Vet Intern Med 2010.
27. Teshima K, Asano K, Sasaki Y, et al. Assessment of left ventricular function using pulsed tissue Doppler imaging in healthy dogs and dogs with spontaneous mitral regurgitation. J Vet Med Sci 2005;67:1207-1215.
28. Thomas W, Gaber C, Jacobs G, et al. Recommendations for standards in transthoracic two-dimensional echocardiography in the dog and cat. Echocardiography committee of the Specialty of Cardiology, American College of Veterinary Internal Medicine. J Vet Intern Med 1993;7:247-252.
29. Technologies GEMSI. Hi-Res physician's guide for the MAC5500. In: 2005.
30. Sen-Chowdhry S, Morgan RD, Chambers JC, et al. Arrhythmogenic cardiomyopathy: Etiology, diagnosis, and treatment. Annu Rev Med 2010;61:233- 253.
181 31. McKenna WJ, Thiene G, Nava A, et al. Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy. Task Force of the Working Group Myocardial and Pericardial Disease of the European Society of Cardiology and of the Scientific Council on Cardiomyopathies of the International Society and Federation of Cardiology. Br Heart J 1994;71:215-218.
32. Marcus F, McKenna WJ, Sherrill D, et al. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia. Eur Heart J 2010;31:806-814.
33. Hauer RNW. Toward early diagnosis in arrhythmogenic right ventricular dysplasia/cardiomyopathy. J Interv Card Electrophysiol 2009;26:1-2.
34. Corrado D, Basso C, Thiene G. Arrhythmogenic right ventricular cardiomyopathy: an update. Heart 2009;95:766-773.
35. Hamilton RM. Arrhythmogenic right ventricular cardiomyopathy. Pacing Clin Electrophysiol 2009;32 Suppl 2:S44-51.
36. Marcus FI, Zareba W, Calkins H, et al. Arrhythmogenic right ventricular cardiomyopathy/dysplasia clinical presentation and diagnostic evaluation: results from the North American Multidisciplinary Study. Heart rhythm 2009;6:984-992.
37. Meurs K, Spier A, Wright N, et al. Comparison of in-hospital versus 24-hour ambulatory electrocardiography for detection of ventricular premature complexes in mature Boxers.J Am Vet Med Assoc 2001;218:222-224.
38. Baumwart R, Meurs K, Raman S. Magnetic resonance imaging of right ventricular morphology and function in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy. J Vet Intern Med 2009;23:271-274.
39. Teske AJ, Cox MG, De Boeck BW, et al. Echocardiographic tissue deformation imaging quantifies abnormal regional right ventricular function in arrhythmogenic right ventricular dysplasia/cardiomyopathy. J Am Soc Echocardiogr 2009;22:920- 927.
40. Sorrell VL, Kumar S, Kalra N. Cardiac imaging in right ventricular cardiomyopathy/dysplasia~how does cardiac imaging assist in understanding the morphologic, functional, and electrical changes of the heart in this disease? J Electrocardiol2009;42:137.el31-110.
182 41. Barahona-Dussault C, Benito B, Campuzano 0, et al. Role of genetic testing in arrhythmogenic right ventricular cardiomyopathy/dysplasia. Clin Genet 2010;77:37-48.
42. Oyama MA, Reiken S, Lehnart SE, et al. Arrhythmogenic right ventricular cardiomyopathy in Boxer dogs is associated with calstabin2 deficiency. J Vet Cardiol 2008;10:1-10.
43. Meurs KM, Lacombe VA, Dryburgh K, et al. Differential expression of the cardiac ryanodine receptor in normal and arrhythmogenic right ventricular cardiomyopathy canine hearts. Hum Genet 2006;120:111-118.
44. Lindqvist P, Calcutteea A, Henein M. Echocardiography in the assessment of right heart function. Eur J Echocardiogr 2008;9:225-234.
45. Aneq MA, Lindstrom L, Fluur C, et al. Long-term follow-up in arrhythmogenic right ventricular cardiomyopathy using Tissue Doppler Imaging. Scand Cardiovasc J 2008;42:368-374.
46. Park Y, Cho Y, Lee D-Y, et al. Correlation between the parameters of signal- averaged ECG and two-dimensional echocardiography in patients with arrhythmogenic right ventricular cardiomyopathy. Ann Noninvasive Electrocardiol 2009;14:50-56.
47. Teshima K, Asano K, Iwanaga K, et al. Evaluation of right ventricular Tei index (index of myocardial performance} in healthy dogs and dogs with tricuspid regurgitation. J Vet Med Sci 2006;68:1307.
48. Baumwart R, Meurs K, Bonagura J. Tei index of myocardial performance applied to the right ventricle in normal dogs. J Vet Intern Med 2005;19:828-832.
49. Harpster N. Boxer cardiomyopathy. Vet Clin North Am Small Anim Pract 1991;21:989-1004.
50. Herren T, Gerber PA, Duru F. Arrhythmogenic right ventricular cardiomyopathy/dysplasia: a not so rare "disease of the desmosome" with multiple clinical presentations. Clin Res Cardiol 2009;98:141-158.
183 51. Harvey AM, Battersby IA, Faena M, et al. Arrhythmogenic right ventricular cardiomyopathy in two cats. J Small Anim Pract 2005;46:151-156.
52. Fox P, Maron B, Basso C, et al. Spontaneously occurring arrhythmogenic right ventricular cardiomyopathy in the domestic cat: A new animal model similar to the human disease. Circulation 2000;102:1863-1870.
53. Simpson KW, Bonagura JD, Eaton KA. Right ventricular cardiomyopathy in a dog. J Vet Intern Med 1994;8:306-309.
54. Santilli RA, Bontempi LV, Perego M, et al. Outflow tract segmental arrhythmogenic right ventricular cardiomyopathy in an English Bulldog. J Vet Cardiol 2009;11:47-51.
55. Calvert CA, Kraus M, Jacobs G, et al. Possible late potentials in 4 dogs with sustained ventricular tachycardia. J Vet Intern Med 1998;12:96-102.
56. Calvert CA, Jacobs GJ, Kraus M, et al. Signal-averaged electrocardiograms in normal Doberman pinschers. J Vet Intern Med 1998;12:355-364.
57. Calvert CA. High-resolution electrocardiography. Vet Clin North Am Small Anim Pract 1998;28:1429-1447, viii.
58. Stern J, Meurs KM, Spier AW, et al. Ambulatory electrocardiographic evaluation of clinically normal adult Boxers. J Am Vet Med Assoc 2010;236:430-433.
184 Study of Bom»r Cardiomyopathy Telephone Questionnaire Prior to Anooirttment Scheduling
Participant Name;
Phone.,n,qmpe.n
Number of Does Interested in Submitting to the Study jeath dog should have a separate questionnaire filed putl:
Questionnaire:
1. Is your dog a Boxer |ie: can you provide Pedigree for your pet?)
YesClNon
If yes, continue to next question, If no {or cannot provide pedigree), dog is BQl eligible for the study and thaftk ihe/n for their interest
2. Are all participating dogs older than one (1) year of age and less than 10 years of age?
YesQ NoQ
// yes, continue to next question. If no, dog is not eligible for study and thank them for their interest.
3. Has your veterinarian ever reported that your dog {•=) has a heart murmur?
Yes (J No U
// yes, continue to questfon 4. If no, proceed to question 5.
4. If your veterinaran has reported a murmur, of what intensity was it graded'
Mild (1 3/6) • Moderate or Severe (4-6/6 J Q
If a mildly intense murmur was heard, proceed to question 4. If moderate or severe murmur was heard, dog is not eligible far the study. If awiwrs are unaware oftne murmur intensity but would still like to participate, have their primary care veterinarian forward the animals records to us.
i. Has your dog received anti-an hythmnc medication (such as aotaiol, atenolol, me*ilitiner etc} tn the prpvious 2 months?
YesONoQ
185 if yes, dog is oat eligible for the VVSc portion of the project - however, we moy contact them of a inter dote for am? further participation irt the Hamiitan (Sick Kids) portion of the study. If no, proceed tc the next question.
6. Has your dog currently been diagnosed with systemic disease or is being treated for systemic disease jie: liver disease, pancreatic disease, renal disease)? Or has your dcg been treated with steroids in the past two months?
YesQ No (J
If yes to either, dog is not ekgtfetefor the study and thank them for their participation, if no, proceed to the next step, if on steroids for seasonal allergies, we may contact them in the future once they hove been off steroids for two months.
7. Based on these preliminary questions, your dog qualifies at this time to participate therefore an appointment may be scheduled. Please note that dogs may be excluded from the study if other exdusmn criteria are noted at their assessment (e.g. congenita) or acquired cardiac disease other than ARVC, high blood pressure, abnormal blood work)
Reminders:
Please bring the pedigree of tie animal If bringing multiple dogs fcr the stucy, please ask that there is several extra sets of "hands" to help If their pet has had a previous rioiter, ask them to bring the results
Thank you!
OVC Cardiology Service
Appendix 3.1: Telephone Questionnaire used to pre-screen Boxer dogs for study participation.
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