The development and preliminary testing of an instrument to measure the perceived economic burden of a subtype of arrhythmogenic right ventricular cardiomyopathy (ARVC) on patients and families in Newfoundland

The goal of this thesis was to develop, construct, and validate the Perceived Economic Burden scale to quantitatively measure the burden associated with a subtype Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) in families from the island of Newfoundland. An original 76 item self-administered survey was designed using content from existing literature as well as themes from qualitative research conducted by our team and distributed to individuals of families known to be at risk for the disease. A response rate of 37.2% (n = 64) was achieved between December 2013 and May 2014. Tests for data quality, Likert scale assumptions and scale reliability were conducted and provided preliminary evidence of the psychometric properties of the final constructed perceived economic burden of ARVC scale comprising 62 items in five sections. Findings indicated that being an affected male was a significant predictor of increased perceived economic burden in the majority of economic burden measures. Affected males also reported an increased likelihood of going on disability and difficulty obtaining insurance. Affected females also had an increased perceived financial burden. Preliminary results suggest that a perceived economic burden exists within the ARVC population in Newfoundland.

Right Ventricular Involvement and Recovery after Acute Stress-Induced (Tako-tsubo) Cardiomyopathy

We would like to thank all NHS Consultant Colleagues at Aberdeen Royal Infirmary for their help with prompt recruitment of these patients (Dr M Metcalfe, Dr AD Stewart, Dr A Hannah, Dr A Noman, Dr P Broadhurst, Dr D Hogg, Dr D Garg) and to Dr Gordon Prescott for help and advice with the Statistical Methods.

Computational Methods for Identifying Hypertrophic Cardiomyopathy using 12-lead ECG

Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease where the heart muscle is partially thickened and blood flow is - potentially fatally - obstructed. It is one of the leading causes of sudden cardiac death in young people. Electrocardiography (ECG) and Echocardiography (Echo) are the standard tests for identifying HCM and other cardiac abnormalities. The American Heart Association has recommended using a pre-participation questionnaire for young athletes instead of ECG or Echo tests due to considerations of cost and time involved in interpreting the results of these tests by an expert cardiologist. Initially we set out to develop a classifier for automated prediction of young athletes’ heart conditions based on the answers to the questionnaire. Classification results and further in-depth analysis using computational and statistical methods indicated significant shortcomings of the questionnaire in predicting cardiac abnormalities. Automated methods for analyzing ECG signals can help reduce cost and save time in the pre-participation screening process by detecting HCM and other cardiac abnormalities. Therefore, the main goal of this dissertation work is to identify HCM through computational analysis of 12-lead ECG. ECG signals recorded on one or two leads have been analyzed in the past for classifying individual heartbeats into different types of arrhythmia as annotated primarily in the MIT-BIH database. In contrast, we classify complete sequences of 12-lead ECGs to assign patients into two groups: HCM vs. non-HCM. The challenges and issues we address include missing ECG waves in one or more leads and the dimensionality of a large feature-set. We address these by proposing imputation and feature-selection methods. We develop heartbeat-classifiers by employing Random Forests and Support Vector Machines, and propose a method to classify full 12-lead ECGs based on the proportion of heartbeats classified as HCM. The results from our experiments show that the classifiers developed using our methods perform well in identifying HCM. Thus the two contributions of this thesis are the utilization of computational and statistical methods for discovering shortcomings in a current screening procedure and the development of methods to identify HCM through computational analysis of 12-lead ECG signals.

Cardiomyopathy in Offspring of Pregestational Diabetic Mouse Pregnancy

PURPOSE: To investigate cardiomyopathy in offspring in a mouse model of pregestational type 1 diabetic pregnancy.

METHODS: Pregestational diabetes was induced with STZ administration in female C57BL6/J mice that were subsequently mated with healthy C57BL6/J males. Offspring were sacrificed at embryonic day 18.5 and 6-week adolescent and 12-week adult stages. The size and number of cardiomyocyte nuclei and also the extent of collagen deposition within the hearts of diabetic and control offspring were assessed following cardiac tissue staining with either haematoxylin and eosin or Picrosirius red and subsequently quantified using automated digital image analysis.

RESULTS: Offspring from diabetic mice at embryonic day 18.5 had a significantly higher number of cardiomyocyte nuclei present compared to controls. These nuclei were also significantly smaller than controls. Collagen deposition was shown to be significantly increased in the hearts of diabetic offspring at the same age. No significant differences were found between the groups at 6 and 12 weeks.

CONCLUSIONS: Our results from offspring of type 1 diabetic mice show increased myocardial collagen deposition in late gestation and have increased myocardial nuclear counts (hyperplasia) as opposed to increased myocardial nuclear size (hypertrophy) in late gestation. These changes normalize postpartum after removal from the maternal intrauterine environment.

Heart failure in young adults

Heart failure (HF) is a major health concern affecting 15 million people in Europe and around 900 000 people in the U.K. HF predominantly affects the elderly, with the mean age of patients with a diagnosis of HF between 70 and 80 years. Most previous HF studies have accordingly focused on older patients. Although HF is less common in younger adults (<65 years), 15% to 20% of patients hospitalised with HF are younger than 60 years of age. Very few studies have described the characteristics of younger adults with HF and its outcome. The aims of this thesis are to describe the clinical characteristics of younger adults with HF, explore the epidemiology of HF in younger adults and determine their short- and long-term outcomes. This was made possible by access multiple databases consisting of large patient cohorts with HF. The first chapter is a systematic literature review of younger adults with HF. Gaps in the current literature were identified and the thesis focused on some of these. The CHARM study allows detail characterisations of younger adults with HF. It recorded characteristics of patients with HF, including symptoms and signs of HF, electrocardiographic changes, chest radiographic findings, and also left ventricular ejection fraction. HF hospitalisations and its precipitating factors were also recorded systematically. Younger adults were more likely to have a third heart sound and hepatomegaly, but less likely to have pulmonary crackles and peripheral oedema. Similarly, radiological findings in younger adults were less likely to show interstitial pulmonary oedema or pleural effusion. Interestingly, younger adults aged <40 years not only have similar HF hospitalisation rate to older patients, however during their presentation with decompensated HF, they were less likely to have clinical pulmonary oedema and radiological signs of HF. Physicians managing younger adults with HF need to be aware of this. Younger adults were also less compliant with medications and lifestyle restriction resulting in hospitalisation with decompensated HF. Fortunately, despite these challenges, mortality rates in younger adults with HF were lower compared to older patients. To further substantiate the findings from the CHARM study, the MAGGIC study, a meta-analysis consists of over 40 000 patients with HF from large observational studies and randomised controlled trials, was examined. In both databases, the commonest aetiology of HF in younger adults was dilated cardiomyopathy. The ejection fraction was the lowest in younger adults. Similar to the CHARM study, mortality rates in younger adults were lower compared to older patients. However, in the MAGGIC study, by stratifying mortality into patients with preserved ejection fraction and with reduced ejection fraction, younger patients with preserved ejection fraction have a much lower mortality rate compared to patients with reduced ejection fraction. Findings from clinical trials are not always reflective of the real life clinical practice. The U.K. Clinical Practice Research Datalink (CPRD), a large and well-validated primary care database with 654 practices contributing information into the database representing approximated 8% of the U.K. population, is a rich dataset offering a unique opportunity to examine the characteristics, treatments, and outcomes of younger adults with HF in the community. In contrast to the CHARM and MAGGIC studies, younger adults aged <40 years were stratified into 20-29 and 30-39 years in the CPRD analysis. This is possible due to the larger number of younger adults with HF. Further stratifying the younger age groups demonstrated heterogeneity among younger adults with HF. In contrast to previous data showing younger adults have lower co-morbidities, the proportions of depression, chronic kidney disease, asthma, and any connective tissue disease were high among patients aged 20-29 years in the analysis from the CPRD. Surprisingly, the treatment rates for angiotensin converting enzyme (ACE) inhibitor, and aldosterone antagonist were the lowest in patients aged 20-29 years. With the exception of patients aged ≥80 years, treatment rate with beta-blocker was also the lowest in patients aged 20-29 years. With over two decades of follow up, long-term mortality rates in younger adults with HF can be determined. The mortality rates continued to decline from 1988 to 2011. Physicians managing younger adults with HF can now use this contemporary data to provide prognostic information to patients and their family. A hospital administrative database is the logical next platform to explore younger adults with HF. The Alberta Ministry of Health database links an outpatient database to a hospitalisation database providing ample data to examine the relationship between outpatient clinic visits and hospital admissions in younger adults with HF. Following a diagnosis of HF in the outpatient setting, younger adults were admitted to the hospital with decompensated HF much sooner than older patients. Younger adults also presented to emergency department more frequently following their first hospitalisation for HF. In conclusion, this thesis presented the characteristics and outcomes of younger adults with HF, and helped to extend our current understanding on this important topic. I hope the data presented here will benefit not only physicians looking after younger adults with HF, but also patients and their family.

A Nine-Year-Old Girl With Left Ventricle Non-Compaction and Skin Lesions (Carvajal Syndrome)

Introduction: Arrhythmogenic right ventricular dysplasia (ARVD), a cardiomyopathy characterized by fibrofatty degeneration of the myocardium with progressive dysfunction, electrical instability, and sudden death, occurs in approximately 1 in 5000 people in the United States. Case Presentation: We present a nine-year-old girl complaining of dyspnea, easy fatigability and skin lesions. She had a history of an occasional epistaxis and weakness since 20 days before her admission, accompanied by the symptoms and signs of common cold, specially cough, during the last two days. Conclusions: This case does confirm that dilated cardiomyopathy’s spectrum is wider than ever known and that like what happened at the congress of Boston in 2006, a more comprehensive approach to its genetic types needs to be done.

Le pore oméga, un défaut biophysique commun aux mutations des canaux Nav1.5 causant le développement des arythmies associées à la cardiomyopathie dilatée

Les canaux sodiques dépendants du voltage (Nav) sont des protéines transmembranaires largement exprimées au sein de l’organisme. Ils sont responsables de l’initiation des potentiels d’action au niveau des cellules excitables et régissent ainsi de nombreuses fonctions physiologiques telles que les fonctions cognitives et sensorielles, les fonctions motrices et la fonction cardiaque. Au niveau du coeur, le sous-type Nav1.5 est majoritairement exprimé à la surface des cardiomyocytes. Leurs dysfonctions sont traditionnellement associées à de nombreux troubles électriques cardiaques. Des mutations de ces canaux ont récemment été reliées au développement d’un phénotype clinique complexe associant diverses arythmies et la cardiomyopathie dilatée (DCM), une atteinte morphologique. L’objectif de mon doctorat a donc été l’identification mais aussi la caractérisation d’un potentiel défaut biophysique commun à l’ensemble des mutations Nav1.5 associées au développement de ce phénotype clinique atypique. Premièrement, nous nous sommes intéressés à deux mutations des canaux Nav1.5 retrouvées chez des patients atteints de DCM, et dont les altérations biophysiques ont été décrites comme divergentes. L’étude parallèle de ces deux mutants nous a amenés à identifier une caractéristique commune : la création d’une nouvelle voie de perméation alternative au sein des canaux Nav1.5, le pore oméga. Dans un second temps, nous avons souhaité consolider l’association entre la création du pore oméga et le développement pathologique. Cette seconde étude portant sur deux autres mutants Nav1.5 a permis de confirmer l’apparition d’un pore oméga et ainsi d’accroître la suspicion du caractère délétère de ce pore oméga. Finalement, à l’aide d’une cinquième mutation des canaux Nav1.5, nous avons investigué les conséquences physiopathologiques de la création d’un pore oméga. Cette étude, a clairement démontré les conséquences néfastes d’un tel pore au niveau de l’homéostasie ionique cellulaire. Ces perturbations se répercutent par la suite sur les signaux électriques, les propriétés morphologiques mais aussi fonctionnelles des cardiomyocytes. Les études menées lors de mon doctorat ont ainsi abouti à l’identification du pore oméga comme étant une caractéristique biophysique commune aux mutations des canaux Nav1.5 associées au développement des arythmies et de la dilatation cardiaque.

Repeatability and validity of lens densitometry measured with Scheimpflug imaging

Purpose To assess the repeatability and validity of lens densitometry derived from the Pentacam Scheimpflug imaging system. Setting Eye Clinic, Queensland University of Technology, Brisbane, Australia. Methods This prospective cross-sectional study evaluated 1 eye of subjects with or without cataract. Scheimpflug measurements and slitlamp and retroillumination photographs were taken through a dilated pupil. Lenses were graded with the Lens Opacities Classification System III. Intraobserver and interobserver reliability of 3 observers performing 3 repeated Scheimpflug lens densitometry measurements each was assessed. Three lens densitometry metrics were evaluated: linear, for which a line was drawn through the visual axis and a mean lens densitometry value given; peak, which is the point at which lens densitometry is greatest on the densitogram; 3-dimensional (3D), in which a fixed, circular 3.0 mm area of the lens is selected and a mean lens densitometry value given. Bland and Altman analysis of repeatability for multiple measures was applied; results were reported as the repeatability coefficient and relative repeatability (RR). Results Twenty eyes were evaluated. Repeatability was high. Overall, interobserver repeatability was marginally lower than intraobserver repeatability. The peak was the least reliable metric (RR 37.31%) and 3D, the most reliable (RR 5.88%). Intraobserver and interobserver lens densitometry values in the cataract group were slightly less repeatable than in the noncataract group. Conclusion The intraobserver and interobserver repeatability of Scheimpflug lens densitometry was high in eyes with cataract and eyes without cataract, which supports the use of automated lens density scoring using the Scheimpflug system evaluated in the study

Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene

Hexachlorobenzene (HCB) is a persistent environmental contaminant that has the potential to interfere with steroid hormone regulation. The prostate requires precise control by androgens to regulate its growth and function. To determine if HCB impacts androgen action in the prostate, we used a number of methods. Our in vitro cell-culture-based assay used a firefly luciferase reporter gene driven by an androgen-responsive promoter. In the presence of dihydrotestosterone, low concentrations (0.5-5 nM) of HCB increased the androgen-responsive production of firefly luciferase and high concentrations of HCB (> 10 microM) suppressed this transcriptional activity. Results from a binding assay showed no evidence of affinity between HCB and the androgen receptor. We also tested HCB for in vivo effects using transgenic mice in which the transgene was a prostate-specific, androgen-responsive promoter upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. In 4-week-old mice, the proportion of dilated prostate acini, a marker of sexual maturity, increased in the low HCB dose group and decreased in the high HCB dose mice. In the 8-week-old mice, there was a significant decrease in both CAT activity and prostate weight upon exposure to 20 mg/kg/day HCB. Therefore, in vitro and in vivo data suggest that HCB weakly agonizes androgen action, and consequently, low levels of HCB enhanced androgen action but high levels of HCB interfered. Environmental contaminants have been implicated in the rising incidence of prostate cancer, and insight into the mechanisms of endocrine disruption will help to clarify their role.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

Temporal summation of intrinsically photosensitive retinal ganglion cell (ipRGC) contributions to the human pupil.

Purpose: IpRGCs mediate non-image forming functions including photoentrainment and the pupil light reflex (PLR). Temporal summation increases visual sensitivity and decreases temporal resolution for image forming vision, but the summation properties of nonimage forming vision are unknown. We investigated the temporal summation of inner (ipRGC) and outer (rod/cone) retinal inputs to the PLR. Method: The consensual PLR of the left eye was measured in six participants with normal vision using a Maxwellian view infrared pupillometer. Temporal summation was investigated using a double-pulse protocol (100 ms stimulus pairs; 0–1024 ms inter-stimulus interval, ISI) presented to the dilated fellow right eye (Tropicamide 1%). Stimulus lights (blue λmax = 460 nm; red λmax = 638 nm) biased activity to inneror outer retinal inputs to non-image forming vision. Temporal summation was measured suprathreshold (15.2 log photons.cm−2.s−1 at the cornea) and subthreshold (11.4 log photons.cm−2.s−1 at the cornea). Results: RM-ANOVAs showed the suprathreshold and subthreshold 6 second post illumination pupil response (PIPR: expressed as percentage baseline diameter) did not significantly vary for red or blue stimuli (p > .05). The PIPR for a subthreshold red 16 ms double-pulse control condition did not significantly differ with ISI (p > .05). The maximum constriction amplitude for red and blue 100 ms double- pulse stimuli did not significantly vary with ISI (p > .05). Conclusion: The non-significant changes in suprathreshold PIPR and subthreshold maximum pupil constriction indicate that inner retinal ipRGC inputs and outer retinal photoreceptor inputs to the PLR do not show temporal summation. The results suggest a fundamental difference between the temporal summation characteristics of image forming and non-image forming vision.

Peripheral refraction patterns out to large field angles

Purpose To investigate hyperopic shifts and the oblique (or 45-degree/135-degree) component of astigmatism at large angles in the horizontal visual field using the Hartmann-Shack technique. Methods The adult participants consisted of 6 hypermetropes, 13 emmetropes and 11 myopes. Measurements were made with a modified COAS-HD Hartmann-Shack aberrometer across T60 degrees along the horizontal visual field in 5-degree steps. Eyes were dilated with 1% cyclopentolate. Peripheral refraction was estimated as mean spherical (or spherical equivalent) refraction, with/against the rule of astigmatism and oblique astigmatism components, and as horizontal and vertical refraction components based on 3-mm major diameter elliptical pupils. Results Thirty percent of eyes showed a pattern that was a combination of type IV and type I patterns of Rempt et al. (Rempt F, Hoogerheide J, Hoogenboom WP. Peripheral retinoscopy and the skiagram. Ophthalmologica 1971;162:1Y10), which shows the characteristics of type IV (relative hypermetropia along the vertical meridian and relative myopia along the horizontal meridian) out to an angle of between 40 and 50 degrees before behaving like type I (both meridians show relative hypermetropia). We classified this pattern as type IV/I. Seven of 13 emmetropes had this pattern. As a group, there was no significant variation of the oblique component of astigmatism with angle, but about one-half of the eyes showed significant positive slopes (more positive or less negative values in the nasal field than in the temporal field) and one-fourth showed significant negative slopes. Conclusions It is often considered that a pattern of relative peripheral hypermetropia predisposes to the development of myopia. In this context, the finding of a considerable portion of emmetropes with the IV/I pattern suggests that it is unlikely that refraction at visual field angles beyond 40 degrees from fixation contributes to myopia development.

Dying feet in ICU : why might extracorporeal membrane oxygenation machines cause necrotic feet?

Background Extracorporeal membrane oxygenation (ECMO) is used for severe lung and/or heart failure in intensive care units (ICU). The Prince Charles Hospital (TPCH) has one of the largest ECMO units in Australia. Its use rapidly increased during the H1N1 (“swine flu”) pandemic and an increase in pedal complications resulted. The relationship between ECMO and pedal complications has been described, particularly in children, though no strong data exists. This paper presents a case series of foot complications in patients having received ECMO treatment. Methods We present nine cases of severe foot complications resulting from patients receiving ECMO treatment at TPCH in 2009–2012. Results Case ages ranged from 16 - 58 years and three were male. Six cases had an unremarkable medical history prior to H1N1 or H1N2 infection, one had Cardiomyopathy, one had received a lung transplant, and one had multi-organ failure post-sepsis. Common medications prescribed included vasopressors, antibiotics, and sedatives. All cases showed signs of markedly impaired peripheral perfusion whilst on ECMO and seven developed increasing areas of foot necrosis. Outcomes include two bilateral below knee amputations, two multiple digital amputations, one Reflex Sympathetic Dystrophy Syndrome, three pressure injuries, and three deaths. Conclusion Necrosis of the feet appears to occur more readily in younger people requiring ECMO treatment than others in ICU. The authors are conducting further studies to investigate associations between particular infections, medical history, medications, or machine techniques and severe foot complications. Some of these early results will also be presented at this conference.

Pupil shape as viewed along the horizontal visual field

Purpose: Changes in pupil size and shape are relevant for peripheral imagery by affecting aberrations and how much light enters and/or exits the eye. The purpose of this study is to model the pattern of pupil shape across the complete horizontal visual field and to show how the pattern is influenced by refractive error. Methods: Right eyes of thirty participants were dilated with 1% cyclopentolate and images were captured using a modified COAS-HD aberrometer alignment camera along the horizontal visual field to ±90°. A two lens relay system enabled fixation at targets mounted on the wall 3m from the eye. Participants placed their heads on a rotatable chin rest and eye rotations were kept to less than 30°. Best-fit elliptical dimensions of pupils were determined. Ratios of minimum to maximum axis diameters were plotted against visual field angle. Results: Participants’ data were well fitted by cosine functions, with maxima at (–)1° to (–)9° in the temporal visual field and widths 9% to 15% greater than predicted by the cosine of the field angle . Mean functions were 0.99cos[( + 5.3)/1.121], R2 0.99 for the whole group and 0.99cos[( + 6.2)/1.126], R2 0.99 for the 13 emmetropes. The function peak became less temporal, and the width became smaller, with increase in myopia. Conclusion: Off-axis pupil shape changes are well described by a cosine function which is both decentered by a few degrees and flatter by about 12% than the cosine of the viewing angle, with minor influences of refraction.