Introduction: Excitability in cardiomyocytes is dependent on the subthreshold current required to raise transmembrane potential to the activation threshold and subsequent recruitment of voltage gated sodium channels to trigger an action potential. Conduction in cardiomyocytes is dependent on the robustness and speed of action potential propagating through tissue. Both are equally important for normal heart function and claim to be linear correlated (i.e if conduction decreases, excitability decreases) Cardiac sodium channels are densely expressed in the intercalated disc within the perinexus, which is two orders of magnitude narrower than bulk extracellular interstitium. The biphasic relationship between conduction and perinexus is well-researched and consistent between computations models. We hypothesized a biphasic relationship between Excitability and perinexal width (Wp). In addition, we hypothesize that the relationship between excitability and conduction is not linear but dependent on the original width of the perinexus. Methods/and Results: Ex vivo guinea pig hearts were epicardially paced and optically mapped to assess ventricular conduction and excitability. Strength-duration curves were constructed for pacing stimuli to measure rheobase (inversely correlated to excitability). Computation models incorporating ephaptic coupling and sodium channel localization to cleft widths between cardiomyocytes demonstrate these findings. Conclusion: Models and experiments reveal that the excitability and perinexus relationship is biphasic where narrowing and widening perinexus decreases conduction and excitability thus showing a linear relationship between excitability and conduction. However, the excitability and conduction become overly complex in the transition phase from release of self-attenuation to reduced self-activation. Therefore, targeting ephaptic coupling and monitoring plasma ions may be a novel strategy for increasing the efficacy and efficiency of cardiac pacemakers. / Doctor of Philosophy / The heart is a muscular organ that uses electrical impulses to function. The heart is made of cells called cardiomyocytes that allow for electricity to flow through the cells. They are connected via different junctions such as gap junctions, adherens, etc. Any loss of electrical coordination leads to irregular heartbeats which can lead to heart death. There are two ways to study electrical coordination, excitability, how easy is for the current to start in the tissue, and conduction, how easy can that current travel through the tissue. Since the 1900s researchers have stated that if excitability decreases conduction decreases. In other words, if you need more current to start the heart (excitability decreases) then that current will travel slower through the tissue (conduction decreases) thus increasing one chances of irregular heartbeats. However, the understanding of how conduction works has changed but not of excitability. For example, originally current was thought to travel through channels called gap junctions. If you have limited availability of gap junctions, current increases (aka excitability decreases) and conduction decreases. However, other species such as frogs, fishes have limited number of gap junctions and can survive. Therefore, a new mechanism was proposed called ephaptic coupling. There is space next to the gap junctions called perinexus which is rich in a channel called Na channels, which is the main driving force for excitability and conduction. The lab has shown that if you change that space between cells, you can change the conduction response. In other words, if you decrease the space between the cells, conduction will not change therefore reducing the chances of irregular heartbeats. Therefore, my project is to understand if by changing this space between cells, is excitability and conductions are still correlates of each other. Using mathematical and animal models, this dissertation shows excitability and conduction have a very complicated relationship.

ECG-gated multidetector computed tomography (MDCT) is an imaging modality widely utilized for the evaluation of cardiac pathology by physicians. However, there has been little research of cardiac MDCT imaging in veterinary patients. Presently, ECG-gating is an upgrade for MDCT, which few veterinary institutions currently possess. The purpose of this study was to compare image quality between a 16 non ECG-gated and 64 ECG-gated MDCT for clinically important cardiac anatomy in dogs. In a crossover trial, six dogs were scanned using 16 non ECG-gated and 64 ECG-gated MDCT. A standardized anesthetic protocol, designed to induce bradycardia (mean HR 45 bpm ± 12.6) was used. Five post-contrast sequential scans through the heart were performed for each patient when utilizing the 16 non ECG-gated MDCT, in attempt to obtain a motion free series of images of the heart. For each scan, assessment of cardiac morphology was performed by evaluating a group of 21 cardiac structures, using a 3-point scale. Each of the images were scored as 0 (motion present, scan non-diagnostic), 1 (motion present, scan diagnostic), and 2 (no motion, therefore diagnostic scan of high quality). Quality scores (QS) from all scans within a dog (30 scans total) were assigned for each cardiac structure. QS from the six ECG-gated MDCT scans were of high diagnostic quality, generating diagnostic images for all of the 21 cardiac structures evaluated for each of the 6 scans. Individual non ECG-gated scans were of variable quality, primarily generating QS of 1 or 2. A complete set of diagnostic images for all 21 structures was not achieved from an individual scan. Minimum number of non ECG-gated scans to identify a single structure was calculated, and ranged from 1-2 scans for all structures. Cumulative number of sequential non ECG-gated scans needed to achieve images of all cardiac structures was calculated and determined to be 5. A 16 non ECG-gated MDCT scanner can produce cardiac images that are similar in quality, to those of 64 ECG-gated MDCT. Cardiac motion negatively impacts image quality in studies acquired without ECG-gating. However, this can be overcome by performing multiple sequential scans through the heart. / Master of Science

Background: Hyperthyroidism has substantial effects on the circulatory system. The cardiac biomarkers NT-proBNP and troponin I (cTNI) have proven useful in identifying cats with myocardial disease but have not been as extensively investigated in hyperthyroidism. Hypothesis: Plasma NT-proBNP and cTNI concentrations are higher in cats with primary cardiac disease than in cats with hyperthyroidism and higher in cats with hyperthyroidism than in healthy control cats. Animals: Twenty-three hyperthyroid cats, 19 cats with HCM without congestive heart failure, and 19 euthyroid, normotensive healthy cats eight years of age or older. Fourteen of the hyperthyroid cats were re-evaluated three months after administration of 131I. Methods: A complete history, physical examination, complete blood count, serum biochemistries, urinalysis, blood pressure measurement, serum T4 concentration, plasma concentrations of NT-proBNP and cTNI, and echocardiogram was prospectively obtained from each cat. Results: Hyperthyroid and HCM cats had plasma NT-proBNP and cTNI concentrations that were significantly greater than healthy older cats, but there was no significant difference between hyperthyroid and HCM cats with respect to concentration of either biomarker. In hyperthyroid cats that were re-evaluated three months after 131I treatment, plasma NT-proBNP and cTNI concentrations as well as ventricular wall thickness decreased. Conclusions and Clinical Relevance: Although there may be a role for NT-proBNP in monitoring the cardiac response to treatment of hyperthyroidism, neither NT-proBNP nor cTNI can be used to distinguish hyperthyroid cats from cats with HCM. Therefore, the thyroid status of older cats should be ascertained prior to interpreting results of cardiac biomarker testing. / Master of Science

Determinations of cardiac output (CO) by M-mode echocardio-graphy were compared with simultaneous determinations by thermodilution in 2 conscious and 5 anesthetized horses. A range of cardiac outputs was induced by use of a pharmacological protocol (dopamine, 4 ug/kg/min, dobutamine, 4 ug/kg/min, and 10 ug/kg detomidine plus 20 ug/kg butorphanol, in sequence). Changes from baseline CO in response to each drug were evaluated, and data was analyzed to determine whether there were any interactions between drug treatment and measurement method. The mathematical relationship between CO as determined by M-mode echocardio-graphy (COecho) and as determined by thermodilution (COTD) was described and used to predict COTD from COecho. The 2 methods were compared with respect to bias and variability in order to determine the suitability of COecho as a substitute for COTD . Sources of the variability for each method were determined. Determination of CO by either method in standing horses was prohibitively difficult due to patient movement. The pharmacologi-cal protocol was satisfactory for inducing a range of cardiac outputs for the purpose of method comparison; however, use of dopamine did not offer any additional benefit over the use of dobutamine and was generally less reliable for increasing CO. Inclusion of detomidine provided an additional change in CO but did not increase the overall range of CO over that produced by halothane and dobutamine. COecho and COTD were significantly related by the predictive equation COTD = (0.63 +/- 0.157) x COecho + (16.6 +/- 3.22). The relatively large standard errors associated with COecho measurements resulted in a broad 95% prediction interval such that COecho would have to change by more than 100% in order to be 95% confident that the determined value represents true hemodynamic change. COecho underestimated COTD by a mean of 10 +/- 6.3 l/min/450 kg. The large standard deviation of the bias resulted in broad limits of agreement (-22.3 to +2.3 l/min/450 kg). Measurement-to-measurement variability accounted for 28% of the total variation in COTD values and 64% of the total variation in COecho values. Results might be improved if the mean of 3-5 consecutive beats was used for each measurement, but as determined in this experiment, COecho is too variable to have confidence in its use for precise determinations of CO. / Master of Science

No / The aim of this research was to optimize x-ray image quality to dose ratios in the cardiac catheterization laboratory. This study examined independently the effects of peak x-ray tube voltage (kVp), copper (Cu), and gadolinium (Gd) x-ray beam filtration on the image quality to radiation dose balance for adult patient sizes. Methods: Image sequences of polymethyl methacrylate (PMMA) phantoms representing two adult patient sizes were captured using a modern flat panel detector based x-ray imaging system. Tin and copper test details were used to simulate iodine-based contrast medium and stents/guide wires respectively, which are used in clinical procedures. Noise measurement for a flat field image and test detail contrast were used to calculate the contrast to noise ratio (CNR). Entrance surface dose (ESD) and effective dose measurements were obtained to calculate the figure of merit (FOM), CNR2/dose. This FOM determined the dose efficiency of x-ray spectra investigated. Images were captured with 0.0, 0.1, 0.25, 0.4, and 0.9 mm Cu filtration and with a range of gadolinium oxysulphide (Gd2O2S) filtration. Results: Optimum x-ray spectra were the same for the tin and copper test details. Lower peak tube voltages were generally favored. For the 20 cm phantom, using 2 Lanex Fast Back Gd2O2S screens as x-ray filtration at 65 kVp provided the highest FOM considering ESD and effective dose. Considering ESD, this FOM was only marginally larger than that from using 0.4 mm Cu at 65 kVp. For the 30 cm phantom, using 0.25 mm copper filtration at 80 kVp was most optimal; considering effective dose the FOM was highest with no filtration at 65 kVp. Conclusions: These settings, adjusted for x-ray tube loading limits and clinically acceptable image quality, should provide a useful option for optimizing patient dose to image quality in cardiac x-ray imaging. The same optimal x-ray beam spectra were found for both the tin and copper details, suggesting that iodine contrast based imaging and visualization of interventional devices could potentially be optimized for dose using similar x-ray beam spectra.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Cardiac Magnetic Resonance Imaging (CMRI) is an important and valuable tool in the routine management of patients with cardiovascular disease. Contrast enhanced CMRI allows detection of ischemic and scarred myocardium, including rare infiltrative processes that cause cardiomyopathies. Amyloidosis comprises a group of diseases that are characterized by the extracellular deposition of insoluble fibrillar proteins in organs. The deposition of amyloid material in the heart leads to presentations of congestive heart failure mainly of the restrictive infiltrative pattern. There may be also conduction abnormalities. Dilated cardiomyopathy is a form of cardiomyopathy that is characterized by abnormal dilatation of the heart and subsequent heart failure. One of the characteristics of the cardiomyopathies and amyloidosis is the diffuse interstitial or replacement myocardial fibrosis. Myocardial fibrosis leads to impaired cardiac diastolic and systolic function and can lead to increased cardiovascular morbidity and mortality. Cardiac Magnetic Resonance Imaging (CMRI) with contrast can be used to characterize the extent of myocardial fibrosis through T1 mapping and as such it can be used as a prognostic indicator in amyloidosis and dilated cardiomyopathy. / 2999-01-02

The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

Since 1933, studies have explored the concept of trauma induced secondary cardiac injury (TISCI), yet till 2012, it had not been defined as the incidence of cardiac events and rise in cardiac biomarkers following traumatic injury. Despite, improvements in early outcomes, trauma patients have reduced long-term mortality with cardiac disease being the major contributor. Although many putative mechanisms have been suggested for TISCI, the underpinning pathophysiology still remains unclear. In this thesis, a prospective study of 290 critically injured patients identifies a 13% incidence of adverse cardiac events (ACE) with consistently raised serum h-FABP levels in these patients. H-FABP was found to be a good predictor of ACE through ROC analysis and a h-FABP of 16.8 ng/ml used to define trauma induced secondary cardiac injury (TISCI). TISCI was associated with longer hospital stay and higher mortality. Patients who developed ACE had higher plasma levels of adrenaline and noradrenaline with a correlating increase in plasma h-FABP. On multivariate analysis, hypertension was the only independent risk factors for ACE. The increase in serum cardiac biomarkers was reflected by an increase in serum h- FABP in our group's trauma hemorrhage murine models. The hearts of these models were used in the experiments that form the last experimental chapter of this thesis. Protein expression studies confirm this increase in serum h-FABP by evidence of concurrent leaching in the cardiac tissue, along with Troponin I. Myocardial injury was evident on electron microscopy with evidence of interstitial and organelle oedema, myofibrillar degeneration, nuclear condensation and changes in mitochondrial morphology. Immunohistochemistry and western blotting protein studies demonstrate the translocation of the mitochondrial death-related protein AIF to the cytosol and nucleus, where it becomes its active pro-apoptotic form. This thesis propositions the utility of the cardiac biomarker h-FABP in predicting ACE and outcomes in critically injured patients. Although increasing serum noradrenaline and adrenaline levels are associated with higher incidence of ACE and biochemical evidence of cardiac injury with rising h-FABP levels, multivariate analysis negates their value as independent predictors of ACE. Leaching out of the proteins h-FABP and Troponin I in the murine cardiac tissue confirmed the value of serum measurements of these proteins as markers of cardiac injury. This was associated with widespread ultrastructural myocardial damage in the TH mice with changes in mitochondrial morphology. The mitochondrial damage seen is associated with the translocation of the mitochondrial death-related protein AIF to the cytosol and the nucleus where I propose its canonical signaling leading to nuclear degradation and cell death is the driver of cardiac dysfunction.

Proper heart function results from the periodic execution of a series of coordinated interdependent mechanical, chemical, and electrical processes within the cardiac tissue. Central to these processes is the action potential - the electrochemical event that initiates contraction of the individual cardiac myocytes. Many models of the cardiac action potential exist with varying levels of complexity, but none account for the electrophysiological role played by caveolae - small invaginations of the cardiac cell plasma membrane. Recent electrophysiological studies regarding these microdomains reveal that cardiac caveolae function as reservoirs of 'recruitable' sodium ion channels. As such, caveolar channels constitute a substantial and previously unrecognized source of sodium current that can significantly influence action potential morphology. In this thesis, I formulate and analyze new models of cardiac action potential which account for these caveolar sodium currents and provide a computational venue in which to develop and test new hypotheses. My results provide insight into the role played by caveolar ionic currents in regulating the electrodynamics of cardiac myocytes and suggest that in certain pathological cases, caveolae may play an arrhythmogenic role.

Angiotensin II signals cardiovascular disease through metalloproteinases including MMP-2, MMP-7 and ADAM-17/TACE. We hypothesized that these metalloproteinases regulate each other at the transcriptional level. Further, MMP-2, being a major gelatinase in cardiac and vascular tissue, could mediate angiotensin II-induced cardiovascular disease. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography and qRT-PCR analysis of hypertrophy marker genes) and fibrosis (by collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. Angiotensin II induced hypertension, cardiac hypertrophy and fibrosis which correlated with an upregulation of MMP-2. Downregulation of MMP-2 by pharmacological inhibition and RNA interference attenuated hypertension but not cardiac hypertrophy or fibrosis. Downregulation of MMP-7 or ADAM-17/TACE by RNA interference attenuated angiotensin II-induced MMP-2 upregulation as well as hypertension, cardiac hypertrophy and fibrosis. We conclude that MMP-2 selectively mediates angiotensin II-induced hypertension under the transcriptional control of MMP-7 and ADAM-17/TACE.