Дисертації з теми "Congenital Cardiovascular Disease"

Cardiac magnetic resonance (CMR) is a non-invasive imaging modality highly reliable for studying cardiovascular morphology and function. Cardiac computed tomography (CCT) can give valuable anatomic information on CHD in children but implies radiation exposure, a relevant issue in children and newborns who are more radiosensitive than adult patients and have a longer lifetime to develop stochastic effects from radiation. We contributed to show the possibility to obtain an impressively low ionizing dose reduction in CHD patients also using standard 64-slice CT scanners. Conversely, CMR holds a pivotal role when functional and flow imaging is required. We showed the role of CMR in evaluating of patients percutaneously implanted with a pulmonary valve. Moreover, we proposed two new approaches for post-processing CMR images, regarding volume estimation of patients with a single ventricle, a rare CHD and a method for quantifying the paradoxical septal motion. CMR and CCT are two fundamental imaging techniques to evaluate patients with complex CHD. Both imaging modalities have limitations and advantages. CMR can evaluate heart function vessel flow but require a long acquisition time and in same patients a long sedation time. CCT has a very high spatial resolution and short acquisition time but implies ionizing radiation exposure. On the one side, we confirming the crucial role of CMR when function analysis is required but also showed the relevant possibilities of x-ray dose reduction in CCT, also using standard 64-slice scanners in the study of CHD patients.

Aim To characterise and explore the contemporary outcomes at key points in the life of individuals in New South Wales (NSW), Australia, who have undergone a procedure for structural heart disease. Methods The population with structural heart disease was identified and outcomes at key points in life were evaluated using different methodologies; record linkage, retrospective cohort study and systematic review. Health and education outcomes of children with structural heart disease were evaluated via population-based record-linkage cohort studies. The role of advanced imaging methods in managing structural heart disease in paediatrics was evaluated via literature review. A retrospective cohort study was used to evaluate individuals who had undergone a percutaneous pulmonary valve implantation for structural heart disease. Health outcomes of women (and their offspring) who had undergone a prosthetic heart valve placement prior to pregnancy were evaluated using population-based record-linkage studies, systematic review and meta-analysis Results The main findings of this thesis include: -Structural heart disease in NSW requiring procedural management in the first year of life affects 2.5 per 1 000 births. Immediate health outcomes such as length of stay and mortality are similar to other centers. -Children who have had a cardiac procedure demonstrate a greater incidence of poor education outcome. Sociodemographic risk factors and ongoing health status are the major predictors of educational outcomes. -Novel imaging strategies can aid diagnosis, monitoring and management in complex structural heart disease. -Percutaneous pulmonary valve implantation, for rehabilitation of the right ventricular outflow tract in structural heart disease, has a low risk of a serious adverse event and shorter length of stay than historical surgical options. -The risk of severe maternal morbidity, a cardiovascular event, preterm birth and small-for-gestation age infants remains higher for women with a heart valve prosthesis than a pregnancy in the general population. Conclusions From early childhood to adult life, including child bearing years, individuals who have undergone a cardiac procedure for structural heart disease remain at risk of altered health status. This includes additional hospitalisations, maternal morbidity as well as adverse developmental and educational outcomes compared to the general population. Contemporary data demonstrates improvements in some domains. Evolution in multi-disciplinary, life-long care provides hope for further ameliorating outcomes.

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Developing countries have been dealing with several difficulties concerning congenital heart diseases. Among them is lack of control of results through some specific database. The participation in the International Quality Improvement Collaborative Database for Congenital Heart Disease (IQIC) - Improving care in low- and middle-income countries provides an opportunity to improve quality of care targeting morbidity and mortality reduction, facilitated by the establishment of parameters and objective data to evaluate treatment offered. Objective: To analyze factors in the International Quality Improvement Collaborative Database for Congenital Heart Disease (IQIC) database of a single center of cardiology and pediatric cardiovascular surgery that influenced the quality of care to patients with congenital heart disease. Casuistic and Methods: Data collection from January 2011 to December 2017 independently and with external audit by IQIC database partnership. Data included preoperative information such as demographic data, nutritional status, associated chromosomal abnormalities, Risk Adjustment for Congenital Heart Surgery (RACHS-1) score, as well as postoperative information such as infections, complications in the first 30 days or until hospital discharge and / or patient death. Results: In the preoperative period, there was a clear trend of increasing newborn patient cases, in detriment of those 1 to 18 years of age. There was a reduction in cases of malnutrition from 70% in 2013 to 55% in 2017. The postoperative period reveled significant variation between groups’ surgical procedures in RACHS-1 risk category (P= 0.003), prevalence of risk categories 2 and 3, as well as an increase in cases of risk categories 4,5 and 6, mainly in the last two years. Infection and mortality showed favorable results for reduction, with statistical significance for surgical site infection (P= 0.03), bacterial sepsis and other infections (both P <0.001). The 30-day postoperative follow-up showed a satisfactory evolution for discrete reduction in mortality, but not statistically significant difference in both in-hospital death (P=0.16) and 30 days (P=0.14). Conclusion: The analysis of the seven years of the IQIC database showed significant decrease in infection, increase in complexity of cases and reduction of mortality of patients with congenital heart disease in our environment.
Países em desenvolvimento enfrentam diversas dificuldades em relação às cardiopatias congênitas, dentre elas a falta de controle de resultados por meio de banco de dados específico. A participação no banco de dados International Quality Improvement Collaborative for Congenital Heart Disease (IQIC) - Improving care in low- and middle-income countries forneceu oportunidade de melhoria da qualidade na assistência para a redução de morbidade e mortalidade infantil, facilitada pelo estabelecimento de parâmetros e dados objetivos para avaliação de tratamentos oferecidos. Objetivo: Analisar os fatores do banco de dados International Quality Improvement Collaborative for Congenital Heart Disease (IQIC) – Improving care in low and middle income countries de um centro único de cardiologia e cirurgia cardiovascular pediátrica que influenciaram a qualidade de atendimento aos pacientes com cardiopatias congênitas. Casuística e Método: Coleta de dados no período de Janeiro de 2011 a Dezembro de 2017 de forma independente e com auditoria externa em parceria com banco de dados IQIC. Os dados incluíram informações pré-operatórias, tais como: dados demográficos, estado nutricional, síndromes associadas e categoria de risco cirúrgico (Risk Adjustment for Congenital Heart Surgery - RACHS-1), assim como, informações pós-operatórias como infecções, complicações nos primeiros 30 dias até a alta hospitalar e ou óbito do paciente. Resultados: No período pré-operatório, observou-se nítida tendência de aumento de casos de pacientes recém-nascidos em detrimento aos de 1 a 18 anos. Encontrou-se redução de casos com desnutrição de 70% em 2013 para 55% em 2017. No período pós-operatório os procedimentos cirúrgicos classificados na categoria de risco RACHS-1 revelaram variação significante entre os grupos (P=0,003), prevalecendo as categorias de grau 2 e 3, assim como, aumento de casos de categorias de risco 4,5 e 6, principalmente nos dois últimos anos do estudo. A infecção e mortalidade demonstraram resultados favoráveis para a redução, com significância estatística para infecção de sítio cirúrgico (P=0,03), sepse bacteriana e outras infecções (P<0,001). O acompanhamento de 30 dias de pós-operatório mostrou evolução satisfatória para discreta redução dos óbitos, porém sem diferença estatística tanto para morte intra-hospitalar (P=0,16) como em 30 dias (P=0,14). Conclusão: A análise dos sete anos do banco de dados IQIC permitiu demonstrar a diminuição significante de infecção, aumento da complexidade das doenças e redução da mortalidade dos pacientes com cardiopatias congênitas em nosso meio.

Congenital heart disease is the most common congenital anomaly, affecting approximately 1% of all live births each year. Although clinical interventions are improving, many affected infants do not survive to adulthood. Congenital cardiac defects originate from disturbances during development, making the study of mammalian cardiogenesis critical to improving outcomes for infants with congenital heart disease. Development of the mammalian heart involves epigenetically-driven specification and commitment of a diverse landscape of cardiac progenitors. Recent studies determined that chromatin modifying enzymes play a previously underappreciated role in the pathogenesis of congenital heart defects. This thesis investigates the functions of Hdac1 and Hdac2, highly homologous Class I histone deacetylases, during early murine cardiac development. We establish that Hdac1 and Hdac2 cooperatively regulate cardiogenesis in distinct cardiac progenitor populations during development. Together, our findings demonstrate that Hdac1 and Hdac2 are critical mediators of the earliest stages of mammalian cardiogenesis through a variety of spatiotemporally specific, redundant, and dose-sensitive roles and indicate they may play important roles in the pathogenesis of human congenital cardiac defects.

Cardiovascular prostheses are routinely used in surgical procedures to address congenital malformations, for example establishing a pathway from the right ventricle to the pulmonary arteries (RV-PA) in pulmonary atresia and truncus arteriosus. Currently available options are fixed size and have limited durability. Hence, multiple re-operations are required to match the patients’ growth and address structural deterioration of the conduit. Moreover, the pre-set shape of these implants increases the complexity of operation to accommodate patient specific anatomy. The goal of the research group is to address these limitations by 3D printing geometrically customised implants with growth capacity. In this study, patient-specific geometrical models of the heart were constructed by segmenting MRI data of patients using Mimics inPrint 2.0. Computational Fluid Dynamics (CFD) analysis was performed, using ANSYS CFX, to design customised geometries with better haemodynamic performance. CFD simulations showed that customisation of a replacement RV-PA conduit can improve its performance. For instance, mechanical energy dissipation and wall shear stress can be significantly reduced. Finite Element modelling also allowed prediction of the suitable thickness of a synthetic material to replicate the behaviour of pulmonary artery wall under arterial pressures. Hence, eliminating costly and time-consuming experiments based on trial-and-error. In conclusion, it is shown that patient-specific design is feasible, and these designs are likely to improve the flow dynamics of the RV-PA connection. Modelling also provides information for optimisation of biomaterial. In time, 3D printing a customised implant may simplify replacement procedures and potentially reduce the number of operations required over a life time, bringing substantial improvements in quality of life to the patients

Cardiovascular disease and congenital heart disease impose a massive burden on society around the world. From the cost in terms of lost human lives and diminished quality of life, to the financial expense of ongoing medical treatment, the heart’s inability to effectively repair and regenerate itself presents a major challenge for medical research. The research conducted within this thesis hoped to contribute to our knowledge of the molecular pathways of myocardial development, and to explore the potential of olfactory derived stem cells to repopulate insulted myocardium. A combination of molecular biology and classical embryology techniques were first used to characterise two novel cDNAs identified in an earlier study as being upregulated in the regions of cardiac development within the chick embryo. cDNA and genomic library screening along with RACE (rapid amplification of cDNA ends) produced products which were sequenced to identify both the transcript and genomic sequence for both of the genes. Protein expression constructs were then used to identify the localisation of the encoded proteins, and whole mount in situ hybridisation utilised to identify the temporal and spatial expression patterns of the genes. The first cDNA was identified as the vertebrate homologue of the Drosophila e(y)2 gene, and produces a transcript of approximately 600 bp in the chick with a genomic structure consisting of 5 exons covering approximately 6 Kb. The encoded protein localises to the nucleus. Its expression is ubiquitous both temporally and spatially, which is at odds with its initial method of identification. The second cDNA remains novel at the time of submission, and shows no homology to any characterised genes. This cDNA, named C1-3C, produces two alternative transcripts, one of approximately 700 bp, and a second of 9.9 Kb, with a genomic structure showing no introns within the 2 Kb of analysed sequence. The encoded protein again localises to the nucleus. Expression of the C1-3C gene demonstrated a discrete pattern, though this pattern is again contrary to an up-regulation within the cardiogenic regions. Whilst unfortunately neither of the investigated genes appear to play a direct role in cardiac development, the aim of characterisation of these novel cDNAs was achieved.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
Full Text

Background: Single ventricle congenital heart defects with cyanotic mixing between systemic and pulmonary circulations afflict 2 per 1000 live births. Following the atriopulmonary connection proposed by Fontan and Baudet in 1971, the present procedure is the total cavopulmonary connection (TCPC), where the superior vena cava (SVC) and inferior vena cava (IVC) are sutured to the left pulmonary artery (LPA) and right pulmonary artery (RPA). However, surgeon preference dictates the implementation of the extra-cardiac and intra-atrial varieties of the TCPC. Overall efficiency and hemodynamic advantage of the competing methodologies have not been determined. Hypothesis: It is hypothesized that an understanding of the experimental fluid dynamic differences between various Fontan surgical methodologies in the TCPC allows for power loss evaluation toward improved surgical planning and design. Methods: Toward such analysis, a previously developed data processing methodology is applied to create an anatomic database of single ventricle patients from in vivo magnetic resonance imaging (MRI) to examine the gamut of TCPC anatomies. From stereolithographic models of representative cases, pressure and flow data are used to quantify control volume power loss to measure overall efficiency. particle image velocimetry (PIV) is employed to detail flow structures in the vasculature. Results are validated with dye injection flow visualization and 3-D phase contrast magnetic resonance imaging (PC-MRI) velocimetry, highlighting flow phenomena that cannot be captured with in vivo MRI due to prohibitively long scanning times. Preliminary results illustrate the variation of control volume power loss over several TCPC anatomies with varying flow conditions, the application of PIV, and validation approaches with 3-D PC-MRI velocimetry. Data from control volume power loss evaluation demonstrate a correlation with TCPC anatomy, providing added clinical knowledge of optimal TCPC design. Findings from PIV and 3-D PC-MRI velocimetry reveal a means for quantitatively comparing flow structure. Dye injection flow visualization offers qualitative insight into limitations of the selected velocimetry techniques.

Dilated cardiomyopathy (DCM) is the third most common cause of heart failure, often associated with arrhythmias and sudden cardiac death if not controlled. Metabolic and/or environmental factors, such as alcohol abuse, obesity, diabetes and Chagas disease, alter glycoprotein glycosylation, can lead to DCM. Inherited genetic disease, such as the human congenital disorders of glycosylation (CDG), causes multi-system manifestations including DCM. Non-congenital changes in glycosylation are also occurred in humans with and in animal models of DCM and heart failure. However, mechanisms responsible for glyco-dependent DCM are not understood. Here we sought to investigate the impact of sialylation and N-glycosylation in cardiac function. Partial reduction of N-α2,3-sialylation achieved through ST3Gal4 deletion (ST3Gal4-/-) led to adult late-onset DCM. The DCM symptoms progressed gradually, developing thinner left ventricular walls and dilation of all four chambers by 18-month old, but with preserved systolic function. Transverse aortic constriction (TAC) was used as a chronic stressor on 16-20 week old mice to determine whether the ability of the ST3Gal4-/- heart to compensate against pathologic insult is compromised. TAC’d ST3Gal4-/- mice presented with insufficient hypertrophy and reduced systolic function that deteriorated into congestive HF within six weeks post-surgery, while constricted WT hearts remained well-adapted throughout (ejection fraction, ST3Gal4-/-=34±5.2%; WT =53.8±7.4%; p<0.05).Calcineurin expression was decreased in ST3Gal4-/- (compared to TAC’d WT), contributed to the maladaptation of TAC’d ST3Gal4-/-. In order to better understand the role of glycosylation on cardiac function, we generated a cardiomyocyte specific knockout (αMHC-Cre) of glycosyltransferase responsible for synthesizing complex and hybrid N-glycans, Mgat1, (Mgat1CKO). Similar to but much more severe than that observed in ST3Gal4-/-, Mgat1CKO developed early-onset of DCM, late adult mortality, severely impaired cardiac systolic and diastolic function and frequent arrhythmias. Marked sex-difference in cardiac phenotype was observed in this autosomal gene (Mgat1) deletion, with male Mgat1CKO more severely affected. Both ST3Gal4 and Mgat1 did not participate in murine cardiogenesis, evidenced by normal litter size, Mendelian distribution of genotypes, no septal defect or vessel deformation under autopsy or echocardiography. In conclusion, we provided here the first and direct evidence of desialylation-elicited idiopathic dilated cardiomyopathy (DCM), reporting the cardiac phenotype of ST3Gal4-/-and cardiac-specific knockout of Mgat1. Our data showed sialylation and complex N-glycosylation are essential for cardiac function, and reduced N-glycosylation or sialylation leads to DCM development, contractile dysfunction and arrhythmia.

A system of 32 first-order differential equations is formulated and solved for the LP model using a fourth-order adaptive Runge-Kutta solver. The output pressure and flow waveforms obtained from the LP model are imposed as boundary conditions on the CFD model. Coupling of the two models is done through an iterative process where the parameters in the LP model are adjusted to match the CFD solution. The CFD model domain is a representative HLHS anatomy of an infant after undergoing the Hybrid Norwood procedure and is comprised of the neo-aorta, pulmonary roots, aortic arch with branching arteries, and pulmonary arteries. The flow field is solved over several cardiac cycles using an implicit-unsteady RANS equation solver with the k-epsilon turbulence model.; Hypoplastic left heart syndrome (HLHS) is a complex cardiac malformation in neonates suffering from congenital heart disease and occurs in nearly 1 per 5000 births. HLHS is uniformly fatal within the first hours or days after birth as the severely malformed anatomies of the left ventricle, mitral and aortic valves, and ascending aorta are not compatible with life. The regularly implemented treatment, the Norwood operation, is a complex open heart procedure that attempts to establish univentricular circulation by removing the atrial septum (communicating the right and left ventricle), reconstructing the malformed aortic arch, and connecting the main pulmonary artery into the reconstructed arch to allow direct perfusion from the right ventricle into the systemic circulation. A relatively new treatment being utilized, the Hybrid Norwood procedure, involves a less invasive strategy to establish univentricular circulation that avoids a cardiopulmonary bypass (heart-lung machine), deliberate cardiac arrest, and circulatory arrest of the patient during the procedure. The resulting systemic-pulmonary circulation is unconventional; blood is pumped simultaneously and in parallel to the systemic and pulmonary arteries after the procedure. Cardiac surgeons are deeply interested in understanding the global and local hemodynamics of this anatomical configuration. To this end, a multiscale model of the entire circulatory system was developed utilizing an electrical lumped parameter model for the peripheral or distal circulation coupled with a 3D Computational Fluid Dynamics (CFD) model to understand the local hemodynamics. The lumped parameter (LP) model is mainly a closed loop circuit comprised of RLC compartments that model cardiac function as well as the viscous drag, flow inertia, and compliance of the different arterial and venous beds in the body.
ID: 030423155; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2011.; Includes bibliographical references (p. 59-61).
M.S.
Masters
Mechanical, Materials, and Aerospace Engineering
Engineering and Computer Science

Com o objetivo de avaliar o atendimento dos pacientes submetidos à intervenção cirúrgica cardiovascular no protocolo de atendimento na via rápida (fast track recovery) em relação ao protocolo convencional, foi comparada a movimentação dos pacientes atendidos em ambos os protocolos nas diferentes unidades hospitalares. O estudo foi realizado em hospital público universitário especializado em cardiologia de 400 leitos, de referência terciária para o Sistema Único de Saúde. Foram estudados 175 pacientes, 107 (61%) homens e 68 (39%) mulheres, de idades entre 2 meses a 81 anos, dos quais 107 operados no protocolo da via rápida e 68 no protocolo convencional. Foram avaliadas variáveis demográficas, clínicas e, para avaliar a movimentação dos pacientes nas diferentes unidades hospitalares, as taxas de alta por unidade de tempo em cada unidade. A análise estatística foi feita por meio de análise exploratória, método de Kaplan Meier e modelo de riscos proporcionais de Cox. A análise de variância foi empregada para comparar o faturamento das despesas. A taxa de alta das diferentes unidades hospitalares por unidade de tempo dos portadores de cardiopatia congênita atendidos no protocolo da via rápida em relação ao protocolo da via convencional foi: a) 11,3 vezes a taxa de alta quando assistidos no protocolo na via convencional quanto ao tempo de permanência no centro cirúrgico; b) 6,3 vezes quanto à duração da intervenção cirúrgica; c) 6,8 vezes quanto à duração da anestesia; d) 1,5 vezes quanto à duração da perfusão; e) 2,8 vezes quanto à permanência na unidade de recuperação pós-operatória I; f) 6,7 vezes quanto à duração da hospitalização; g) 2,8 vezes quanto à permanência na unidade de internação pré-operatória; h) 2,1 vezes quanto à permanência na unidade de internação após a alta da unidade de terapia intensiva de recuperação pós-operatória. Para os portadores de cardiopatia isquêmica, as taxas de alta das unidades hospitalares para os protocolos de atendimento no protocolo da via rápida e no protocolo convencional não demonstraram diferença estatisticamente significante. Os valores de faturamento das despesas de internação dos portadores de cardiopatia congênita decorrentes de exames e procedimentos realizados nas fases pré- e pós-operatória e dos exames da fase trans-operatória foram menores quando os pacientes foram assistidos no protocolo da via rápida. Portanto, os portadores de cardiopatias congênita apresentaram menor permanência hospitalar nos recursos médicos hospitalares instalados, quando assistidos no protocolo de atendimento na via rápida, bem como menores despesas nas fases pré- e pós- operatória da internação.
Objective - To evaluate patient assistance in pre, per and postoperative phases of cardiac surgical intervention under fast track recovering protocol compared to the conventional way. Patients - 175 patients were studied, 107 (61%) men and 68 (39%) women. Ages 2 months to 81 years old. Patients included: first surgical intervention, congenital and ischemic cardiopathy without complexity, normal ventricular function and with at least 2 preoperative ambulatory consultations. Patients submitted to emergency surgeries were excluded. Interventions - assistance submitted by fast track and conventional protocol. Statistical analysis (measures) - exploratory, uni-varied (Kaplan Meier) and multi-varied (Cox) of the time in each admission unit. Hospital installations were classified in ambulatory, preoperative admission unit, surgical center, postoperative recovery unit and postoperative admission unit; the expression of this use was the discharge rate by unit of time from the significant interaction observed between assistance protocol and the kind of cardiopathy for the stay in the surgical center, surgical intervention time, stay in postoperative recovery unit, anesthesia time and time between admission and surgery dates. Results - the patients of congenital cardiopathy who underwent the protocol of conventional way recovery in relation to the fast track protocol, in the reliability range of 95% allows one to state that discharge rate by unit of time of the congenital cardiopathy patients assisted by the fast track protocol was: 11.3 times the discharge rate when assisted by the conventional way protocol as to the time of staying in the surgical center; 6.3 times as to the duration of the surgical intervention; 6.8 times as to duration of the anesthesia; 1.5 times as to the duration of the perfusion; 2.8 times as to the stay in the postoperative recovery unit; 6.7 times as to the stay in the hospital (period of time between the admission and the discharge date); 2.8 times as to the stay in the preoperative admission unit ( period of time between the admission date and the surgery date); 2.1 times as to the stay in the postoperative unit (period of time between the date of leaving the postoperative recovery unit and the date of discharge from the hospital). For the ischemia cardiopathy patients the risks concerning the protocols of recovery by the traditional way and the fast track were the same. CONCLUSIONS - The data concerning this study allows one to suggest that the assistance can be more efficient if one takes into consideration some variables studied in the protocol of fast track recovery. The congenital and ischemic cardiopathy patients presented shorter interval of time (concerning hospital stay in doctor-hospital installed facilities) when assisted in the fast track recovery protocol as well as fewer expenses with medical and hospital assistance.

Cardiac arrhythmias are caused by a disruption of the normal initiation or propagation of electrical impulses in the heart. Hundreds of mutations in genes encoding ion channels or ion channel regulatory proteins are linked to congenital arrhythmia syndromes that increase the risk for sudden cardiac death. This dissertation focuses on how mutations in a gene (KCNQ1) that encodes a voltage-gated K+ ion channel (Kv7.1) can disrupt proper channel function and lead to abnormal repolarization of atrial and ventricular cardiomyocytes. In the heart, Kv7.1 coassembles with a regulatory protein to conduct the slowly activating delayed rectifier K+ current (IKs). Loss-of-function KCNQ1 mutations are linked to type 1 long QT syndrome (LQT1), and typically decrease IKs, which can lead to ventricular action potential (AP) prolongation. In patients, LQT1 is often characterized by an abnormally long corrected QT (QTc) interval on an electrocardiogram (ECG), and increases the risk for polymorphic ventricular tachycardias. KCNQ1 mutations are also linked to atrial fibrillation (AF), but cause a gain-of-function phenotype that increases IKs. Surprisingly, patients diagnosed with both LQT1 and AF are increasingly identified as genotype positive for a KCNQ1 mutation. The first aim of this dissertation was to determine a unique functional phenotype of KCNQ1 mutations linked to both arrhythmia syndromes by functional analyses via the whole-cell patch clamp technique in HEK293 cells. A proportion of patients with LQT1-linked KCNQ1 mutations do not have abnormal QTc prolongation known as latent LQT1. Interestingly, exercise can reveal abnormal QTc prolongation in these patients. During exercise, beta-adrenergic activation stimulates PKA to phosphorylate Kv7.1, causing an increase in IKs to prevent ventricular AP prolongation. Therefore, the second aim of this dissertation was to determine a molecular mechanism of latent LQT1 through functional analyses in HEK293 cells while incorporating pharmacological and phosphomimetic approaches to study PKA regulation of mutant Kv7.1 channels. The findings in this dissertation provide new insight into how KCNQ1 mutations disrupt the function of Kv7.1 in a basal condition or during beta-adrenergic activation. Also, this dissertation suggests these approaches will improve patient management by identifying mutation specific risk factors for patients with KCNQ1 mutations.

The KCNE1 and KCNE3 type I transmembrane-spanning β-subunits assemble with the KCNQ1 voltage-gated K+ channel to afford membrane-embedded complexes with dramatically different properties. Assembly with KCNE1 produces the very slowly activating and deactivating IKs current that shapes the repolarization phase of cardiac action potentials. Genetic mutations in KCNQ1 or KCNE1 that reduce IKs current cause long QT syndrome and predispose affected individuals to potentially fatal cardiac arrhythmias. In contrast, complexes formed between KCNQ1 and KCNE3 produce rapidly activating and mostly voltage-independent currents, properties that are essential for function in K+ recycling and Cl−secretion in gastrointestinal epithelia. This thesis addresses how these two homologous accessory peptides impart their distinctive effects on KCNQ1 channel gating by examining two important protein regions: 1) a conserved C-terminal motif in the β-subunits themselves, and 2) the voltage sensing domain of KCNQ1 channels. Sequences in both the transmembrane domain and C-terminus of KCNE1 and KCNE3 have been identified as contributing to the divergent modulatory effects that these β-subunits exert. The homology of transmembrane-abutting C-terminal residues within the KCNE family and the presence of long QT-causing mutations in this region highlight its importance. A bipartite model of modulation was proposed that suggests the transmembrane domain of KCNE1 is passive, allowing the C-terminal domain to control modulation. Chapter II builds on this model by investigating the effect of mutating specific amino acids in the KCNE1 C-terminal domain. Point mutants that produce ‘high impact’ perturbations in gating were shown to cluster in a periodic fashion, suggesting an alpha-helical secondary structure that is kinked by a conserved proline residue and interacts with the Q1 channel complex. In Chapter III, the voltage sensing domain of Q1 channels is examined in the presence of either KCNE1 or KCNE3. To determine the influence of these two peptides on voltage sensing, the position of the S4 voltage sensor was monitored using cysteine accessibility experiments. In the slowly opening KCNQ1/KCNE1 complexes, voltage sensor activation appears to occur much faster than the onset of current, suggesting that slow channel activation is not due to slowly moving voltage sensors. KCNE3, on the other hand, shifts the voltage sensor equilibrium to favor the active state, producing open channels even at negative voltages. Taken together, these findings provide mechanistic detail to illustrate how two homologous peptides radically alter the gating properties of the same K+ channel and present a structural scaffold to map protein-protein interactions.

KCNE1 (E1) peptide is the founding member of the KCNE family (1-5), which is a class of type I transmembrane ß-subunits. KCNE1 peptides assemble with and modulate the gating, ion conducting properties and pharmacology of a variety of voltage-gated K+ channel a-subunits, including KCNQ1 (Q1). Mutations that interfere with the function of either E1 and/or Q1 and disrupt the assembly and trafficking of KCNE1- KCNQ1 channel complexes give rise to diseases such as Romano-Ward (RW) and Jervell Lange Nielsen Syndrome (JLNS), two different forms of Long QT Syndrome (LQTS). Using enzymatic deglycosylation assays, immunofluorescence techniques and quantitative cell surface labeling, we showed that KCNE1 peptides are retained in the early stages of the secretory pathway as immaturely N-linked glycosylated proteins. KCNE1 co-assembly with KCNQ1 leads to E1 progression through the secretory pathway and glycan maturation, resulting in cell surface expression. N-linked glycosylation of some membrane proteins is critical for proper folding, co-assembly and subsequent trafficking through the biosynthetic pathway. Previous studies have shown that genetic mutations that disrupt one of the two N-linked glycosylation sites on KCNE family members lead to LQTS (T7I, KCNE1 and T8A, KCNE2) (Schulze-Bahr et al., 1997; Sesti et al., 2000a; Park et al., 2003). Having confirmed that KCNE1 proteins acquire N-linked glycans, we examined the kinetics and efficiency of N-linked glycan addition to KCNE1. We showed that KCNE1 has two distinct N-linked glycosylation sites. The N-terminal sequon is a traditional co-translational site. The internal sequon (which is only ~ 20 residues away from the N-terminal sequon) acquires N-linked glycans primarily after protein synthesis (post-translationally). Surprisingly, mutations that prevent N-glycosylation at the cotranslational site also reduce the glycosylation efficiency of post-translational glycosylation at the internal sequon, resulting in a large population of unglycosylated KCNE1 peptides that are retained in the early stages of the secretory pathway and do not reach the cell surface with their cognate K+ channel. We showed that KCNE1 post-translational N-glycosylation in the endoplasmic reticulum is a cellular mechanism that ensures E1 proteins acquire the maximal number of glycans needed for proper channel assembly and trafficking. Our findings provide a new biogenic mechanism for human disease by showing that the JLNS mutation, T7I, not only inhibits glycosylation of the N-terminal sequon, but also indirectly prevents the glycosylation of the internal sequon, giving rise to a large population of assembly incompetent hypoglycosylated KCNE1 peptides. To further investigate the two N-linked glycosylation sites on KCNE1, we generated structure-function deletion scans of KCNE1 and performed positional glycosylation scanning mutagenesis. We examined the glycosylation pattern of glycosylation mutants in an effort to define the glycosylation window important for proper KCNE1 assembly and trafficking. Our findings suggested a nine amino acid periodicity to serve as a desirable glycosylation site and a better substrate for N-glycosylation. Appendix II shows work on the characterization of the C-terminally HA-tagged KCNE1 protein, which was used throughout the experiments presented in Chapter II, Chapter III and Chapter IV. Analysis of the C-terminally HA-tagged KCNE1 protein revealed that in heterologous expression systems KCNE1 had an internal translational start site, a methionine at position 27. A proteolytic cleavage site was also identified at the arginine cluster spanning residues 32 through 38 bearing the two known Long QT mutations (R32H and R36H) (Splawski et al., 2000; Napolitano et al., 2005). My work in Professor Craig C. Mello’s lab during the first four years of my graduate study is presented in Appendix I. The highly conserved Wnt/Wingless glycoproteins regulate many aspects of animal development. Wnt signaling specifies endoderm fate by controlling the fate of EMS blastomere daughters in 4-cell stage Caenorhabditis elegans embryos. A suppressor genetic screen was performed using two temperature sensitive alleles of mom-2/Wnt to identify additional regulators of the Wnt/Wingless signaling pathway during C. elegans endoderm specification. Five intragenic suppressors and three extragenic suppressors of mom-2/Wnt embryonic lethality were identified. We cloned ifg-1, eIF4G homologue, as one of the extragenic suppressors suggesting an intriguing connection between the Wnt signaling pathway and the translational machinery.

Indiana University-Purdue University Indianapolis (IUPUI)
MCTP2 (multiple C2 domain transmembrane containing protein 2) encodes a protein with poorly understood roles in lipid metabolism and lipid droplet biogenesis. Genetic studies previously identified variations in MCTP2 in conjunction with left ventricular outflow tract obstructive forms of congenital heart disease (CHD). This dissertation research aimed to delineate the biomedical significance of Mctp2 by investigating its expression and consequences of its genetic deletion in mouse models. Temporal and spatial expression of Mctp2 was investigated by RT-PCR and in-situ hybridization. A novel isoform, designated as isoform 2 in mice, results from alternative pre-mRNA splicing. Similar levels of Mctp2 isoforms 1 and 2 are present in embryonic tissues, whereas isoform 1 is preferentially expressed in adult tissues with high lipid metabolism. During mouse embryonic development, in-situ hybridization suggests expression of Mctp2 at the gut tube, liver bud and near the pharyngeal arches from E8.5 – E10.5. Given association of MCTP2 with CHD, the biological significance of Mctp2 was addressed using gene trap (GT) and conditional mouse models. Survival of Mctp2 GT mice was dependent on the genetic background strain, suggesting a role for strain-specific modifiers. Conditional knockout of Mctp2 in cardiac progenitor cells displayed no effect on survival. The role of Mctp2 in cardiac development remains to be delineated. The role of Mctp2 in cardiac function was addressed in both mouse models. Initial findings suggest Mctp2 allele dosage effects on the development of heart failure. GT mice lacking one, or both, copies of Mctp2 display cardiac systolic dysfunction, with upregulation of heart failure markers at 50 weeks of age in heterozygotes and increases in cardiac fibrosis in homozygotes. Systemic conditional deletion of Mctp2 did not show heart failure phenotypes using the strain protective from lethality. However, cardiac specific deletion of Mctp2 using the Nkx2.5-Cre driver, a line that is sensitized for cardiac dysfunction, led to decreased ejection fraction and fractional shortening in mice with conditional deletion of both copies of Mctp2 as well as Mctp2 dosage dependent penetrance of cardiac dilation. These studies of knockout mice suggest a role for Mctp2 in maintenance of cardiac function and possible genetic interaction with Nkx2.5.
2022-02-02

Thesis (M. Med.(Paediatrics and Child Health)) -- University of Limpopo, 2019
Background: Congenital heart disease (CHD) is a significant contributor to Under 5 Mortality rate(U5MR) in Limpopo. Atrioventricular septal defect (AVSD) is the best ascertained lesion in Limpopo and is strongly associated with Down syndrome. Few children from Limpopo with CHD including AVSD access cardiac diagnostic and surgical services. Objectives: The study aimed to enumerate, describe syndromes associated with AVSD and outcomes of children with AVSD at Pietersburg hospital. Methods: This is a Retrospective study of all children (n=80) diagnosed with AVSD from 1 January 2010 to 31 December 2014 at Pietersburg hospital. Data were drawn from echocardiogram reports and patient records. District Health Information Software (DHIS) data was used to obtain the number of live births per district during the 5 years study period. Results: Eight hundred and sixty six (n=866) patients had a first diagnosis of CHD confirmed on echocardiography and 80 (9.2%) of these had AVSD (an estimated 31.5% of expected cases of AVSD). Eighty four per cent (84%) of AVSD patients were associated with Down syndrome. 42/67 (63%) AVSD patients were referred for surgical assessment and of those 15/42 (36%) had surgery. The median interval between diagnosis and surgery was 13 months. Seventy five percent (n=50/67) of patients defaulted follow up. Conclusion: The study confirmed that most children with AVSD had associated Down syndrome and that the majority of children with AVSD from Limpopo do no access surgery. There is under referral of children with Down syndrome for screening of CHD.

The cardiac mechanical function plays a critical role in governing and regulating its performance under both normal and pathological conditions. The left ventricle has historically received more attention in both congenital and acquired heart diseases and was considered as the mainstay of normal hemodynamics. However, over the past few decades, there has been increasing recognition of the pivotal role of the right ventricle in determining functional performance status and prognosis in multiple conditions. Nonetheless, the ventricles should not be considered separately as they share the septum, are encircled with common myocardial fibers and are surrounded by the pericardium. Thus, changes in the filling of one ventricle may alter the mechanical function of its counterpart. This ventricular interdependence remains even after the removal of the pericardium because of constrictive pericarditis or during open chest surgery. Interestingly, during open chest surgery, only the right ventricle mechanical activity is visually checked by the surgeon and cardiologist due to the absence of an intraoperative imaging technique able to evaluate its complex function. Noteworthy, most of the imaging techniques available to clinicians are established for the assessment of the left ventricle, with the ejection fraction being the most used parameter. However, this value is a measure of global systolic function which comes short in identifying regional myocardial impairment and the mechanical contraction. Therefore, new approaches are needed to deeply investigate the mechanics of both ventricles and correctly assess the cardiac mechanical performance. In this thesis, I studied the mechanical function of the left ventricle through different modalities of cardiac magnetic resonance and employed an innovative imaging technique for the assessment of the right ventricle mechanical function during open chest surgery.

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
De acordo com os dados de 2017 da Organização Mundial da Saúde, as doenças cardiovasculares são a principal causa de morte a nível mundial. Se estes tipos de doenças não forem diagnosticadas e tratadas atempadamente, podem levar a insuficiências cardíacas ou outras complicações irreversíveis. As duas doenças cardiovasculares congénitas estudadas neste trabalho são a coarctação aórtica (CoA), caracterizada por uma estenose, habitualmente, na zona do arco da artéria aorta, e a doença da válvula aórtica (AvD), uma malformação ao nível da válvula aórtica. Estas doenças são responsáveis por cerca de 50,000 intervenções por ano. Deste modo, a melhoria métodos de diagnóstico e de intervenção adequados e eficientes é uma prioridade e pode levar ao decréscimo no número das intervenções, bem como reduzir a morbilidade e a mortalidade. A área de imagiologia médica de diagnóstico tem tido uma evolução significativa ao longo dos anos e é de extrema importância nas tentativas de substituição de métodos de diagnóstico invasivos. As imagens médicas são adquiridas e posteriormente processadas e analisadas, com recurso a programas adequados. Atualmente, é possível obter os valores de gradientes de pressão relativa a partir de Ecocardiografia Doppler e Ressonância Magnética. Contudo, os gradientes de pressão medidos no cateterismo cardíaco, o método gold standard para o diagnóstico de CoA e AvD, são gradientes de pressão absoluta. Nesta dissertação desenvolveu-se um método de diagnóstico de CoA e AvD, a partir dos mapas de pressão relativa no estreitamento da aorta e na válvula aórtica, respectivamente. O método matemático desenvolvido tem por base as equações de Poisson, resolvida com a condição de fronteira de Neumann utilizando os métodos de elementos finitos, e a de Navier Stokes para a conservação do momento. O método desenvolvido também tem em conta a informação proveniente da função de Windkessel da artéria aorta, uma artéria distensível. Esta função dá-nos o comportamento da propagação do pulso de pressão com uma velocidade de pulso de propagação. Deste modo, é observado um desfasamento temporal entre as curvas de fluxo da pressão e da velocidade, entre as duas regiões de interesse escolhidas. Deste modo, o método, denominado de Time-shift Corrected Pressure Maps (TCPM, sigla em inglês), permite obter os mapas de pressão absoluta, isto é, mapas de pressão que têm em conta o intervalo de tempo entre os picos de pressão na aorta descendente e ascendente, no caso do primeiro estudo, e antes e depois da válvula aórtica, no caso do segundo estudo. Os pacientes de ambos os estudos tinham indicação clínica para cateterismo cardíaco e foram submetidos a ressonância magnética cardiovascular de contraste de fase em tempo real (4D PC MRI, em inglês), para recolher as imagens ao nível da aorta e da válvula aórtica e os respectivos campos de velocidade da corrente sanguínea. O primeiro estudo tem como objetivo a aplicação do método TCPM a 27 pacientes de CoA (n=16 masculinos, n=11 femininos, faixa etária de 4 a 52 anos, idade média de 20±15 anos). Após aquisição das imagens, estas foram processadas usando programas específicos. Em primeiro lugar foi necessário segmentar a aorta, seguiu-se a seleção das regiões de interesse e, finalmente, a obtenção dos campos de velocidade e dos mapas de pressão relativa entre as duas regiões de interesse selecionadas. Após aplicação do método TCPM, foram aplicados testes estatísticos (correlação, teste t e Bland-Altman) para comparar os valores obtidos a partir de TCPM com os valores obtidos no cateterismo cardíaco. Após processamento das imagens dos 27 pacientes, 6 pacientes foram retirados do estudo. N=3 pacientes foram retirados porque a percentagem de fluxo que passa pelo estreitamento é insuficiente para calcular o gradiente de pressão a partir de TCPM e N=3 pacientes foram retirados porque a aorta não estava inserida por completo no FOV. As medições obtidas a partir de TPCM e cateterismo cardíaco têm uma correlação linear significante (R²=0,90; p<0,001). A partir dos gráficos Bland-Altman é possível verificar uma boa concordância entre as medições de ambos os métodos, com bias de -2,69 mmHg e os limites de concordância de ±4,74 mmHg. O teste de equivalência mostrou uma relação significante entre os métodos (p=0,007). O segundo estudo tem como objetivo a aplicação do método TPCM e o método da Área de Gorlin a 4 pacientes de AvD (n=4 masculinos, faixa etária 17 a 36 anos, idade média 27±7 anos). O método da Área de Gorlin permite obter o gradiente de pressão absoluta a partir da área geométrica da válvula e do fluxo total que passa nessa área. Após a aquisição das imagens, foi feito o processamento das mesmas. Numa primeira fase, as imagens foram segmentadas na região da válvula aórtica. Depois, as imagens segmentadas foram analisadas em dois programas distintos. O primeiro foi utilizado de forma a obter os campos de velocidade e os mapas de pressão relativa entre dois pontos antes e depois da válvula aórtica. O segundo permitiu definir a região da válvula como região de interesse e exportar os valores de velocidade, área, pressão relativa e fluxo absoluto nessa região. Os resultados mostram uma correlação linear significativa entre os valores de cateterismo cardíaco e de TCPM (R²=0,99; p<0,001). Os gráficos de Bland-Altman mostram uma boa concordância entre os valores de TCPM (24,75±22,50 mmHg) e de cateterismo (20,88±19,51 mmHg), com um bias de -3,87 mmHg e limites de concordância de ±3,64 mmHg. Os resultados também sugeriram uma ligeira subestimação dos valores do cateterismo cardíaco a partir do método da Área de Gorlin (14,47±13,00 mmHg), com um bias de 6,41 mmHg e limites de concordância de ±7,15 mmHg. Este estudo foi feito com uma amostra diminuta de 4 pacientes, o que não é suficiente para retirar conclusões com significância. Contudo, foi uma primeira abordagem positiva, que mostra a potencialidade que este método pode vir a apresentar. O método TCPM proposto neste projeto permite a medição não invasiva de gradientes de pressão absoluta a partir de mapas de pressão relativa em pacientes de CoA e AvD. Vários aspectos têm que ser tidos em conta de forma a garantir a eficácia deste método. Por exemplo, as regiões de interesse escolhidas têm que se cuidadosamente selecionadas de forma a serem perpendicular à direção do fluxo naquele local. Só desta maneira é possível obter o fluxo, os campos de velocidade e as pressões relativas corretas. Também, se o raio da estenose for menor que 2 voxéis, a relação sinal-ruído aumenta substancialmente, e a resolução especial da aquisição é insuficiente. Contudo, a aplicação do método TPCM a casos de grande estreitamento não é necessária visto que estes casos já são tipicamente identificados em imagens anatómicas de ressonância magnética e que o paciente segue automaticamente para intervenção quando a área do estreitamente representa cerca de 50% do valor de área típico da aorta. O método não invasivo TCPM apresenta uma boa concordância com o cateterismo cardíaco em termos da medição dos gradientes de pressão em CoA e AvD. Os bias e os limites de concordância entre cateterismo e TCPM foram substancialmente mais pequenos que os bias e os limites de concordância entre cateterismo e ecocardiografia Doppler e entre o cateterismo e o método da Área de Gorlin. Com os resultados apresentados já é possível ver o potencial desta técnica no processo de diagnóstico e decisão de intervenção em casos de CoA e AvD. Contudo, estudos com populações maiores será extremamente benéfico para validar clinicamente este método.
This dissertation aims to validate MRI-based time-shift corrected pressure mapping (TCPM) against cardiac catheterization in CoA and AvD patients. Also, in AvD patients, catheterization will be compared against Gorlin Area method. This project is divided in two independent studies: the first one for CoA patients and the second one for AvD patients, all with clinical indication for cardiac catheterization. In both CoA and AvD, clinical guidelines recommend treatment in the presence of a relevant pressure gradient. While reliable non-invasive measurement approaches would be crucial, the accuracy of currently available methods has been limited. In both studies, 4D PC-MRI was performed to compute relative pressure maps via Pressure-Poisson equation. To consider the patient-specific peak pressure time-shift from the ascending to the descending aorta and before and after the aortic valve, relative pressure gradient maps were corrected by the inertial term. Comparison between TCPM and invasive peak-to-peak measurements was performed using correlation, Bland-Altman plots and mean-equivalence t-test. In the first study, with a cohort of 21 patients with CoA, TCPM and catheter measurements showed significant linear correlation (R²=0.90; p<0.001). Bland-Altman plots demonstrated good agreement between TCPM and catheter derived pressure gradients with mean differences of -2.69 mmHg and 95% limits of agreement between -6.38 and 1.00 mmHg between methods. The mean-equivalence test was significant (p=0.007). In the second study, with a cohort of 4 patients with AvD, the catheterization measurements were compared against TPCM measurements. The results showed significant linear correlation (R²=0.99; p<0.001). Bland Altman plots showed a good agreement between TCPM (24.75±22.50 mmHg) and catheter derived peak-to-peak pressure gradients (20.88±19.51 mmHg), and suggested slight underestimation of the pressure gradients by the Gorlin Area method (14.47±13.00 mmHg). Non-invasive TCPM showed equivalence to pressure gradients from invasive heart catheterization in patients with CoA and AvD. However, in the AvD study, they were obtained for a very small cohort of patients and do not have sufficient statistical significance to validate the method for AvD patients.