published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

Introduction There is a pressing clinical need to treat patients with acute coronary syndrome (ACS) timely and efficiently in order to improve their prognosis. Standard tools available in ED, while useful, do not comprehensively characterize ACS for either diagnosis or risk stratification. The role of CMR in ACS is emerging because it allows assessment of both myocardial composition and function. Newer CMR techniques such as: a) T2 W imaging for assessing myocardial oedema and area at risk B) pre contrast T1 mapping techniques for quantitative characterization of the tissue composition, are adding further utility for CMR in ACS. At present the clinical use of these techniques is still limited and further investigations are needed to assess their clinical applicability in ACS patients. Aims The aims of this thesis were several. Firstly we sought to establish a CMR protocol for imaging ACS patients on a 3T CMR scanner. In order to do so, we validated a novel T2 W technique for oedema imaging (T2 prep SSFP) at 3T. Second, we aimed to perform a detailed study of the time course of oedema in ACS patients in order to establish the appropriate imaging time for the assessment of area at risk. Third, by applying T2W acute oedema imaging, we sought to investigate the functional and pathological meaning of complicated remote plaques in patients with multivessel disease. Finally, we aimed to establish whether, in comparison to standard CMR techniques, novel precontrast Tl mapping allows better characterisation of the acutely injured myocardium and whether it can predict long-term functional recovery. Methods The research studies were all performed on a 3T Trio Siemens scanner. In the initial stage of the research, we validated the T2 W technique performing phantom work and scanning both volunteers and patients to assess the uniformity of signal intensity in the myocardium and to establish a threshold based method to post process the images. We then established a CMR protocol for ACS including oedema imaging, T1 mapping imaging, perfusion, functional and late gadolinium enhancement imaging. Patients with acute myocardial infarction (both ST elevation myocardial infarction (STEMI) and non STEMI) were scanned at 4 different time points after the acute event (3 scans within 2 weeks and one at 6 months). All STEMI patients underwent primary percutaneous coronary intervention (PCI) while the non-STEMI patients underwent coronary angiography and for PCI. Results We validated the T2prep SSFP technique at 3T, highlighting its limitations and establishing a threshold of mean ± 2SD to assess myocardial oedema. We found that the optimal imaging window to assess the maximal expression of myocardial oedema was within 1 week from the acute event in patients with ST elevation MI. Also, our results showed a reduction of LGE over time (from acute to chronic) in segments which also showed improvement in contractile function indicating that even segments with transmural LGE assessed in the early hours post event could be viable. By applying these techniques in acute patients with bystander disease undergoing percutaneous coronary intervention, we found that: l) T2W imaging can detect myocardial injury downstream from a vessel identified as "non culprit" 2) in 20% of NSTEMI patients, the angiographic assessment alone failed to identify the culprit vessel. Finally, we found that the diagnostic performance of acute pre-contrast Tl-mapping was at least as good as that ofT2W CMR for detecting myocardial injury. There was a significant relationship between the segmental damaged fraction assessed by either by LGE or T2W, and mean segmental Tl values and the likelihood of improvement of segmental function at 6 months decreased progressively as acute Tl values increased. Conclusions In summary, we defined a stable imaging window for the retrospective evaluation of area at risk and we also indicated that acutely detected LGE does not necessarily equate with irreversible injury and may severely underestimate salvaged myocardium. Furthermore, in NSTEMI patients with multivessel disease, by revealing acute myocardial damage in territories pertaining to vessels not treated acutely, we raised the issue of the need for better tools for the correct identification of the culprit vessel and to stratify patients rather than by angiographic assessment alone. Finally, we demonstrated how pre-contrast Tl mapping allows for assessment of the extent of myocardial damage and how Tl mapping might become an important complementary technique to LGE and T2W for the identification of reversible myocardial injury and the prediction of functional recovery in acute MI.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, South Africa 2017. / Almost half of all cases of heart failure have a preserved ejection fraction. However, therapy targeting the mechanisms of this disorder has not improved outcomes. Left ventricular (LV) diastolic dysfunction is a characteristic feature of heart failure with a preserved ejection fraction. A more sound understanding of the mechanisms responsible for LV diastolic dysfunction produced by risk factors may lead to better approaches to preventing this syndrome. Although obesity is thought to be a major risk factor for LV diastolic dysfunction, this does not occur in all obese individuals. In the present thesis I have demonstrated in 737 randomly recruited participants from a community sample of African ancestry, that the relationship between insulin resistance (homeostasis model) and LV diastolic function, as assessed from trans-mitral velocity (E/A) and tissue Doppler imaging of the lateral and septal walls of the LV (e’ and E/e’), is markedly altered by the presence of a more concentrically remodelled LV (as indexed by LV relative wall thickness [RWT]). Importantly, insulin resistance was only associated with LV diastolic function or dysfunction in those with an RWT above a threshold value. In contrast no interactive effects on LV diastolic function between either blood pressure or age and RWT were noted. These data therefore suggest that obesity will only translate into LV diastolic dysfunction if it is associated with insulin resistance and a concentrically remodeled LV. Although hypertension is thought to play an important role in contributing to LV diastolic dysfunction, the pulsatile hemodynamic change primarily responsible for this effect is uncertain. In 524 randomly selected individuals from a community sample I have demonstrated that independent of confounders including left ventricular mass and RWT, aortic backward wave pressure effects (as determined using wave separation analysis), antedate the impact of aortic stiffness (indexed by aortic pulse wave velocity) or the factors determined by aortic stiffness (the time of backward wave return or forward wave pressures) on LV filling pressures (E/e’). These data therefore suggest that to adequately prevent LV diastolic dysfunction, targeting aortic backward wave pressures may be required. As conventional risk factors account for only a portion of the inter-individual variations in LV diastolic function, it is thought that the genetic factors may play a iv significant role. In 694 randomly recruited participants of African ancestry belonging to nuclear families, I demonstrated that independent of conventional risk factors, heritability accounts for approximately 50% of the variation in LV RWT, an important LV structural determinant of LV diastolic function. Moreover, in 442 randomly recruited individuals of African ancestry belonging to nuclear families, I also demonstrated that heritability accounts for approximately 50% of the variation in the index of LV filling pressures, E/e’, independent of LV mass or RWT remodeling and aortic function. These data provide strong evidence that genetic factors responsible for LV diastolic dysfunction and the structural determinants thereof should be sought. In conclusion, the results provided in the present thesis have advanced our knowledge of possible pathophysiological mechanisms that play a role in the development of LV diastolic dysfunction and hence possibly heart failure with a preserved ejection fraction. / MT2017

Echocardiography has an prominent role in the era of CRT by virtue of its non-invasive nature with high feasibility and reproducibility. The clinical applications include not only quantification of the change in systolic function, hemodynamics, LV volume, or mitral regurgitation, but also assessment of systolic dyssynchrony. A number of new echocardiographic techniques were employed in this study, such as tissue Doppler imaging (TDI) and its post-processing modalities including strain, strain rate and displacement mappings, tissue synchronization imaging (TSI), as well as three-dimentional (3D) echocardiography. / For heart failure patients with wide QRS complexes who received CRT, LV volumes, cardiac function and synchronicity were shown to change acutely between CRT-on and CRT-off modes by both 2D and 3D echocardiography methods. Furthermore, the usefulness of 3D echocardiography and its accuracy in assessing volumetric changes / Systolic dyssynchrony, which illustrates discoordinated contraction of the heart, is relatively common in heart failure patients, in particular those with prolonged QRS complexes. It is caused by electromechanical delay in some regions of the failing heart and will result in further reduction of cardiac function. Cardiac resynchronization therapy (CRT) is a rapidly evolving pacing modality for advanced heart failure, characterized by implantation of the left ventricular (LV) lead through coronary sinus to the free wall region. It is recommended to patients who have refractory heart failure despite optimal medical treatment, LV dilatation with ejection fraction lower than 35%, and prolonged QRS duration on surface ECG. / The main findings were as followed: The Ts-SD was 17.0+/-7.8ms in normal control, 33.8+/-16.9ms in narrow QRS group and 42.0+/-16.5ms in wide QRS group, respectively. The prevalence of systolic dyssynchrony in heart failure population was 43% in the narrow QRS group, and 64% in the wide QRS group, when a Ts-SD of > 32.6 ms (+2 SD of normal controls) was used to define significant dyssynchrony. QRS duration does not have a linear relationship with systolic dyssynchrony. / TSI was useful to predict a reverse remodeling and gain in ejection fraction after CRT. Qualitative identification of the latest peak systolic contraction at the lateral wall was a quick and specific guide to predict a favorable reverse remodeling response while quantitative computation of "Asynchrony Index" from 12 LV segments in ejection phase was beneficial in the absence of lateral wall delay. In conclusion, the improvement of cardiac function and LV reverse remodeling after CRT is more obvious in heart failure patients with wide QRS complex and echocardiographic evidence of significant systolic dyssynchrony. Reverse remodeling is not only an objective measure of favorable responses, but also a prognosticator of disease outcomes. "Asynchrony Index" is a strong predictor of LV reverse remodeling response after CRT. Assessment of systolic dyssynchrony by various echocardiographic tools is promising, however, further studies are needed to compare the predictive values of different parameters objectively and prospectively. / We performed echocardiography with TDI in 200 subjects, including 67 patients with heart failure and narrow QRS complexes (≤ 120ms), 45 patients with heart failure and wide QRS complexes (>120ms), and 88 normal controls, which served as a polit study. Severity and prevalence of systolic dyssynchrony were assessed by the maximal difference in time to peak myocardial systolic velocity (Ts-dif-12) and the standard deviation (Ts-SD) of the 12 LV segments. / We recruited a group of seventy patients with chronic heart failure who fulfilled the established criteria and received CRT. Serial echocardiographic assessment with clinical evaluation was performed at baseline, predischarge, 1-month, 3-month, 6-month and long-term follow up. The objective was to demonstrate the improvement of cardiac function and dyssynchrony after CRT by echocardiographic parameters, in particular the reduction of LV end-systolic volume (LVESV) which is also known as reverse remodeling, and its relationship with the improvement in clinical status and prognosis. We also attempted to identify dyssynchrony parameters which are useful in predicting LV reverse remodeling after CRT. / Zhang Qing. / "October 2006." / Adviser: Yu Cheuk-Man. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5852. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Cardiac pacing has been the only effective treatment in the management of patients with symptomatic bradycardia caused by sinus node dysfunction or atrioventricular block for decades. Conventional dual-chamber pacing is performed by implanting two leads in right atrial (RA) appendage and right ventricular (RV) apex separately. RV apex is the most commonly applied pacing site because it can be easily reached and allows a chronically stable position and stimulation thresholds. However, large randomized clinical trials have suggested that right ventricular apical (RVA) pacing may cause abnormal ventricular contraction and reduce pump function and lead to myocardial hypertrophy, in particular in patients with impaired left ventricular (LV) function. Recent studies have also reported a reduced LV systolic function in patients with pacing indications and preserved ejection fraction. The deleterious effects of RVA pacing on LV function may be related to the abnormal electrical and mechanical activation pattern or ventricular dyssynchrony. During RVA pacing, conduction of the electrical wave front propagates slowly through ventricular myocardium rather than through the His-Purkinje conduction system, comparable to left bundle branch block (LBBB). In addition , RVA pacing alters ventricular synchrony and loading conditions which may result in diastolic heart failure with abnormal LV relaxation, high filling pressure and low cardiac output state. Furthermore, it is possible that left atrial (LA) remodeling and reduction of atrial function may occur during RVA pacing . However, it is not been carefully studied. / Echocardiography is a convenient, non-invasive and established tool to assess cardiac function in clinical practice. Conventional two-dimensional echocardiography is useful to assess cardiac chamber size, volume and function. With the development of real time three-dimensional echocardiography (RT3DE) and color tissue Doppler imaging (TDI), echocardiography provides further valuable information and more accurate measurements which include myocardial velocity and parameters of dyssynchrony. In the present study, the main echocardiographic parameters including the maximal left atrial volume (LAVmax), pre-atrial contraction volume (LAVpre) and the minimal left atria l volume (LAVmin) were assessed by two-dimensional echocardiography. Peak systolic (Sm-la), peak early diastolic (Em-la), peak late diastolic (Am-la) velocities of left atrium (LA) and atrial conduction time (from onset of P wave on electrocardiogram to onset of atrial velocity) were measured by TDI. / In a cross-sectional study, ninety-eight patients who had been implanted with RVA-based dual-chamber pacemakers were enrolled. Four patients with pacing dependent were excluded. Eventually 94 patients were included in the final analysis. Echocardiography was performed (iE33, Philips) during intrinsic ventricular conduction (V-sense) and RVA pacing (V-pace) modes with 15 minutes between switching modes. We aimed to investigate if RVA pacing has any acute effects on atrial remodeling and function in patients with preserved ejection fraction (LV ejection fraction> 45%). The result showed that during V-pace, LA volumes increased significantly when compared with V-sense (LAVmax: 52.0 +/- 18.8 vs. 55.2 +/- 21.1 ml, p = 0.005; LAVpre: 39.8 +/- 16.4 vs. 41.3 +/- 16.6 ml, p = 0.014; LAVmin: 27.4 +/- 14.0 vs. 29.1 +/- 15.1 ml, p = 0.001) . TDI parameters showed significant reduction in Sm-la (3.0 +/- 1.1 vs. 2.7 +/- 0.9 cm/s, p < 0.01), Em-la (2.7 +/- 1.1 vs. 2.4 +/- 1.0 cm/s, p = 0.001). However, there was no change in Am-la. / In a prospective study, patients with symptomatic bradycardia, preserved ejection fraction, and received RVA pacing were recruited. Echocardiography was performed at both baseline and one year follow up through a standard protocol by experienced echocardiographers. LA volumes and velocities as well as intra- and interatrial dyssynchrony were measured offline with the use of dedicated software. The objectives of this study were to investigate: (1) if RVA pacing has any deleterious effects on LA remodeling and function during long-term follow up; (2) if RA appendage pacing has separate effects on atrial pump function, intra- and interatrial dyssynchrony; (3) if atrial dysfunction and dyssynchrony can predict atrial high rate episodes (AHREs) burden in the first year of RVA pacing. The main findings of this study were: (a) at one year follow up, LA volumes and indexes were increased with reduction in passive emptying fraction and total emptying fraction. Atrial velocities showed significant reduction when compared with baseline; (b) in multivariate regression analysis, the ratio of transmitral early diastolic filling velocity to mitral annular early diastolic velocity (E/e') > 15 at one year and reduction of LV ejection fraction ≥ 5% were independent predictors of reduction of Am-la > 30%; (c) high percent of RA appendage pacing prolonged atrial conduction and induced intra- and interatrial dyssynchrony. (d) Am-la < 5.3 cm/s can predict AHREs burden which had a sensitivity of 71% and specificity of 75%. / In conclusion, our studies suggest even short-term RVA pacing induces LA dilatation and impaired passive atrial function, though it did not have direct effect on active atrial contractility. However, chronic RVA pacing results in LA remodeling and reduces atrial function with decreased contractility. This was more likely to occur in those with impaired LV ejection fraction and evidence of diastolic dysfunction. Atrial dysfunction and interatrial dyssynchrony can predict AHREs burden after chronic RVA pacing. Therefore, measures that may minimize such adverse effect of pacing on atrial function need to considered for patients receiving RVA pacing, such as the use of new pacing modalities. / Xie, Junmin. / "December 2010"--Abstract. / Adviser: Yu Cheuk Man. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 142-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Background: Recent studies have revealed an association between genetic variants at the ADAMTS7 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 7) locus and susceptibility to coronary artery disease (CAD). ADAMTS-7 has been reported to facilitate vascular smooth muscle cell (VSMC) migration and promote neointima formation. We sought to study the functional mechanisms underlying this relationship and to further investigate the role of ADAMTS-7 in atherosclerosis. Methods and Results: In vitro assays showed that the CAD-associated non-synonymous single nucleotide polymorphism rs3825807, which results in a serine to proline (Ser-to- Pro) substitution at residue 214 in the ADAMTS-7 pro-domain, affected ADAMTS-7 prodomain cleavage. Immunohistochemical analyses showed that ADAMTS-7 localised to vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) in human coronary and carotid atherosclerotic plaques. Cell migration assays demonstrated that VSMCs and ECs from individuals who were homozygous for the adenine (A) allele (encoding the Ser214 isoform) had increased migratory ability compared with cells from individuals who were homozygous for the G allele (encoding the Pro214 isoform). Western blot analyses revealed that media conditioned by VSMCs of the A/A genotype contained more cleaved ADAMTS-7 pro-domain and more of the cleaved form of thrombospondin-5 (TSP-5, an ADAMTS-7 substrate that had been shown to be produced by VSMCs and inhibit VSMC migration). In in vitro angiogenesis assays, ECs of the A/A genotype exhibited increased capillary-like network formation. ADAMTS-7 over-expression in ECs by transfection of an ADAMTS7-214Ser expressing plasmid significantly accelerated EC migration and in vitro angiogenesis, whereas ADAMTS-7 knockdown by shRNA had opposite effects. Preliminary proteomics analyses of conditioned media of ECs overexpressing ADAMTS-7 and ECs with ADAMTS-7 knockdown indicated that ADAMTS- 7 can cleave thrombospondin-1 (TSP-1), a well-recognised angiogenesis inhibitor. Conclusion: The results of this study indicate that rs3825807 has a functional effect on ADAMTS-7 maturation, TSP-5 cleavage, VSMC and EC migration, and angiogenesis. As VSMC migration and angiogenesis play important roles in atherosclerosis, these results provide a mechanistic explanation for the association between rs3825807 and CAD.

The development and function of the heart is governed by a conserved set of transcription factors (TFs) that regulate gene expression in a cell-type, time point and stimulus driven manner. Of these core cardiac TFs, the most ubiquitously expressed is the zinc finger protein GATA4. In cardiomyocytes, GATA4 is central to proliferation, differentiation, hypertrophy and induction of pro-survival pathways. In cardiac endothelial cells, it is required for valve and septal development, although the exact mechanisms remain unclear. To regulate such a wide array of functions in a spatially and temporally controlled manner, GATA4 interacts with specific protein partners, the majority of whom have been identified in cardiomyocytes. However, a complete understanding of the protein interactome of GATA4, particularly in cardiac endothelial cells, has not yet been achieved. Using a mass spectrometry-based approach, we have identified a series of novel GATA4 interacting partners in cardiac endothelial cells. 3xFlag GATA4 was stably overexpressed via retroviral transduction in the TC13 cardiac endothelial precursor cell line, immunoprecipitated from nuclear protein extracts and sent for HPLC-ESI-MS/MS. Several novel GATA4 interacting partners were identified including the chaperone protein Heat Shock Protein 70 (HSP70), the inducible orphan nuclear receptor Nerve Growth Factor 1β (NGFIβ, NUR77) and the Drosophila-Binding/Human Splicing protein family members Non-POU Domain Containing Octamer Binding Protein (NONO) and Paraspeckle 1 (PSPC1). Chapter 1 discusses the interaction between GATA4 and HSP70 and its role in cardiomyocyte survival upon exposure to chemotherapeutic agent Doxorubicin (DOX). HSP70 binds directly to GATA4, preventing DOX-mediated cleavage and degradation by Caspase-1, cardiomyocyte cell death and heart failure. Chapter 2 focuses on the cooperative interaction between GATA4 and NUR77 in cardiac microvascular endothelial cells and its central role in myocardial angiogenesis in response to pressure overload. The GATA4-NUR77 complex transactivates the promoter of Angiopoietin-Like 7 (ANGPTL7), a secreted pro-angiogenic chemotactic factor, triggering endothelial cell proliferation and tube formation in cultured cardiac endothelial cells and increasing myocardial capillary density in vivo. Chapter 3 discusses the interaction between GATA4 and the DBHS proteins NONO and PSPC1 in the regulation of cardiac development. These proteins play opposing roles when bound to GATA4 as PSPC1 enhances GATA4 activation of critical cardiac promoter targets and NONO acts as a rheostat to repress GATA4 activity. In vivo, loss of NONO results in left ventricular non-compaction consistent with humans with loss-of-function mutations. However, simultaneous Gata4 haploinsufficiency partially rescues this phenotype. Together, this data identifies multiple novel cell type and time point specific GATA4 protein partners and sheds light on GATA4 regulatory mechanisms in cardiac development and disease.

Chow Tsun Cheung, Louis. / "May 2000." / Thesis (M.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.