Macrophages, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) are key factors in atherosclerotic plaque formation and plaque rupture, both of which are driven by macrophage accumulation and activation. The thesis investigated whether distinct phenotypes of macrophages may play different roles in MMP or TIMP expression during inflammation or plaque formation. The results showed that classically activated Ml macrophages significantly up-regulated MMP-l, MMP-3, MMP-7, MMP-IO, MMP-12, MMP-14 and MMP-25 and down-regulated TIMP-3, which suggests a strong increase in matrix-degrading potential. Patterns of response to classical activation in vitro are dominated by LPS and pro-inflammatory cytokines, only MMP-12, MMP-14 and MMP-25 expressions being partly affected by interferon-y. On the other hand, alternatively activated M2 macrophages strongly up-regulated MMP-II, MMP-12 and MMP-25, and this was accompanied by lower expression of many other MMPs and greater expression of TIMP-3. Hence alternative activation may rather selectively increase the potential for elastolysis by MMP-12, without promoting degradation of other matrix components. The MMP expression stimulated by classical activation depended on activation of mitogen activated protein kinases, phosphinositide-3 kinase, and inhibitor of K.8 kinase-2. In addition, MMP-l, and MMP-I0 co-localised with nuclear localised NF-KB in human atherosclerotic plaques. Furthermore, unstable plaques have higher COX-2, MMP-I, MMP-3, MMP-IO and MMP-14 expression in foam cell macro phages when compared to stable human coronary atherosclerotic plaques. By contrast, stable plaques had higher expression of the M2 marker, CD206, and TIMP-3 than human unstable plaques. In conclusion, Ml macrophages selectively increase the balance of several MMPs over TIMPs in macrophages in vitro and in unstable plaques, an example of inflammation in vivo. However, only limited groups of MMPs and TIMP-3 were significantly over-expressed in M2 macrophages.

Congenital heart disease (CHD) is a leading non-infectious cause of death in children. Dysregulation of heart development is at the root of the disease, which is determined to some extent by genetic factors. TBX5 and MEF2A are two of the transcription factors implicated in the process of heart development. Mutations of TBX5 cause Holt-Oram syndrome with cardiac abnormalities, and mutations of MEF2A were found in coronary heart disease patients. To widen the knowledge of CHD and to investigate more genes involved in cardiac development, we performed micro array analysis on RNA from heart tissues following knockdown of TBX5 and MEF2A in chick embryos. Out of 32,773 transcripts screened, 16 novel chick genes were selected for further analysis. One of them, designated LOC418543, shows specific expression in the inflow and outflow tracts during the early stages of chick embryonic heart development. The expression of this gene remains obvious in the outflow tract during heart tube looping, suggesting a potential involvement in heart morphogenesis. In humans, a predicted gene, hmm296404, has been identified as the potential homolog of LOC418543, and hmm296404 is expressed in human fetal heart RNA samples. During the study, the extracelluar matrix coding genefibulin 2 has attracted our attention as it shares similarity with LOC418543 at the protein level, and its high expression pattern in the outflow tract of the heart. Mutational scanning was performed for the whole coding region of the gene in 174 CHD patients having outflow tract defects. Eight putative non-synonymous mutations were found, and five of them are unique to the CHD patients in my study.

LQTl (Long QT Syndrome 1) and CPVTl (Catacholaminergic polymorphic ventricular tachycardia-i) are two commonly prevalent cardiac channelopathies. Whilst LQTl accounts for ~45-54% of all genotyped cases of LQTS (Medeiros-Domingo et al., 2007), CPVTl is thought to be accountable for ~15% of all cases of sudden cardiac death (Priori et ai, 2002). Limitations to currently available models of these disorders include the inter-specific differences in cardiac electrophysiology associated with animal models, the lack of a multi- ion channel phenotype in recombinant cell models and the limited availability of human cardiac tissue (Rajamohan et ai, 2013). This necessitates the development of humanised hPSC-based models of LQTl and CPVTl that can: 1) help better model and understand their pathogenesis and 2) aid in the development of new strategies for their treatment. Skin fibroblasts were derived from 3x LQTl patients and lx CPVTl patient. These were reprogrammed into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated into functional cardiomyocytes (hiPSC-CMs). Spontaneously beating cells were analysed, by multi-electrode array and patch clamp technologies, for their electrophysiology and response to pharmacology. Initial experiments have shown that, relative to controls, LQTlhiPSC- CMs exhibit prolonged action potential durations under basal conditions, and that CPVTl-afflicted hiPSC-CMs develop putative DADs on treatment with isoprenaline - electrophysiological hallmarks of LQT and CPVT, respectively. Further experiments are ongoing to confirm these observations and to demonstrate that these iPSC models are capable of responding appropriately to a range of clinically relevant pharmaceuticals. This will ascertain the suitability of these cells as humanised assays for the evaluation of new treatments of LQTl and CPVTl, respectively. This study also looked at deriving hiPSCs from a range of different tissue types in order to compare the feasibility and efficiencies of hiPSC generation from different cell sources and to give potential donors the option of making tissue donations in a manner that is less invasive and painful than a skin biopsy. In this regard, hiPSCs were successfully derived from skin, tooth and oral gingival samples. Finally, steps were taken to increase the feasibility of the use of hPSC-CMs as large-scale pharmaceutical screens. First, an improvement in the cardiac differentiation of hiPSCs was achieved by modulation of the BMP/Activin/Nodal cardiac signalling pathways. Next, in collaboration with engineers at Loughborough University, a novel MEA (multi-electrode array) II biochip was designed and fabricated with a view to allow cost-effective and high-throughput MEA-based assessment of hPSC-CM electrophysiology. Lastly, in collaboration with scientists at BioFocus (Cambridge), steps were taken to translate the patch clamp analysis of hPSC-CMs onto a commercially available robotic, planar patch platform - The PatchXpress (Molecular Devices). III

Cardiovascular complications, including cardiac hypertrophy, arrhythmias and sudden death, are commonly associated with muscular dystrophies. Null mutations of the integrin alpha? gene, an essential mediator of cellular attachment to the extracellular matrix in cardiac and skeletal muscle, leads to progressive muscular dystrophy in humans, which is faithfully replicated in the integrin alpha? deficient (a7-/-) mouse model. In the latter, premature sudden death is not directly attributable to the dystrophic phenotype. We therefore analysed the cardiac phenotype in integrin a7-/- mice to determine whether their premature death is associated with altered cardiac structure and function and/or altered cardiac rhythm.

Dilated cardiomyopathy (DCM) is a leading cause for cardiac transplantation with an estimated prevalence of 36.5 per 100,000 individuals in the USA.  DCM is a disorder characterized by ventricular dilation and systolic contractile dysfunction.  It is the result of a heterogenous group of inherited and acquired disorders and is an important cause of heart failure.  Idiopathic DCM is the most common cause of DCM and accounts for <span style='font-family: Symbol'>~50% of cases with a familial basis found in up to 35%. A genome wide screen in four generations of a Puerto Rican kindred with autosomal dominant isolated familial dilated cardiomyopathy (FDCM) was conducted.  Two-point linkage analysis mapped the disease locus to chromosome 14q11.2-13 (lod score = 5.11 at <i><span style='font-family:Symbol'>q </i> = 0).  The <span style='font-family:Symbol'>b-myosin heavy chain (<span style='font-family:Symbol'>b-MHC) candidate gene mapped to this region and direct sequencing identified a single nucleotide T168C mutation (Ser<span style='font-family:Symbol'>®Pro⁵³²) which was highly conserved across a range of different species.  A novel mismatch primer that converted the half-site to a full recognition site for the <i>Sph</i>I endonuclease independently confirmed the mutation and its absence in 192 normal chromosomes.  A further disease causing mutation 2378^C^{<span style='font-family:Symbol'>®G} replacing Phe764Leu in <span style='font-family:Symbol'>b-MHC was identified in an unrelated family.  A further two unrelated FDCM families were found to have deleted one of four tandem lysine residues (<span style='font-family:Symbol'>DLys210) of the cardiac troponin T gene. Understanding the monogenic disorders provides us with important knowledge not only about the rare condition being studied but also about potential mechanisms underlying the more common phenotypes.  This finding may open up novel and perhaps unpredictable therapeutic targets.

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The mutations E361G and E99K in the cardiac actin gene cause dilated and hypertrophic cardiomyopathy respectively. Transgenic mouse models have been developed that over-express these mutations in their hearts. Mutant protein was expressed at 50% of to tal actin. Thin filaments were reconstituted with mouse actin, human cardiac tropomyosin and troponin, and studied by in vitro motility assay. Phosphorylated E361G and non-transgenic thin filaments were indistinguishable at all Ca2+-concentrations (EC50 NTg/E361G =1.07±0.11, p=0.59). E361G-actin reconstituted with dephosphorylated troponin had a lower Ca2+-sensitivity than non-transgenic (EC50 NTg/E361G = 0.47±0.09, p=0.01). When we compared E361G thin filaments containing phosphorylated and dephosphorylated troponin, the Ca2+-sensitivity was indistinguishable (EC50 E361G/E361G.dp = 1.03±0.08, p=0.74). This is in contrast to non-transgenic actin, where the EC50 of the phosphorylated thin filament was 3-times greater than dephosphorylated (p=0.003). The major functional change induced by the E361G mutation was therefore the abolition of the response to troponin phosphorylation. This would blunt the inotropic response in vivo and be the cause of the E361G DCM phenotype. The in vitro motility technique was extended to measure α-actinin binding affinity. The E361G mutant actin had a 5.3-fold reduced affinity for α-actinin, indicating a weaker attachment of filaments at the Z-line. Investigation of the DCM-causing mutation TNNC1 G159D in troponin isolated from human muscle showed a higher Ca2+ sensitivity with native phosphorylation levels (EC50 NF/G159D = 4.67±1.87) and upon dephosphorylation (EC50 NF/G159D = 1.77±0.29). G159D thin filaments also blunted the change in Ca2+-sensitivity when dephosphorylated (EC50 G159D/G159D.dp =1.24±0.17). The E99K mutation produced a strikingly different pattern of results to DCM mutations of an increased Ca2+-sensitivity and faster cross-bridge cycling rate, with phosphorylated (EC50 NTg/E99K = 2.54±0.64, p=0.07) and dephosphorylated troponin (EC50 NTg/E99K =1.2). The E99K mutation also 3 produced a reduced but not completely suppressed response to troponin phosphorylation (EC50 E99K/E99K.dp = 1.14±0.06, p=0.12).

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