Heart failure induced by cardiac hypertrophy is a cause of high mortality in the world and has been the fastest growing cardiovascular disease over the past decade. Cardiac hypertrophy is characterised as a reactive increase in cardiac mass growth with a complex of ventricular remodelling. It occurs initially as a compensatory response to an increased workload but eventually leads to cardiac dysfunction. An in-depth understanding of cardiac hypertrophy and the capacity to regulate it has profound clinical implications. The MAPK pathways provide an important connection between external stimuli and intracellular signals for cardiac hypertrophic response. At least four MAPK subfamilies have been identified: extracellular-regulated protein kinases 1 and 2 (ERK1/2), ERK5, c-Jun NH2-terminal protein kinases (JNKs) and p38 MAPKs. Mitogen-activated protein kinase kinase 4 (MKK4), a vital activator of JNK and p38 is implicated as an important mediator of hypertrophy. ERK5, an atypical MAPK, is also involved in both hypertrophic growth and cardiomyocyte survival. However, conflicting data have been yielded from previously-published studies, since the results are based entirely on experiments conducted in cultured cardiomyocytes or transgenic and conventional knockout mouse models. To elucidate their biological roles and underlying signalling mechanisms in hypertrophy, mice with a cardiomyocyte-specific deletion of MKK4 or ERK5 (MKK4cko and ERK5cko mice) were generated in the present study. In response to pathological hypertrophic stresses including pressure overload or isoprenaline stimulation, MKK4cko mice developed exacerbated pathological hypertrophy with increased cardiomyocyte apoptosis, impaired cardiac function and remarkably upregulated NFAT (nuclear factor of T-cell) transcriptional activity. However, MKK4cko mice exhibited a similar extent of swimming exercise-induced physiological hypertrophy compared with the controls. In response to pathological hypertrophic stimuli, ERK5cko mice were resistant to hypertrophic growth, foetal gene induction and ventricular fibrosis, which is associated with repressed activation of MEF2 (myocyte enhancer factor 2). ERK5 deficiency also caused a profound increase in cardiomyocyte apoptosis which accounted for the impaired cardiac function. In conclusion, the present study provides biological evidence that clarifies in vivo functions of MKK4 and ERK5 in hypertrophy. MKK4 acts a protective role against pathological hypertrophy through inhibiting NFAT signalling, but it is not necessary for the regulation of physiological hypertrophy. ERK5 is essential for pathological hypertrophic remodelling and cardiomyocyte survival and its function in hypertrophic remodelling is mediated through regulation of MEF2 activity. Taken together, these data presented in my thesis advances knowledge about biological functions of MAPK pathways in the heart.

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.