Regulation of RNA polymerase III transcription during cardiomyocyte hypertrophy

In comparison to cell division, the mechanisms underlying cell growth are poorly understood. Progress towards understanding the latter requires systems in which cell growth can be studied independently from proliferation. Cardiomyocytes terminally differentiate and lose the capacity to divide shortly after birth in mammals. Enlargement of existing cardiomyocytes accounts for the increase in heart size that occurs during post-natal development. This increase in cell size, in the absence of proliferation, is known as hypertrophy. Cardiomyocyte hypertrophy also occurs in the adult heart in response to a range of physiological stimuli, allowing the heart to adjust its contractile capacity according to demand. However, if sustained, cardiomyocyte hypertrophy is associated with several serious cardiovascular disorders, and is an independent risk factor for heart disease. One of the key hallmarks of hypertrophy is an increase in protein synthesis; however, the mechanisms responsible for this increased translation are not fully understood. RNA polymerase III (pol III) plays a vital role in protein synthesis by producing several components of the cellular biosynthetic machinery, including transfer (t)RNAs and 5S ribosomal (r)RNA. Thus, this project aimed to investigate whether pol III transcription is upregulated during hypertrophic growth, and if so, to elucidate the mechanisms responsible for such control. Various stimuli induce hypertrophy in primary cultures of rat cardiomyocytes. Data presented in this thesis show that this hypertrophic growth accompanies a substantial increase in transcription by pol III, along with markedly elevated levels of various pol III products, including tRNAs and 5S rRNA. Furthermore, analysis of mouse ventricular tissue demonstrated that pol III transcription is also enhanced during hypertrophy in the myocardium in situ. Several proteins are known to regulate transcription by pol III in proliferating cell lines. These include activators, such as c-Myc and extracellular signal- regulated kinase (ERK), and repressors, such as the retinoblastoma protein (RB). Summary In addition, these molecules have been implicated in the regulation of cardiomyocyte hypertrophy; therefore, their involvement in pol III transcriptional control was investigated in these terminally differentiated cells. This revealed that c-Myc and ERK, which are known to promote hypertrophic growth, can activate pol III transcription in cardiomyocytes. On the other hand, although normally associated with controlling the cell division cycle, RB has recently been implicated as a negative regulator of hypertrophy, and in the present study, RB was shown to attenuate transcription by pol III in the heart. The activation or inhibition of pol III transcription by these proteins is likely to contribute to their ability to induce or repress hypertrophic cell growth. Therefore, proliferating and non-dividing terminally differentiated cells appear to use some common means to regulate pol III transcription, and hence cellular biosynthetic capacity. However, a novel pol III transcriptional control mechanism was also identified in this study, namely the hypertrophy-associated, ERK-mediated induction of the pivotal pol Ill-specific transcription factor Brf1. Further work is required to establish whether Brf1 induction contributes to increased pol III transcription during the growth of other terminally differentiated cell types, or whether this mechanism is unique to cardiomyocytes. Thus, numerous mechanisms contribute to the control of transcription by pol III in cardiomyocytes, suggesting that such regulation is a critical determinant of hypertrophic cell growth. In summary, this thesis has identified a previously undescribed means by which hypertrophic stimuli could increase the protein synthetic capacity of cardiomyocytes, and has delineated the mechanisms responsible. This has important implications for understanding the molecular basis of pathological cardiomyocyte hypertrophy, and cell growth in general