Molecular biology of the circadian clock in the rodent heart

Circadian rhythms are physiological and behavioural patterns with a period of approximately 24 hours that allow almost all organisms to anticipate predictable changes in their environment. In mammals, these rhythms are co-ordinated by a hypothalamic pacemaker, the suprachiasmatic nuclei (SCN) that is synchronised to solar time by retinal signals. Rhythms are generated at the cellular level by interlocking auto-regulatory transcriptional/post-translational feedback loops known as the circadian clock. The same mechanism exists in all mammalian cells investigated, including components of the cardiovascular system (CVS). Signals from the clock are translated into physiological rhythms via the regulation of clock-controlled genes (CCGs) and recent observations suggest that over 10% of the transcriptome is rhythmically expressed. Disruption of the clock is associated with chronic illness such as cardiovascular disease, cancer and diabetes and in the human CVS circadian rhythms in cardiac function and the occurrence of pathological events have been observed. The molecular targets of the clock in the heart are however largely unconfirmed. The aim of this thesis was to identify CCGs in the mouse heart and investigate their regulation by the clock using in vivo and in vitro approaches. SCN-dependent temporal expression and up-regulation by clock factors suggests for the first time that Bnp is a cardiac CCG and, with the putative CCGs Anp and Ms1, implicates the cardiac clock in regulating hypertrophy, and cardio-protection. Treatment of SCN-ablated mice with dexamethasone re-established circadian expression of Bnp and altered expression of most genes investigated, implicating glucocorticoids in synchronisation of the cardiac clock and target processes. Cardiac circadian expression of Pai-1 was confirmed and direct transcriptional regulation by the clock was demonstrated. A novel E-box-containing distal region in the Pai-1 promoter was identified which may be sufficient to generate cycling in dexamethasone-synchronised cells. E-box dependent activation of this distal region and the proximal promoter by clock and hypoxic factors suggests the E-box provides the molecular interface between circadian and stress pathways and that Pai-1 is a key integrator of the circadian clockwork and diverse physiological processes in the CVS. Together these and previous findings suggest the cardiac clock can control complex co-ordination of gene cascades and integrate diverse processes with adaptation to the temporal environment, providing a molecular explanation for the diurnal variation in cardiovascular events and suggesting new therapeutic targets