Beta-adrenergic receptor signalling and excitation-contraction coupling in the heart: impact of circadian rhythms and beta-3 receptors

A time-of-day variation in myocardial contraction and its response to sympathetic stimulation of β-adrenoceptors (b-ADR) exists, which is reflected by time-of-day variations in intracellular calcium [Ca2+]i regulation and electrical activity. There are three isoforms of β-ADR (β1/β2/β3) and the functional outcome of each receptor differs. β1/β2-ADRs result in positive inotropism, whereas β3-ADR induces a negative inotropic effect, associated with nitric oxide (NO) signalling. The aims of this thesis were to determine the role of the β3-ADR in the time-of-day variation in the response to sympathetic stimulation previously shown by our group and to investigate the role β1-ADR, β2-ADR and β3-ADR play the control of Ca2+ regulation in isolated ventricular myocytes. Ventricular myocytes were isolated during the rest-period (ZT3) and active-period (ZT15) of male Wistar rats by enzymatic digestion. [Ca2+]i was measured in myocytes loaded with Fura-2. Arrhythmic activity was determined from video imaging, myocytes were electrically field-stimulated at 1Hz. Measurement of action potentials, transient outward (Ito) and L-type calcium (LTCC) currents were made using whole-cell patch-clamp recordings. Investigation of the time-of-day variation in response to sympathetic stimulation revealed a time-of-day variation in basal systolic [Ca2+]i and in the increase in systolic [Ca2+]i following β-ADR activation with ISO, greatest in rest-period (ZT3) myocytes. This did not appear to be governed by the time-of-day variation in action potential duration I observed or in the response of β3-ADR agonist-induced stimulation. Unlike conventional β1-ADR, β2-ADRs are tightly coupled to phosphodiesterase’s (PDE) through the inhibitory G-protein (Gi). Our data suggests this coupling to Gi and PDE activation is responsible for the reduced response of systolic Ca2+ to β2-ADR agonists, reducing the arrhythmic potential of β2-ADR activation. The reduction in systolic [Ca2+]i by β3-ADR activation with BRL37344 shows a time-of-day variation, with a greater negative inotropic response in rest-period (ZT3) myocytes, mediated via NO and PDE, leading to an increase in Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) activity but a reduction in SR Ca2+ content. This action contributes to an anti-arrhythmic action of the β3-ADR agonist BRL37344 against β1-ADR agonist induced arrhythmia. The ability of β3-ADR activation to reduce arrhythmic activity in response to further sympathetic stimulation is likely to exhibit a time-of-day variation. This work highlights the importance of chrono-pharmacology in the use of β3-ADR agonists as a treatment in arrhythmia management.