Dissecting the mechanisms of pacemaking in the heart using photo-affinity probes and pharmacological intervention

The aim of the work described in this thesis was to dissect the mechanisms underlying pacemaking in the heart using both pharmacological and chemical tools. The mechanisms underlying pacemaker activity in the sino-atrial node are controversial, with some researchers giving prominence to the role of the 'funny' current, If, others emphasising a 'calcium clock', and with still others highlighting the elusive sustained inward current, Ist. Autonomic modulators, particularly adrenaline and noradrenaline regulate all the proposed mechanisms. To investigate which adrenoceptors are responsible for driving positive chronotropic action in the heart, adrenoceptors α1, β1 and β2 were studied using selective blockers (prazosin, CGP20712A and ICI118551, respectively). The results imply that activation of the β1-adrenoceptor pathway is responsible for most positive chronotropic actions in the mouse heart. Selective blockers of If (ZD7288, ivabradine) and sarcoplasmic reticulum (SR) [ryanodine and cylopiazonic acid (CPA)] were used to explore the contributions of If and Ca2+ release from SR to cardiac pacemaking in the absence and presence of adrenoceptor stimulation. The results showed that both If and Ca2+ release from SR significantly contribute to pacemaking both in the presence or absence of β adrenoceptor stimulation. They also show that the effects of ivabradine and ZD7288 on heart rate are substantially reduced following CPA or ryanodine administration; this may reflect a possible effect of reduced activity of Ca2+-stimulated adenylyl cyclases (AC) present in SAN cells. To investigate this phenomenon genetically modified mice lacking Ca2+-stimulated (AC) were used. Contrary to predictions, the resting heart rates and responses to If blockers, CPA blockers and isoprenaline from genetically modified mice showed only small differences relative to control mice. With aim of identifying the sequence of the Ist protein a series of photo-affinity probes were developed to enable covalent photocapture of the Ist ion channel following exposure to UV light. Proof of concept studies involved for photo-crosslinking and pull-down experiments in pursuit of identifying the Cav1.2 channel. Subsequent to cross-linking the photo-affinity probe to its target, Western blot analysis identified a ∼190 kD band, consistent with the L-type Ca2+ channel.