Autonomic reflex control of dominant pacemaker location and impulse propagation in the right atrium

Abstract

Reflex vagal control is a fundamental component of the autonomic regulation of heart rate, and loss of function in the vagal reflex arc is a negative prognostic indicator of cardiovascular health. Vagal activation can cause rapid and profound heart rate slowing and a concomitant caudal shift of the impulse origin within the sino-atrial node (SAN). The research presented in this thesis furthers our understanding of the mechanisms that contribute to pacemaker shift in the SAN. This advance has been accomplished through mapping impulse propagation in the right atrium (RA) during reflex activation with higher precision than previously achieved. For the first time, high-resolution extracellular and optical mapping techniques have been employed in a rat arterially perfused working heart-brainstem preparation (WHBP). Autonomic reflex pathways in the WHBP were kept intact, which enabled stimulation of the baroreceptor reflex under tightly controlled conditions, free of the confounding effects of anaesthetic agents on neural function. TheWHBP spontaneously produced eupneiclike phrenic nerve activity, characteristic of adequate oxygenation of the brainstem, and central cardiorespiratory coupling was preserved. The baroreflex was activated systematically by applying pressure challenges of increasing magnitude. Distinct dominant pacemaker (DP) regions with preferential conduction pathways were identified and related to the underlying distribution of cholinergic nerves fluorescently labelled using antibodies. There is a wide-spread notion that the DP is the SAN region with the highest intrinsic rate, and that ACh-induced pacemaker shift occurs when cells in the central node slow, allowing caudal pacemaker cells less sensitive to ACh to assume control at a higher rate. Our findings for the baroreflex onset are not consistent with this view. Baroreflex induced caudal DP shifts were synchronous with substantial increases in cycle length (CL). On the other hand, rostral pacemaker shifts during baroreflex recovery were coincident with small decreases in CL. We also observed competitive pacemaker activity between rostral and caudal regions immediately before DP shifts in recovery, but during onset only subthreshold depolarisation was detected in the rostral pacemaker region. Activation spread from caudal pacemaker sites through distinct conduction pathways to the crista terminalis (CT), but propagation toward the rostral SAN was slow. Taken together, these results are consistent with low safety factor for electrical propagation in the rostral SAN during baroreflex and suggest caudal DP shift occurs as a result of failure of central pacemaker cells to drive activation in surrounding myocardium. To probe this mechanism further, we compared baroreflex results to responses recorded during stimulation of peripheral chemoreceptors with arterial injection of KCN and homogeneous stimulation of muscarinic (M2) receptors with an arterial bolus of carbachol (CCh). The dynamics of DP shift during onset and recovery of chemoreflex activation were comparable to that recorded during baroreflex responses, but larger fluctuations in CL and DP shifts were seen in synchrony with inspiratory motor drive, consistent with enhanced respiratory coupling as a result of the hypoxic stimulus. Compared to baroreflex and chemoreflex responses, CCh induced more gradual changes in CL, and DP shifts during onset and recovery occurred with small changes in CL that were not significantly different. This result led us to hypothesise that the speed of onset of cholinergic stimulation is an important factor in determining whether DP shift occurs due to failure of impulse propagation from the central SAN. To test this hypothesis, we explored changes in the dynamics of DP shift following inhibition of If with the HCN-channel specific blocker ivabradine (IVB). Post-IVB, baseline CL increased on average by ∼19%, indicating effective If inhibition in the SAN. The onset of CL responses to reflex and CCh stimulation was faster post-IVB, with a shorter time to maximum CL. There was a trend toward larger increases in CL with DP shifts during onset and recovery but this was not significant over all stimulation methods. The trend was most evident for CCh responses, where the range of CL increases with DP shift was augmented. In some reflex responses, the increase in CL was attenuated post-IVB and caudal DP shifts did not occur or occurred with small changes in CL later in the response, suggesting IVB could have altered reflex function. Here, our findings show If acts to buffer the rate slowing effects of cholinergic stimulation and thereby modulates the timing of DP shift during periods of increasing CL. Lastly, we attempted to develop a toxic cardiomyopathy model of atrial structural remodelling in order to assess the effects of SAN fibrosis on reflex control of pacemaker function in the WHBP. We delivered single isoprenaline injections on two consecutive days across a dosage range from 150 to 340 mg kg−1. In vivo cardiac cine-MRI demonstrated a slightly lower left ventricular (LV) ejection fraction at 340 mg kg−1 compared to control (66% vs. 71%), and less contraction of the LV lumen at end systole. Measurements of LV pressure at this dose showed an elevation of mean change in diastolic pressure compared to control (9.58 vs. 3.54 mmHg). Picrosirius red staining of collagen in LV tissue sections showed patchy interstitial fibrosis which was not present on sections from control samples. Our results are consistent with the development of ventricular dysfunction and structural remodelling after isoprenaline injection, but the very low mortality rate observed (5%) suggests the effects of isoprenaline in the juvenile rats used here are more moderate than those reported for adult rats. In conclusion, we have completed the first high-resolution analysis of SAN function during vagal reflex activity in a strictly controlled preparation with intact autonomic reflex pathways. Our most novel finding is that reflex-induced caudal DP shift is not driven by rate entrainment alone and appears instead to be the result of failure of impulse propagation from the central SAN. We hypothesise that the DP is therefore the region with the highest intrinsic rate in the SAN that has the capacity to drive electrical activity in the surrounding atrial myocardium.