Heterogeneity of calcium clock functions in dormant, dysrhythmically and rhythmically firing single pacemaker cells isolated from SA node

Abstract

Current understanding of how cardiac pacemaker cells operate is based mainly on studies in isolated single sinoatrial node cells (SANC), specifically those that rhythmically fire action potentials similar to the in vivo behavior of the intact sinoatrial node. However, only a small fraction of SANC exhibit rhythmic firing after isolation. Other SANC behaviors have not been studied. Here, for the first time, we studied all single cells isolated from the sinoatrial node of the guinea pig, including traditionally studied rhythmically firing cells ('rhythmic SANC'), dysrhythmically firing cells ('dysrhythmic SANC') and cells without any apparent spontaneous firing activity ('dormant SANC'). Action potential-induced cytosolic Ca transients and spontaneous local Ca releases (LCRs) were measured with a 2D camera. LCRs were present not only in rhythmically firing SANC, but also in dormant and dysrhythmic SANC. While rhythmic SANC were characterized by large LCRs synchronized in space and time towards late diastole, dysrhythmic and dormant SANC exhibited smaller LCRs that appeared stochastically and were widely distributed in time. β-adrenergic receptor (βAR) stimulation increased LCR size and synchronized LCR occurrences in all dysrhythmic and a third of dormant cells (25 of 75 cells tested). In response to βAR stimulation, these dormant SANC developed automaticity, and LCRs became coupled to spontaneous action potential-induced cytosolic Ca transients. Conversely, dormant SANC that did not develop automaticity showed no significant change in average LCR characteristics. The majority of dysrhythmic cells became rhythmic in response to βAR stimulation, with the rate of action potential-induced cytosolic Ca transients substantially increasing. In summary, isolated SANC can be broadly categorized into three major populations: dormant, dysrhythmic, and rhythmic. We interpret our results based on simulations of a numerical model of SANC operating as a coupled-clock system. On this basis, the two previously unstudied dysrhythmic and dormant cell populations have intrinsically partially or completely uncoupled clocks. Such cells can be recruited to fire rhythmically in response to βAR stimulation via increased rhythmic LCR activity and ameliorated coupling between the Ca and membrane clocks.

Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

“…We have also recently observed similar non-AP-firing behaviours in a population of enzymatically isolated guinea pig and human SAN cells: these cells have LCRs but do not fire APs [34,35]. A large population of these dormant isolated SAN cells begin to fire spontaneous APs in response to increases in intracellular cAMP [34,36]. Such dormant cell behaviour within SAN tissue can potentially contribute to pacemaker function via (i) having a role in entrainment of oscillators that are heterogeneous both in phase and amplitude to self-organize into synchronized signals that underlie rhythmic impulse that emanate from the SAN; and (ii) their recruitment to fire APs in response to adrenergic receptor activation or AP silencing in response to cholinergic receptor stimulation.…”

“…only a fraction of cells (~25%) embedded in SAN tissue participate in generating AP in any given electrical impulse that emanate from SAN node. We have also recently observed similar non-AP-firing behaviours in a population of enzymatically isolated guinea pig and human SAN cells: these cells have LCRs but do not fire APs [34,35]. A large population of these dormant isolated SAN cells begin to fire spontaneous APs in response to increases in intracellular cAMP [34,36].…”

Functional Microdomains in Heart’s Pacemaker: A Step Beyond Classical Electrophysiology and Remodeling

“…Though such simplification was beneficial for computational modeling and enabled the development of relatively straightforward biophysical models based on non-linear dynamics and oscillatory theory, it recently became evident that this simple “random collision model” is inadequate to explain the emerging experimental results which highlight microdomain-specific regulation of cardiomyocyte physiology (reviewed in details elsewhere, Zaccolo and Pozzan, 2002 ; Warrier et al, 2007 ; Best and Kamp, 2012 ; Balycheva et al, 2015 ; Vinogradova et al, 2018 ). In the SAN, these include findings on a complex spatial-temporal coupling between the membrane- and Ca 2+ clocks confirmed in various species, including human ( Kim et al, 2018 ; Tsutsui et al, 2018 ), synchronization of spontaneous LCRs between discrete RyR clusters ( Stern et al, 2014 ; Torrente et al, 2016 ), compartmentalized autonomic regulation of pacemaker ion channels which relies on tightly confined cAMP signaling ( Barbuti et al, 2004 ; St Clair et al, 2017 ; Vinogradova et al, 2018 ), as well as microdomain-specific remodeling of ion channels secondary to structural alterations including changes in scaffolding proteins ( Le Scouarnec et al, 2008 ; Alcalay et al, 2013 ; Bryant et al, 2018 ). The emerging results demonstrate that the functioning of the complex pacemaking machinery at the cellular level depends on tightly regulated spatiotemporal signals which are restricted to precise subcellular microdomains and associated with discrete clusters of different ion channels, transporters and regulatory receptors.…”

“…These data correlate well with the distribution of transversal-axial tubule system within the SAN: primary pacemakers with the fastest spontaneous beating rate do not have T-tubules, express the smallest I Ca,L which predominantly rely on extratubular LTCCs, and have the highest pacemaker current I f , while subsidiary SAN pacemakers possess a rudimentary T-tubule network which results in a significant increase of I Ca,L and decrease in I f . Subsequently, two recent reports from Lakatta’s group demonstrated in guinea pig ( Kim et al, 2018 ) and human SANCs ( Tsutsui et al, 2018 ) several populations of cells which show rhythmic pacemaking activity, dysrhythmic firing, and no spontaneous activity (i.e., ‘dormant’ cells). Dysrhythmic and dormant SANCs have smaller and desynchronized LCR activity than rhythmic SANCs; however, in response to sympathetic stimulation, all dysrhythmic cells and a third of dormant SANCs increased their LCR activity and developed automaticity resulting in spontaneous electrical beating ( Kim et al, 2018 ).…”

Ca2+and Membrane Potential Transitions During Action Potentials are Self-Similar to Each Other and to Variability of AP Firing Intervals Across the Broad Physiologic Range of AP Intervals During Autonomic Receptor Stimulation

“…Likewise, by convention, the AP firing interval (APFI) in isolated SANC in a given "steady state" is usually referred to as "the" APFI, but the presence of beat-to-beat APFI variability (APFIV) indicates that, as in vivo, true "steady state" of AP firing in single SANC in vitro is never achieved (Kim et al, 2018;Li et al, 2016;Monfredi et al, 2014;Monfredi et al, 2013;Opthof, VanGinneken, Bouman, & Jongsma, 1987;Papaioannou, Verkerk, Amin, & de Bakker, 2013;Rocchetti, Malfatto, Lombardi, & Zaza, 2000;Sirenko et al, 2016;Tsutsui et al, 2018;Wilders & Jongsma, 1993;Yaniv, Ahmet, et al, 2014;Zaniboni, Cacciani, & Lux, 2014;Zaza & Lombardi, 2001). Further, the nonlinear relationship of the APFIV to the APFI in SANC in vitro (Zaza & Lombardi, 2001), suggests that mechanisms intrinsic to pacemaker cells are involved in APFIV.…”