Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping

Abstract

Historically, milestones in science are usually associated with methodological breakthroughs. Likewise, the advent of electrocardiography, microelectrode recordings and more recently optical mapping have ushered in new periods of significance of advancement in elucidating basic mechanisms in cardiac electrophysiology. As with any novel technique, however, data interpretation is challenging and should be approached with caution, as it cannot be simply extrapolated from previously used methodologies and with experience and time eventually becomes validated. A good example of this is the use of optical mapping in the sinoatrial node (SAN): when microelectrode and optical recordings are obtained from the same site in myocardium, significantly different results may be noted with respect to signal morphology and as a result have to be interpreted by a different set of principles. Given the rapid spread of the use of optical mapping, careful evaluation must be made in terms of methodology with respect to interpretation of data gathered by optical sensors from fluorescent potential-sensitive dyes. Different interpretations of experimental data may lead to different mechanistic conclusions. This review attempts to address the origin and interpretation of the "double component" morphology in the optical action potentials obtained from the SAN region. One view is that these 2 components represent distinctive signals from the SAN and atrial cells and can be fully separated with signal processing. A second view is that the first component preceding the phase 0 activation represents the membrane currents and intracellular calcium transients induced diastolic depolarization from the SAN. Although the consensus from both groups is that ionic mechanisms, namely the joint action of the membrane and calcium automaticity, are important in the SAN function, it is unresolved whether the double-component originates from the recording methodology or represents the underlying physiology. This overview aims to advance a common understanding of the basic principles of optical mapping in complex 3D anatomic structures.

Figure 1. Mechanism of the double-components of the Optical Action Potential (AP) recording from canine SAN tissue

Panel A. A simplified model of the SAN based on published data. This “section” cuts along the superior-inferior axis of SAN. Top Left – Optical AP (OAP) recordings from only atrial and only SAN layers of tissue, which closely resemble morphology of microelectrode AP recordings. Top Right – OAP recording from an area encompassing both atrial and SAN tissue layers, thus averaging APs from both structures. Bottom –OAPs from different SAN regions. Panel B. Depth penetration measurements for excitation wavelength. Modified with permission from Bishop et al 2007.

Figure 2. Physiological validation of the multiple components in the rabbit AVN and canine SAN

Panel A – Simultaneous optical recordings from the distal AVN and bipolar electrograms from the atrial input to the AVN and from the bundle of His. A flouting glass microelectrode was utilized to impale cells in different AVN layers (black and white circles). Recording panels illustrate three consecutively paced beats during Wenchebach 2:1 block with simultaneously acquired atrial inputs and His electrograms, along with optical and microelectrode signals. Panel B – Histology shows a section of the rabbit heart through approximately the same area, which was functionally documented in Panel A. Panel C-D - OAPs during transient SAN exit block demonstrate separation of two components: SAN and atrial. Modified from Figure 6. Abbreviations: SVC and IVC, superior and inferior vena cava; RAA, right atrial appendage; IAS, intra-atrial septum.

Figure 3. Analysis of optical transmembrane potentials from canine the SAN

Panel A - OAPs, their first (dV/dt), and second (d²V/dt²) derivatives during normal sinus rhythm from photodiodes recordings shown in Panel B. OAPs were separated into the SAN and atrial components using the d²V/dt²_max of the signal as the transition point. Panel B - Parallel histology section to the epicardial (Epi) surface with 9×9 mm OFV (blue dotted square) is shown by a dark blue dotted rectangle. Activations maps of the SAN and atrial components of the OAPs (modified from Figure 2 and 5,12). Panel C -3D model of SAN. A 3D model of the canine SAN. Modified from Figure 8 12).

Figure 4. Simultaneous voltage and calcium epicardial optical mapping of the canine SAN

Panel A - Epicardial photographs of a perfused canine atrial preparation with two optical field of views (OFVs) for voltage sensitive dye RH237 and Ca²⁺sensitive dye RHOD2. The SAN arteries are shown by blue curves. The red oval shows the approximate border of the SAN region. Abbreviations are the same as in Figure 2. Panel B Optical action potentials and intercellular Ca²⁺ traces from the center of the SAN (1), the atrial breakthrough in CT (2), and Block area (3) from sites 1 -3 in panels A and C during normal SR. Panel C – Separated SAN and atrial activation maps (AP50% and dV/dt_max) obtained by both dyes. Panel D shows optical action potentials and intercellular Ca²⁺ traces three recording sites shown in the activation maps (right) following the termination of atrial pacing with an S1S1=150ms and recovery of spontaneous SAN activity. (Fedorov VV, Glukhov AV, Schuessler RB, Fast VG, Efimov IR. Unpublished data.)

Figure 5

Activation pattern of SAN and surrounding RA during 0.3 μmol/L isoproterenol infusio. A, Isochronal map of V_m. The number on the each isochronal line indicates time (ms). B, The V_m (blue) and Ca_i (red) recordings from the superior (a), middle (b), and inferior (c) SANs and the RA (d) are presented. C, Magnified view of Ca_i and V_m tracings of the superior SAN. Note the robust LDCAE (solid arrow) before phase 0 of the action potential (0 ms), which in turn was much earlier than the onset of the p wave on ECG. D, The V_m and Ca_i maps from −60 ms before to 180 ms after phase 0 of the action potential of C. The LDCAE (broken arrows in frames −40 and −20 ms) was followed by the Ca_i sinkhole during early diastole (solid arrow in frame 180 ms). E. The SAN LDCAE (upper panel) and DD (lower panel) isochronal maps. Note the co-localization of the LDACE and DD. This figure was modified from Joung et al.

Figure 6

Co-localization of LDCAE and the leading pacemaking site. A, Upward shift of the leading pacemaking site with LDCAE during isoproterenol infusion. (a) Ca_i ratio maps of SAN at the respective sinus rate. (b) Corresponding Ca_i tracings from the superior (1, 2), middle (3, 4), and inferior (5, 6) SAN. At 95 bpm, sites 4 and 5 had the most prominent LDCAEs (asterisks). As the sinus rate gradually increased, the sites of Ca_i elevation progressively moved upward. At the maximum sinus rate of 173 bpm, site 2 had the most apparent LDCAE. B, Differential responses of different SAN sites to isoproterenol. (a) The Ca_i and V_m tracings from the inferior, middle, and superior SAN sites at different sinus rates. (b) The LDCAE and DD slopes of the superior SAN at different sinus rates. This figure was reproduced with permission from Joung et al.

Figure 7

Schematic explanation of how the slope of LDCAE (A) and DD (B) were measured. The slopes of LDCAE and DD were measured from the onsets of LDCAE and DD to peak levels of LDCAE and DD, respectively. The onsets of LDCAE and DD were defined by the time of the transition between negative to positive values in dCa_i/dt and dV_m/dt curves (arrows).

Figure 8

Identification of conduction block with double potentials in the sinus node. A, The action potentials (on the right side) were recorded at corresponding points of the activation map (on the left side). The dashed line indicates the time reference. The area in which double potentials (arrows) in the action potential were clearly visible is hatched in the activation map. (Modified from Bleeker et al.26). B, The V_m tracings showing the double potentials (arrows) at the border of SAN.