Redundant and diverse intranodal pacemakers and conduction pathways protect the human sinoatrial node from failure

Abstract

The human sinoatrial node (SAN) efficiently maintains heart rhythm even under adverse conditions. However, the specific mechanisms involved in the human SAN's ability to prevent rhythm failure, also referred to as its robustness, are unknown. Challenges exist because the three-dimensional (3D) intramural structure of the human SAN differs from well-studied animal models, and clinical electrode recordings are limited to only surface atrial activation. Hence, to innovate the translational study of human SAN structural and functional robustness, we integrated intramural optical mapping, 3D histology reconstruction, and molecular mapping of the ex vivo human heart. When challenged with adenosine or atrial pacing, redundant intranodal pacemakers within the human SAN maintained automaticity and delivered electrical impulses to the atria through sinoatrial conduction pathways (SACPs), thereby ensuring a fail-safe mechanism for robust maintenance of sinus rhythm. During adenosine perturbation, the primary central SAN pacemaker was suppressed, whereas previously inactive superior or inferior intranodal pacemakers took over automaticity maintenance. Sinus rhythm was also rescued by activation of another SACP when the preferential SACP was suppressed, suggesting two independent fail-safe mechanisms for automaticity and conduction. The fail-safe mechanism in response to adenosine challenge is orchestrated by heterogeneous differences in adenosine A1 receptors and downstream GIRK4 channel protein expressions across the SAN complex. Only failure of all pacemakers and/or SACPs resulted in SAN arrest or conduction block. Our results unmasked reserve mechanisms that protect the human SAN pacemaker and conduction complex from rhythm failure, which may contribute to treatment of SAN arrhythmias.

Fig. 1. Functional, structural, and molecular features of the 3D human SAN pacemaker complex revealed by integrated ex vivo human heart mapping. (A)

From left to right: Photograph of human SAN preparation of heart 118258 with SAN and atrial OAPs, SAN and atrial activation maps at baseline conditions, and schematic human SAN. Leading pacemaker (LP) is shown with circle, and EAS is shown with asterisk in activation maps and schematic. Arrows indicate the functionally and structurally defined SACP. (B) From left to right: Fluorescence image of Cx43 immunostaining of heart tissue with SAN outlined by dotted line, Masson’s trichrome staining of SAN pacemaker complex with fibrosis (blue) and cardiomyocytes (red), magnification of SAN histology showing the preferential SACP, and 3D reconstruction of the SAN (blue) with five SACPs (yellow). CT, crista terminalis; IAS, interatrial septum; RA, right atrium; SVC, superior vena cava.

Fig. 2. Adenosine-induced SAN dysfunction recorded by optical mapping and SAN electrograms in ex vivo human hearts

(A) Left: Heart 947202 preparation showing the location of the SAN and bipolar catheter. Circles #1 and #2 indicate SAN leading pacemaker and EAS (asterisk), respectively. The orange arrow indicates the preferential SACP. Right: OAPs of extracted SAN (blue) and atria (green) with SAN electrogram (EG) (black) during adenosine (10 μM) perfusion. (B) Atrial ECG and SAN electrogram recordings of adenosine (10 μM)–induced bradycardia in heart 947202, 2:1 exit block followed by complete exit block in heart 118258, and SAN arrest in heart 442404. Purple box in (B) shows SAN beat presented in (A). (C) SAN and atria activation maps and OAPs during baseline, adenosine (10 μM), and adenosine (100 μM) + tertiapin in heart 118258, respectively. (D) Quantification of adenosine-induced SAN conduction and automaticity inhibition restored by tertiapin. Bar graphs summarize the average drug effect; white numbers show the number of hearts used. Data are means ± SD. P values were determined by analysis of variance (ANOVA) after Tukey’s adjustment. Ado, adenosine; HR, heart rate; IVC, inferior vena cava; SACTsr, sinoatrial conduction time during sinus rhythm; SR, sinus rhythm.

Fig. 3. Exit and intranodal block revealed by ex vivo intramural optical mapping and SAN electrogram during adenosine treatment (10 mM) in heart 118258. (A)

OAPs and SAN electrogram show exit block from SAN to atria. Intranodal block seen as a reduction in amplitude on SAN electrogram. (B) From left to right: Activation maps for complete activation of the SAN that activated atria through the superior SACP, partial SAN activation and exit block, and partial SAN activation that activated atria through the inferior (inf) and middle (mid) lateral SACPs. SACT, sinoatrial conduction time.

Fig. 4. Multiple SAN intranodal leading pacemakers and SACPs revealed by adenosine challenge

From left to right: SAN leading pacemaker and SACP shift during baseline (blue), 10 μM adenosine (pink), 100 μM adenosine (red), and tertiapin (green) for heart 118258 and (right) for all hearts during sinus rhythm.

Fig. 5. SAN intranodal automaticity and conduction strongly affected by combination of atrial pacing and adenosine in ex vivo human hearts. (A)

Baseline SAN activation and conduction pattern during and after atrial pacing. (B) Adenosine impaired SAN automaticity and conduction; conversely, entrance block, caused by conduction depression, prevented overdrive suppression of SAN automaticity. (C) Top: Atrial and SAN OAPs during 500-ms CL pacing and post-pacing with 100 μM adenosine. Bottom: SAN exit block (left) and atrial ectopic beat (right) activation maps during 100 μM adenosine. (D) Bar graphs summarizing average drug effect on cSNRTi (left), cSNRTd (middle), and SACTppb (right). White numbers show the number of hearts used. Data are means ± SD.P values were determined by ANOVA after Tukey’s adjustment. CS, coronary sinus; RAA, right atrial appendage; SACTppb, sinoatrial conduction time of first post-pacing SAN beat; SNRTi/d, indirect/direct SAN recovery time; cSNRTi/d, corrected SNRTi/d.

Fig. 6. Heterogeneous A1R and GIRK1/4 protein expression in human SAN and atria revealed by ex vivo molecular mapping

(A) SAN localization and tissue dissection. Left: SAN activation map of heart 987692. SAN leading pacemaker is shown with red circle. Middle: Outline of functionally defined SAN overlaid on photograph of dissected SAN preparation. Blue and red circles indicate the leading pacemaker locations during baseline and 100 μM adenosine, respectively. Lines 1 to 4 indicate the dissection lines for the SAN head, center, and tail regions. Beneath is shown a photograph of transmural section of frozen SAN preparation. Right: Immunostaining showing α-actinin (red)–positive and Cx43 (green)–negative regions of the human SAN indicated by the white dotted line. Blue circles show the locations where tissue was collected for SAN protein isolation. (B) Left: Representative immunoblot from hearts 809108 and 674541. Right: Bar graphs summarizing A1R and GIRK4 protein expression in the SAN central region and the right atria (RA) (n = 10, samples from unmapped human hearts). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–normalized band density is shown as means ± SD; n, number of human hearts; comparison of protein expression in SAN versus RA was done using mixed models in package lme4. P value was determined by pairwise comparisons with Tukey’s adjustment. A.U., arbitrary unit. (C) Left: Representative immunoblot from each SAN region and RA for heart 809108. Right: SAN-to-RA ratio of GAPDH-normalized band density shown as means ± SD; n, number of human hearts (four unmapped and one mapped heart); values of SAN/RA ratios were compared to 1 using two-sided one-sample test; for each protein Max versus Min, P values were determined by two-sided paired t test.