The mechano-electric feedback mediates the dual effect of stretch in mouse sinoatrial tissue

Abstract

The sinoatrial node (SAN) is the primary heart pacemaker. The automaticity of SAN pacemaker cells is regulated by an integrated coupled-clock system. The beat interval (BI) of SAN, and its primary initiation location (inferior vs. superior) are determined by mutual entrainment among pacemaker cells and interaction with extrinsic effectors, including increased venous return which stretches the SAN. We aim to understand the mechanisms that link stretch to changes in BI and to heterogeneity of BI in the SAN. Isolated SAN tissues of C57BL/6 mice were gradually stretched to different degrees [(low (5-10 % lengthening), medium (10-20 %), and high (20-40 %))] using motor controlled with a custom-made Arduino software. Recordings were acquired 30 s following each level of step. In 8/15 tissues, stretch led to a positive chronotropic response, while in 7/15 tissues, a negative chronotropic response was observed. In the positive chronotropic response group, BI was shortened in parallel to shortening of the local Ca²⁺ release (LCR) period, a readout of the degree of clock coupling. In the negative chronotropic response group, in parallel to a prolongation of BI and LCR period, an unsynchronized firing rate was observed among the cells upon application of stretch. Eliminating the mechano-electrical feedback by addition of blebbistatin disabled the stretch-induced chronotropic effect. Reduction of the sarcoplasmic reticulum Ca²⁺ levels, which mediates the mechano-electrical feedback, by addition of cyclopiazonic acid disabled the dual effect of stretch on SAN function and BI heterogeneity. Thus, the mechano-electric feedback mediates the dual effect of stretch in mouse SAN tissue.

Keywords: Calcium cycling; Computational model; Ion channels; Sinoatrial node; Stretch.

Graphical abstract

Fig. 1

(A) SAN preparation comprised of the right and left atria (RA, LA, respectively) flattened down by pins clamping the crista terminalis (CT) and the interatrial septum (IS). (B). Line-scan confocal image captured along a pacemaker cell within the SAN tissue. (C) Identification of the maximal Ca²⁺ transient amplitude (max Ca²⁺), diastolic Ca²⁺ (min Ca²⁺), 50 % relaxation time (T₅₀), 90 % relaxation time (T₉₀) and local Ca²⁺ release (LCR) peak using “Sparkalyzer”. (D) A motor-controlled hook was attached to the CT pin and operated to gradual stretch the SAN tissue.

Fig. 2

Stretch leads to a positive chronotropic response in mouse sinoatrial node tissue. (A) Representative time course of Ca²⁺ transients in pacemaker cells residing in the SAN before (Baseline), during (high Stretch), and upon release and return to the baseline length of (Release) stretch application. Recordings were acquired 30 s after each level. The arrow shows the appearance of local Ca²⁺ release. The effect of gradual stretch and its release on (B) beat interval (C) beat interval variability, (D) Ca²⁺ transient time to peak, (E) 50 % Ca²⁺ transient relaxation time, (F) 90 % Ca²⁺ transient relaxation time and (G) Ca²⁺ transient amplitude. ^⁎p < 0.05 compared to baseline, (number of cells = 8).

Fig. 3

Stretch leads to a negative chronotropic response in mouse sinoatrial node tissue. (A) Representative time course of Ca²⁺ transients in pacemaker cells residing in the SAN before (Baseline), during (high Stretch), and upon release and return to the baseline length of (Release) stretch application. Recordings were acquired 30 s after each level. The arrow shows the appearance of local Ca²⁺ release. The shift shows the shift in beat interval. The effect of gradual stretch and its release on (B) beat interval (C) beat interval variability, (D) Ca²⁺ transient time to peak, (E) 50 % Ca²⁺ transient relaxation time, (F) 90 % Ca²⁺ transient relaxation time and (G) Ca²⁺ transient amplitude. ^⁎p < 0.05 compared to baseline, (number of cells = 7).

Fig. 4

Stretch leads to a change in local Ca²⁺ release (LCR) in mouse sinoatrial node tissues. The effect of gradual stretch and its release on (A) LCR period, (B) LCR normalized amplitude, (C) LCR length and (D) 50 % LCR duration in tissues with a positive chronotropic response (N = 8). The effect of gradual stretch and its release on (E) LCR period, (F) LCR normalized amplitude, (G) LCR length and (H) 50 % LCR duration in tissues with a negative chronotropic response (number of cells = 7). ^⁎p < 0.05 compared to baseline.

Fig. 5

Elimination of mechano-electrical feedback disabled the positive and negative chronotropic response in mouse sinoatrial node tissue. (A) Representative time course of Ca²⁺ transients in blebbistatin-treated pacemaker cells residing in the SAN before (Baseline), during (high Stretch), and upon release and return to the baseline length of (Release) stretch application. Recordings were acquired 30 s after each level. The arrow shows the appearance of local Ca²⁺ release. The effect of gradual stretch and its release on (B) beat interval, (C) beat interval variability, (D) Ca²⁺ transient time to peak, (E) 50 % Ca²⁺ transient relaxation time, (F) 90 % Ca2⁺ transient relaxation time, (G) Ca²⁺ transient amplitude, (H) LCR period, (I) LCR normalized amplitude, (J) LCR length and (K) 50 % LCR duration. ^⁎p < 0.05 compared to baseline, ^#p < 0.05 compared to blebbistatin, (number of cells = 7).

Fig. 6

Disabling Ca²⁺ cycling does not affect the positive chronotropic response to stretch. (A) Representative time course of Ca²⁺ transients in CPA-treated pacemaker cells residing in the SAN before (CPA), during (high Stretch) and upon release and return to the baseline length of (Release) stretch application. Recordings were acquired 30 s after each level. The arrow shows the appearance of local Ca²⁺ release. The effect of gradual stretch and its release on (B) beat interval, (C) beat interval variability, (D) Ca²⁺ transient time to peak, (E) 50 % Ca²⁺ transient relaxation time, (F) 90 % Ca²⁺ transient relaxation time, (G) Ca²⁺ transient amplitude, (H) LCR period, (I) LCR normalized amplitude, (J) LCR length and (K) 50 % LCR duration. ^⁎p < 0.05 compared to baseline, ^#p < 0.05 compared to CPA, (number if cells = 7).

Fig. 7

Disabling Ca²⁺ cycling eliminates the negative chronotropic response to stretch. (A) Representative time course of Ca²⁺ transients in CPA-treated pacemaker cells residing in the SAN before (CPA), during (high Stretch), and upon release and return to the baseline length of (Release) stretch application. Recordings were acquired 30 s after each level. The arrow shows the appearance of local Ca²⁺ release. The shift shows the shift in beat interval. The effect of gradual stretch and its release on (B) beat interval, (C) beat interval variability, (D) Ca²⁺ transient time to peak, (E) 50 % Ca²⁺ transient relaxation time, (F) 90 % Ca²⁺ transient relaxation time, (G) Ca²⁺ transient amplitude, (H) LCR period, (I) LCR normalized amplitude, (J) LCR length and (K) 50 % LCR duration. ^⁎p < 0.05 compared to baseline, ^#p < 0.05 compared to CPA, (number of cells = 5).

Fig. 8

Inhibition of PKA activity does not affect the chronotropic response to stretch. (A) Representative time course of Ca²⁺ transients in H-89-treated pacemaker cells residing in the SAN before (H-89), during (high Stretch), and upon release and return to the baseline length of (Release) stretch application. Recordings were acquired 30 s after each level. The arrow shows the appearance of local Ca²⁺ release. The effect of gradual stretch and its release on beat interval (B) and beat interval variability (C) in tissues with a positive chronotropic response to stretch (number of cells = 8). (D) Representative time course of Ca²⁺ transients in pacemaker cells residing in the SAN before (H-89), during (Stretch (high level)) and after (Release) stretch application in a tissue with a negative chronotropic response to stretch. The effect of gradual stretch and its release on beat interval © and beat interval variability (F) in tissues with negative chronotropic response to stretch (N = 4). ^⁎p < 0.05 compared to baseline, ^#p < 0.05 compared to H-89.