Identifying sex similarities and differences in structure and function of the sinoatrial node in the mouse heart

Abstract

Background: The sinoatrial node (SN) generates the heart rate (HR). Its spontaneous activity is regulated by a complex interplay between the modulation by the autonomic nervous system (ANS) and intrinsic factors including ion channels in SN cells. However, the systemic and intrinsic regulatory mechanisms are still poorly understood. This study aimed to elucidate the sex-specific differences in heart morphology and SN function, particularly focusing on basal HR, expression and function of hyperpolarization-activated HCN4 and HCN1 channels and mRNA abundance of ion channels and mRNA abundance of ion channels contributing to diastolic depolarization (DD) and spontaneous action potentials (APs).

Methods: Body weight, heart weight and tibia length of 2- to 3-month-old male and female mice were measured. Conscious in-vivo HR of male and female mice was recorded via electrocardiography (ECG). Unconscious ex-vivo HR, stroke volume (SV) and ejection fraction (EF) were recorded via echocardiography. Ex-vivo HR was measured via Langendorff apparatus. Volume of atria, ventricles and whole hearts were measured from the ex-vivo hearts by microcomputed tomography (micro-CT). Immunohistochemistry targeting HCN4 and HCN1 was conducted in the SN and RA tissues from both male and female hearts. The funny current (I _f) of SN cells in 1 nM and following wash-on of 1 μM isoproterenol (ISO) were recorded via whole cell patch clamp. The APs of SN tissue were recorded via sharp microelectrode and optical mapping of membrane voltage. The relative abundance of mRNAs was measured in male and female mice by qPCR.

Results: Heart weight to tibia length ratio and heart volume of females were significantly smaller than males. Unconscious in-vivo HR in male mice was higher than that in females. Conscious in-vivo HR, ex-vivo HR, SV, and EF showed no notable difference between male and female mice. Immunohistochemistry revealed HCN4, HCN1, and the sum of HCN4 and HCN1, expression in the SN was notably elevated compared with the RA in both male and females, but there was no sex difference in these channels expression. There were also no significant sex differences in the V _0.5 of I _f in SN cells in the presence of 1 nM ISO, however wash-on 1 μM ISO in the same cells induced a significantly increased shift of V _0.5 to more positive voltages in males than in females. The expression of mRNA coding for adrenergic receptor beta-1 (Adrb1) and cholinergic receptors muscarinic 2 (chrm2) in male mice was higher compared with that in female mice. Early diastolic depolarization (EDD) rate in APs from peripheral SN (pSN) from male mice were higher than these in female mice. Mice of both sexes showed equivalent frequency of SN APs and spatial localization of the leading site in control, and similar significant response to ISO 100 nM superfusion.

Conclusion: Males display faster in-vivo HR, but not ex-vivo HR, than females associated with increased expression of Adrb1 in male versus female. This suggests a possible difference in the β-adrenergic modulation in males and females, possibly related to the greater ISO response of I _f observed in cells from males. The role of hormonal influences or differential expression of other ion channels may explain these sex-specific variations in HR dynamics. Further investigations are necessary to pinpoint the precise molecular substrates responsible for these differences.

Keywords: diastolic depolarization; electrophysiology; heart rate; ion channels; sex; sinoatrial node.

Figure 1

Phenotype data for male and female mice. (A) Body weight, heart weight and tibia length of female and male mice. (B) Heart weight to body weight and to tibia length ratio of female and male mice. N = 13. All values are shown as Mean ± SEM. ^*p < 0.05, ^***p < 0.001, and ^****p < 0.0001. Unpaired t-test was used to analyse the data between two groups.

Figure 2

Comparison of cardiac function between male and female mice. (A,B) Comparison of conscious in-vivo HR and unconscious in-vivo HR between male and female mice recorded by ECG and echocardiography. N = 6 for males and N = 7 for females. (C,D) Comparison of stroke volume and ejection fraction between male and female mice via echography. (E) Comparison of ex-vivo HR between male and female mice. All values are shown as mean ± SEM. ^*p < 0.05. Unpaired t-test was used to analyse the data between two groups.

Figure 3

The anatomy of male and female mice heart. (A,B) Different aspects of the male and female mouse heart including ventral view, dorsal view and lateral right view are shown. The area where the SN is located is indicated by the red box and RA is marked by the dotted green line. (C,D) An illustration of segmented ventricle (blue), RA (pink) and LA (yellow) of young male and female mice from ventral and dorsal views are shown. (E) LA, RA, both atria, ventricular (LV, RV and septum), whole heart, and atria/ventricular volume ratio in male and female mice are shown. N = 4 for males and N = 4 for females. All values are shown as mean ± SEM. ^**p < 0.01 and ^****p < 0.0001. Unpaired t-test was used to analyse the data between two groups. Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; SVC, superior vena cave.

Figure 4

Immunohistochemical characterization of SN and RA in male and female mice. (A) Segmentation of the SN (shown in yellow and framed with the red box). (B) Example of SN/RA preparation with the SN region outlined. Level of leading pacemaker site shown by red dotted line, determined by sharp microelectrode measurement. (C) Immunohistochemistry targeting HCN4 within one SN/RA tissue section. (D) Immunohistochemistry targeting HCN4 within the SN and RA area of male and female mice. (E) Immunohistochemistry targeting HCN1 within the SN and RA area of male and female mice. RA, right atrium; SN, sinus node.

Figure 5

Semi-quantitative analysis of HCN4 and HCN1 signal intensity in SN and RA in male and female mice. (A) Semi-quantitative analysis of HCN4 between SN and RA in male and female mice. (B) Semi-quantitative analysis of HCN1 between SN and RA in male and female mice. (C) Semi-quantitative analysis of HCN4 and HCN1 between SN and RA in male and female mice. N = 4 for each. All values were shown as mean ± SEM. ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001. Unpaired t-test was used to analyse the data between two groups. RA, right atrium; SN, sinus node.

Figure 6

Different responses of SN cells from male and female mice to ISO treatment. (A,B) Current–voltage relationship of I_f in female (A) and male (B) SN cells in 1 nM and 1 μM ISO. Asterisks indicate p < 0.05 for the comparison between current densities at the indicated potentials in 1 nM and 1 μM ISO (paired t-test). There were no significant differences in current density between cells from male and female animals. (C,D) Voltage dependence of activation for I_f in female (C) and male (D) SN cells in 1 nM and 1 μM ISO. (E) Half-activation voltages (V_0.5) for I_f in SN cells in 1 nM and 1 μM ISO. N = 3, n = 10 (female); N = 4, n = 10 (male). (F) ISO-dependent shifts in the midpoint activation voltage for I_f in individual cells compared by paired t-tests. All values are shown as mean ± SEM. SN, sinus node.

Figure 7

The measurements of AP parameters in cSN and pSN from male and female mice. (A) Representative AP from male and female SN (a,b, EDD; b,c, LDD; c,d, dV/dt; b–f, APD; a–e, APA; a, MDP). (B) AP parameters including MDP, APD, APA, dV/dt, EDD, LDD from cSN of male and female mice. N = 3, n = 6 (male); N = 4, n = 21 (female). (C) AP parameters including MDP, APD, APA, dV/dt, EDD, LDD from pSN of male and female mice. N = 4, n = 10 (male) and n = 14 (female). All values are shown as mean ± SEM. ^**p < 0.01. Unpaired t-test was used to analyse the data between two groups. APD, action potential duration; APA, dV/dt, upstroke velocity; EDD, early diastolic depolarization rate; LDD, late diastolic depolarization rate; MDP, maximum diastolic depolarization.

Figure 8

Comparison of mRNA for SN from male and female mice via qPCR for HCN, Ca²⁺, K⁺ channel subunits, calcium handling proteins, adrenergic receptors and cholinergic receptor. (A) Abundance of mRNA for HCN channel subunits in SN from male and female mice. (B) Abundance of mRNA for Ca²⁺ channel subunits in SN from male and female mice. (C) Abundance of mRNA for K⁺ channel subunits in SN from male and female mice. (D) Abundance of mRNA for calcium handling proteins mRNA in SN from male and female mice. (E) Abundance of mRNA for adrenergic and cholinergic receptor in SN from male and female mice. $\Delta {\mathrm{C}}_{\mathrm{T}}$ values shown as mean ± SEM. N = 5 (male); N = 6 (female). ^*p < 0.05. SN sinus node.

Figure 9

Automaticity and distribution of pacemaker LR in SN tissue from male and female mice. (A) Representative traces of voltage signal from male and female SN tissue without or with 100 nM ISO. (B) Comparison between depolarisation rates from male and female SN tissue without or with 100 nM ISO. (C) Sample snapshots of SN/RA tissues with points showing the position of the pacemaking LR. (D) Counting of the LRs into four categories depending on the alpha angle value calculated. N = 8 (male); N = 8 (female). All values are shown as mean ± SEM. ^*p < 0.05 and ^**p < 0.01. LR, leading region.