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. 2023 Nov 9;15(21):12551-12569.
doi: 10.18632/aging.205196. Epub 2023 Nov 9.

D-galactose causes sinoatrial node dysfunction: from phenotype to mechanism

Heng Zhang  1 , Chen Chen  2 , Yue Liu  3 , Wei Chen  4 , Jing Qi  4 , Yue Xu  4 , Lu Ren  4 , Guanlin Yang  4 , Dongyu Min  4 , Zhuang Liu  4 , Xintong Cai  4 , Miao Hao  4 , Guanzhen Xu  4 , Ping Hou  4
Affiliations

D-galactose causes sinoatrial node dysfunction: from phenotype to mechanism

Heng Zhang et al. Aging (Albany NY). .

Abstract

With the population aging, age-related sinoatrial node dysfunction (SND) has been on the rise. Sinoatrial node (SAN) degeneration is an important factor for the age-related SND development. However, there is no suitable animal modeling method in this field. Here, we investigated whether D-galactose could induce SAN degeneration and explored the associated mechanism. In vivo, twelve C57BL/6 mice were divided into Control and D-galactose group to receive corresponding treatments. Senescence was confirmed by analyzing the hair and weight; cardiac function was evaluated through echocardiography, cerebral blood flux and serum-BNP; the SAN function was evaluated by electrocardiogram; fibrotic change was evaluated by Masson's trichrome staining and oxidative stress was assessed through DHE staining and serum indicators. Mechanism was verified through immunofluorescence-staining and Western blotting. In vitro, mouse-atrial-myocytes were treated with D-galactose, and edaravone was utilized as the ROS scavenger. Senescence, oxidative stress, proliferation ability and mechanism were verified through various methods, and intuitive evidence was obtained through electrophysiological assay. Finally, we concluded that D-galactose can be used to induce age-related SND, in which oxidative stress plays a key role, causing PITX2 ectopic expression and downregulates SHOX2 expression, then through the downstream GATA4/NKX2-5 axis, results in pacing-related ion channels dysfunction, and hence SND development.

Keywords: D-galactose; ion channels dysfunction; oxidative stress; senescence; sinoatrial node dysfunction.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Structural formula of D-galactose.
Figure 2
Figure 2
D-galactose induces senescence in mice. (A) D-galactose causes hair senescence in mice. (B) D-galactose causes weight gain in mice. NS represents P>0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group.
Figure 3
Figure 3
D-galactose decreased SAN and heart function in mice. (A) Electrocardiogram. (B) Echocardiogram. (C) Cerebral blood flux. (D) Heart rate in the indicated groups. (E, F) LVEF and LVFS in the indicated groups. (G) BNP assay results for the indicated groups. (H) Cerebral blood flux results for the indicated groups. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group.
Figure 4
Figure 4
D-galactose causes fibrosis of sinoatrial node and myocardium. (A) Masson’s trichrome staining of sinoatrial node (Scale = 50 μm) and myocardium (Scale = 20 μm). (B) CVF of sinoatrial node. (C) CVF of myocardium. Black arrow represents the artery of sinoatrial node; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group.
Figure 5
Figure 5
D-galactose induces oxidative stress in the sinoatrial node. (A, B) DHE staining results for the indicated groups. Scale = 50 μm. (C, D) SOD and MDA assay results for the indicated groups. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group.
Figure 6
Figure 6
Oxidative stress causes If channel abnormality. (A) Immunofluorescence staining of the SAN. Scale = 100 μm. (B) Western blotting assay. (C) Results of immunofluorescence staining. (D, E) Results of Western blotting assay. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group.
Figure 7
Figure 7
D-galactose causes cell senescence. (A, B) Results of P16 immunofluorescence staining. Scale = 20 μm. (C, D) Results of β-galactosidase staining. Scale = 100 μm. ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group; Edaravone: D-galactose+Edaravone administered group.
Figure 8
Figure 8
D-galactose causes cell peroxidation and decreased mitochondrial function. (A) Mito-tracker and DCFH-DA staining results for the indicated groups. Scale = 20 μm. (B, C) Staining results for the indicated groups. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group; Edaravone: D-galactose+Edaravone administered group.
Figure 9
Figure 9
D-galactose causes cell cycle and proliferation abnormalities. (A, B) Results of cell cycle assay. (C) CCK-8 assay results for the indicated groups. NS represents P>0.05; * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group; Edaravone: D-galactose+Edaravone administered group.
Figure 10
Figure 10
D-galactose causes abnormal expression of pacing-related gene. (A–D) RT-qPCR results for the indicated groups. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group; Edaravone: D-galactose+Edaravone administered group.
Figure 11
Figure 11
ROS scavenging and Pitx2 silencing improves pacemaker ion channel. (A, B) Immunofluorescence staining results for the indicated groups. Scale = 20 μm. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose administered group; Edaravone: D-galactose+Edaravone administered group; si-NC: si-NC transfection+D-galactose administered group; si-Pitx2: si-Pitx2 transfection+D-galactose administered group.
Figure 12
Figure 12
Results of Western blotting assay. (A, B) Results of Western blotting and statistical analysis. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose-administered group; Edaravone: D-galactose+Edaravone-administered group; si-NC: si-NC transfection + D-galactose-administered group; si-Pitx2: si-Pitx2 transfection + D-galactose-administered group.
Figure 13
Figure 13
ROS scavenging and PITX2 silencing improves pulsatile function in hiPSC-CMs. (A) Field potential traces of hiPSC-AMs at 48h. (B, C) Relative trend of beat rate and field potential duration. (D, E) Beat rate and field potential duration of hiPSC-AMs at 48h. * represents P<0.05; ** represents P<0.01; Control: control group; D-galactose: D-galactose-administered group; Edaravone: D-galactose+Edaravone-administered group; si-NC: si-NC transfection+D-galactose administered group; si-PITX2: si-PITX2 transfection+D-galactose-administered group.
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