Shenxian-Shengmai Oral Liquid Improves Sinoatrial Node Dysfunction through the PKC/NOX-2 Signaling Pathway

Abstract

Sick sinus syndrome (SSS) is one of the common causes of cardiac syncope and sudden death; the occurrence of SSS is associated with the accumulation of ROS in the sinoatrial node (SAN). Shenxian-shengmai (SXSM) is a traditional Chinese medicine available as oral liquid that causes a significant increase in heart rate. The objective of this study is to observe the improvement of SXSM on SAN function in SSS mice and explore its potential mechanism. In the current study, SSS was simulated in mice by inducing SAN dysfunction using a micro-osmotic pump to inject angiotensin II (Ang II). The mouse model with SSS was used to determine the effect of SXSM on SAN function and to explore its potential mechanism. Furthermore, the HL-1 cell line, derived from mouse atrial myocytes, was used to simulate SAN pacemaker cells. Our results indicated that SXSM significantly increased the heart rate of SSS mice by reducing the AngII-induced accumulation of ROS in the SAN and by inhibiting the expression of HDAC4, thereby reducing the loss of HCN4, a critical component of the cardiac conduction system. MASSON staining revealed a reduction of SAN damage in SSS mice that were treated with SXSM compared with controls. In vitro experiments showed that AngII treatment caused an upregulation of the PKC/NOX-2 signaling pathway in HL-1 cells which could be prevented by pretreatment with SXSM. The protective effect of SXSM was attenuated upon treatment with the PCK agonist PMA. In conclusion, SXSM reduced the AngII-induced accumulation of ROS in the SAN through the PKC/NOX2 signaling pathway, improving the functioning of the SAN and preventing the decrease of heart rate in SSS mice.

Figure 1

SXSM reduces Ang II-induced SAN dysfunction and increases heart rate in SSS mice. (a) Representative ECGs of mice in each group. (b) Mean heart rates in each group of mice. (c) Masson staining of the SAN of mice in each group. (d) Collagen volume fraction in the SAN of mice in each group. SHAM: mice with sham surgery; SSS: mice with Ang II treatment; SXSM: mice with Ang II and SXSM treatment; ^∗p < 0.05, ^∗∗p < 0.01. SAN, sinoatrial node; ECG, electrocardiogram; SXSM, Shenxian-shengmai oral liquid; SSS, sick sinus syndrome, Ang II, angiotensin II.

Figure 2

SXSM reduces ROS accumulation in the SAN and oxidative stress factors in serum of SSS mice. (a) Representative fluorescence images of ROS in the SAN of mice from each group. (b) ROS analysis using DHE. (c) Oxidative stress factors in serum of mice. SHAM: mice underwent sham surgery; SSS: mice underwent Ang II treatment; SXSM: mice underwent Ang II treatment in the presence of SXSM; ^∗∗p < 0.01. SAN, sinoatrial node; SXSM, Shenxian-shengmai oral liquid; SSS, sick sinus syndrome, Ang II, angiotensin II; ROS, reactive oxygen species; DHE, dihydroethidium.

Figure 3

SXSM treatment prevents the reduction in HCN4 expression in the SAN of SSS mice. (a) HCN4/NF160 immunofluorescence staining of heart tissues of mice from the three groups. NF160 (green fluorescence) marks the location of the SAN. DAPI marks the location of the nuclei. (b) Western blot showing the expression of HCN4 in the SAN of mice from the different groups. β-tubulin was a loading control. (c) Quantitative analysis of the western blot results in (b). SHAM: mice underwent sham surgery; SSS: mice underwent Ang II treatment; SXSM: mice underwent Ang II treatment in the presence of SXSM; ^∗∗p < 0.01. SAN, sinoatrial node; SXSM, Shenxian-shengmai oral liquid; SSS, sick sinus syndrome, Ang II, angiotensin II.

Figure 4

SXSM reduces Ang II-induced ROS accumulation in HL-1 cells. (a) A CCK-8 cell viability assay was used to determine the optimal concentration of SXSM. (b) ROS accumulation in HL-1 cells was assayed using green-fluorescent DCFH-DA. (c) ROS fluorescence photos statistical analysis. CON: control group cells; Ang II: cells treat with Ang II (1 μM); SXSM: cells treated with Ang II and SXSM (1 ml/L); SXSM + PMA: cells treat with Ang II, SXSM (1 mL/L) and the PKC activator PMA (1 μM); ^∗∗p < 0.01. SAN, sinoatrial node; SXSM, Shenxian-shengmai oral liquid; SSS, sick sinus syndrome, Ang II, angiotensin II; ROS, reactive oxygen species.

Figure 5

SXSM prevents Ang II-induced HCN4 loss in HL-1 cells. (a) Immunofluorescence staining of HCN4 (red). DAPI staining indicates the nuclei. (b) Western blot showing the expression of HCN4 and HDAC4. (c) Quantitative analysis of the western blot results in (b). CON: control group cells; Ang II: cells treated with Ang II(1 μM); SXSM: cells treated with Ang II (1 μM) and SXSM (1 mL/L); SXSM + PMA: cells treated with Ang II (1 μM), SXSM (1 mL/L) and PMA (1 μM); ^∗∗p < 0.01. SXSM, Shenxian-shengmai oral liquid; Ang II, angiotensin II; PMA, PKC activator.

Figure 6

Protein assay of the PKC/NOX2 signaling pathway. (a) Western blot was used to assay the expression of NOX-2 and p47^phox. GAPDH was used as a loading control. (b) Western blot quantitative analysis. CON: control group cells; Ang II: cells treated with Ang II (1 μM); SXSM: cells treated with Ang II (1 μM) and SXSM (1 mL/L); SXSM + PMA: cells treated with Ang II (1 μM), SXSM (1 mL/L) and PMA (1 μM); ^∗∗p < 0.01. SXSM, Shenxian-shengmai oral liquid; Ang II, angiotensin II; PMA, PKC activator.