Effect of Shenfu Injection on Differentiation of Bone Marrow Mesenchymal Stem Cells into Pacemaker-Like Cells and Improvement of Pacing Function of Sinoatrial Node

Abstract

Sick sinus syndrome (SSS), a complex type of cardiac arrhythmia, is a major health threat to humans. Shenfu injection (SFI), a formula of traditional Chinese medicine (TCM), is effective in improving bradyarrhythmia. However, the underlying mechanism of SFI's therapeutic effect is subject to few systematic investigations. The purpose of the present research is to examine whether SFI can boost the differentiation effectiveness of bone marrow mesenchymal stem cells (BMSCs) into pacemaker-like cells and whether the transplantation of these cells can improve the pacing function of the sinoatrial node (SAN) in a rabbit model of SSS. BMSCs from New Zealand rabbits were extracted, followed by incubation in vitro. The flow cytometry was utilized to identify the expression of CD29, CD44, CD90, and CD105 surface markers. The isolated BMSCs were treated with SFI, and the whole-cell patch-clamp method was performed to detect hyperpolarization-the activated cyclic nucleotide-gated potassium channel 4 (HCN4) channel current activation curve. The SSS rabbit model was established using the formaldehyde wet dressing method, and BMSCs treated with SFI were transplanted into the SAN of the SSS rabbit model. We detected changes in the body-surface electrocardiogram and recorded dynamic heart rate measurements. Furthermore, transplanted SFI-treated BMSCs were subjected to HE staining, TUNEL staining, qPCR, western blotting, immunofluorescence, immunohistochemistry, and enzyme-linked immunosorbent assay to study their characteristics. Our results indicate that the transplantation of SFI-treated BMSCs into the SAN of SSS rabbits improved the pacing function of the SAN. In vitro data showed that SFI induced the proliferation of BMSCs, promoted their differentiation capacity into pacemaker-like cells, and increased the HCN4 expression in BMSCs. In vivo, the transplantation of SFI treated-BMSCs preserved the function of SAN in SSS rabbits, improved the expression of the HCN4 gene and gap junction proteins (Cx43 and Cx45), and significantly upregulated the expression of cAMP in the SAN, compared to the SSS model group. In summary, the present research demonstrated that SFI might enhance the differentiation capacity of BMSCs into pacemaker-like cells, hence offering a novel approach for the development of biological pacemakers. Additionally, we confirmed the effectiveness and safety of pacemaker-like cells differentiated from BMSCs in improving the pacing function of the SAN.

Figure 1

Characteristics and surface markers of bone marrow mesenchymal stem cells (BMSCs). (a) Morphological features of BMSCs exhibiting fibroblast-like morphology under microscopic observations. Scale bar = 100 μm. (b) The expression of the fluorescent protein was observed under the fluorescence microscope. GFP: green fluorescent protein. Scale bar = 100 μm. (c) Flow cytometry was used to detect the expression of surface markers of BMSCs. Positive markers CD90, CD105, CD29, and CD44 as well as negative markers CD45, HLA-DR, CD34, and CD31 were identified on the surface of BMSCs.

Figure 2

Shenfu injection (SFI) promotes the differentiation of BMSCs into pacemaker-like cells. (a) Cell Counting Kit-8 was utilized to detect the effect of SFI on the viability of BMSCs. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. 0 μL/mL. (b) BMSCs were clamped at -70 mV and stimulated with hyperpolarizing voltages ranging from -70 to -150 mV for 3 s in 10 mV steps to record cellular I_f currents. (c) Whole-cell patch-clamp electrophysiology was conducted and used for the purpose of recording the I_f current on BMSCs with different treatments. (d) SFI treatment could induce the expression of HCN4 channel in BMSCs. ^∗P < 0.05 vs. LV-NC, ^#P < 0.05 vs. SFI-H under ZD7288. Data represented mean ± SEM, n = 6. SFI-L: Shenfu injection-low dose (10 μL/mL); SFI-M: Shenfu injection-medium dose (20 μL/mL); SFI-H: Shenfu injection-high dose (40 μL/mL).

Figure 3

Expression of HCN4 in BMSCs treated with Shenfu injection (SFI). (a) Western blot was conducted to examine the HCN4 expression in SFI-treated BMSCs at protein levels. (b) Real-time PCR was used to detect HCN4 mRNA expression. Data are expressed as the mean ± SEM, n = 3 − 6, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. LV-NC. (c) Representative transmission electron microscopy (TEM) images of the ultrastructure derived from BMSCs. Scale bar = 2 μm. (d) ELISA was performed to detect the expression of cAMP. Data are expressed as the mean ± SEM, n = 6, ^∗P < 0.05, ^∗∗P < 0.01 vs. LV-NC. SFI-L: Shenfu injection-low dose (10 μL/mL); SFI-M: Shenfu injection-medium dose (20 μL/mL); SFI-H: Shenfu injection-high dose (40 μL/mL).

Figure 4

Impacts of SFI-treated BMSCs on the sinoatrial node (SAN) tissue. (a) After establishing the rabbit model of sick sinus syndrome (SSS) with SAN dysfunction, green fluorescent protein- (GFP-) expressing BMSCs (1 × 10⁷ cells in 1 mL of PBS) were transplanted into the head, body, and tail of the SAN tissue. Photograph of the implantation procedure for transduced BMSCs. (b) The rabbits' mean heart rate prior to and after SFI-treated BMSC transplantation. Data are expressed as the mean ± standard error of the mean (SEM). bpm: beats per minute. ^∗P < 0.05. (c) Typical electrocardiogram (ECG) image of the sinus rhythm before and after the transplantation of SFI-treated BMSCs, and the middle ECG represents the electrogram of the SAN. A relatively small QRS and deep reversed P wave were observed in the ECG. (d) Tissue of the SAN. (e) Microscopic images of GFP-expressing BMSCs transplanted into the SAN tissue of rabbits with SSS. (f) TUNEL assay was performed to detect apoptosis of SAN cells. Scale bar = 100 μm. Data are expressed as the mean ± SEM. ^∗∗∗P < 0.001 vs. sham; ^#P < 0.05 vs. LV-NC. (g) Masson's trichome staining was conducted to detect local fibrosis in the SAN tissue. The area of blue represented the fibrotic tissue. Scale bar = 50 μm. Data are articulated as the mean ± SEM. ^∗∗∗P < 0.001 vs. sham; ^#P < 0.05 vs. LV-NC. SFI-L: Shenfu injection-low dose (10 μL/mL); SFI-M: Shenfu injection-medium dose (20 μL/mL); SFI-H: Shenfu injection-high dose (40 μL/mL).

Figure 5

Evaluation of the HCN4 expression and gap junction protein expression in the SAN of BMSC-transplanted rabbits with SSS. (a, b) Detection of HCN4 in the SAN of BMSC-transplanted rabbits at protein and mRNA levels by real-time PCR and western blot, respectively. Data are expressed as the mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01 vs. sham; ^#P < 0.05, ^###P < 0.001 vs. LV-NC. (c, d) Immunochemistry was conducted to determine the Cx43 and Cx45 protein expression in the SAN tissue of the SSS rabbit model. Scale bar = 50 μm. (e) ELISA was performed to detect the cAMP content in the SAN tissue of the SSS rabbit model. ^∗P < 0.05 vs. sham; ^#P < 0.05, ^##P < 0.01 vs. LV-NC.

Figure 6

Transplantation of Shenfu injection-treated BMSCs into the sinoatrial node of rabbits with sick sinus syndrome.