TBX18 gene induces adipose-derived stem cells to differentiate into pacemaker-like cells in the myocardial microenvironment

Abstract

T-box 18 (TBX18) plays a crucial role in the formation and development of the head of the sinoatrial node. The objective of this study was to induce adipose-derived stem cells (ADSCs) to produce pacemaker-like cells by transfection with the TBX18 gene. A recombinant adenovirus vector carrying the human TBX18 gene was constructed to transfect ADSCs. The ADSCs transfected with TBX18 were considered the TBX18-ADSCs. The control group was the GFP-ADSCs. The transfected cells were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). The results showed that the mRNA expression of TBX18 in TBX18-ADSCs was significantly higher than in the control group after 48 h and 7 days. After 7 days of co-culturing with NRVMs, there was no significant difference in the expression of the myocardial marker cardiac troponin I (cTnI) between the two groups. RT-qPCR and western blot analysis showed that the expression of HCN4 was higher in the TBX18-ADSCs than in the GFP-ADSCs. The If current was detected using the whole cell patch clamp technique and was blocked by the specific blocker CsCl. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) showed approximately twice the current density compared with the ADSCs. Our study indicated that the TBX18 gene induces ADSCs to differentiate into pacemaker‑like cells in the cardiac microenvironment. Although further experiments are required in order to assess safety and efficacy prior to implementation in clinical practice, this technique may provide new avenues for the clinical therapy of bradycardia.

Figure 1

Transfection rate and expression of T-box 18 (TBX18). (A) Adipose-derived stem cells (ADSCs) in passage 3 observed under a light microscope. (B) TBX18-ADSCs observed under a fluorescence microscope. (C) Transfection rate of different multiplicity of infection (MOI) values detected by flow cytometric analysis. (D) Expression of TBX18 mRNA at two and 7 days after transfection. Scale bar, 200 µm. ^*P<0.05 vs. GFP-ADSCs group.

Figure 2

Expression of cardiac troponin I (cTnI) after co-culture for 7 days. (A) GFP-ADSCs detected by immunofluorescence assay (green). (B) Representative positive staining of cTnI (red). (C) Overlay of images (A and B). The nuclei were counterstained with DAPI (blue). (D) T-box 18 (TBX18)-ADSCs detected by immunofluorescence assay (green). (E) Representative positive staining of cTnI (red). (F) Overlay of images (D and E). The nuclei were counterstained with DAPI (blue). (G) Degree of positive staining of cTnI in TBX18-ADSCs and GFP-ADSCs. Scale bar, 50 µm.

Figure 3

(A) Transfected adipose-derived stem cells (ADSCs) form connections with neonatal rat ventricular cardiomyocytes (NRVMs) and generate synchronous beating. (A) Observed under a light microscope in GFP-ADSCs group. The beating rate is 56 bpm. (B) Observed under a fluorescence microscope in GFP-ADSCs group. (C) Observed under a light microscope in T-box 18 (TBX18)-ADSCs group. The beating rate is 75 bpm. (D) Observed under a fluorescence microscope in TBX18-ADSCs group. Scale bar, 100 µm.

Figure 4

Expression of hyperpolarization activated cyclic nucleotide gated potassium channel 4 (HCN4) after co-culture for 7 days. (A) Western blot analysis of HCN4 protein expression in the transfected groups and neonatal rat ventricular cardiomyocytes (NRVMs). GAPDH was used as the protein control. (B) Quantitative assessment of HCN4 protein levels by integrated optical density analyses. (C) Expression of HCN4 mRNA in the transfected groups. ^*P<0.05 vs. other groups.

Figure 5

Detection of I_f current using the patch clamp technique. (A) The spindle-shaped cell was used for electropysiological recordings. (B) Hyperpolarization-activated inward current in T-box 18 (TBX18)-adipose-derived stem cells (ADSCs) and was blocked by CsCl (4 mM/l). (C) Average current-voltage association of the I_f current. Scale bar, 100 µm.

Figure 6

Identification of I_f channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs). (A) Macroscopic I_f currents a in single beating hiPSCM using hyperpolarization-activated inward current. (B) Current-voltage relationship for I_f channels was studied using whole-cell patch clamp techniques.