Abstract
This study investigated whether overexpression of paired-related homeobox 1 (prrx1) can successfully induce differentiation of brown adipose-derived stem cells (BADSCs) into sinus node-like cells. The experiments were performed in two groups: adenovirus-green fluorescent protein (Ad-GFP) group and Ad-prrx1 group. After 5-7 days of adenoviral transfection, the expression levels of sinus node cell-associated pacing protein (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 [HCN4]) and ion channel (calcium channel, voltage-dependent, T type, alpha 1G subunit [Cacna1g]), as well as transcription factors (T-box 18 [TBX18], insulin gene enhancer binding protein 1 [ISL-1], paired-like homeodomain transcription factor 2 [pitx2], short stature homeobox 2 [shox2]), were detected by western blot and reverse transcription-quantitative polymerase chain reaction. Immunofluorescence assay was carried out to detect whether prrx1 was coexpressed with HCN4, TBX18, and ISL-1. Finally, whole-cell patch-clamp technique was used to record pacing current hyperpolarization-activated inward current (If). The isolated cells were CD90+, CD29+, and CD45-, indicating that pure BADSCs were successfully isolated. After 5-7 days of Ad transfection into cells, the mRNA levels and protein levels of pacing-related factors (TBX18, ISL-1, HCN4, shox2, and Cacna1g) in Ad-prrx1 group were significantly higher than those in Ad-GFP group. However, the expression level of pitx2 was decreased. Immunofluorescence analysis showed that prrx1 was coexpressed with TBX18, ISL-1, and HCN4 in the Ad-prrx1 group, which did not appear in the Ad-GFP group. Whole-cell patch clamps were able to record the If current in the experimental group rather than in the Ad-GFP group. Overexpression of prrx1 can successfully induce sinus node-like cells.