Shox2 influences mesenchymal stem cell fate in a co-culture model in vitro

Abstract

Sinoatrial node (SAN) dysfunction is a common cardiovascular problem, and the development of a cell sourced biological pacemaker has been the focus of cardiac electrophysiology research. The aim of biological pacemaker therapy is to produce SAN-like cells, which exhibit spontaneous activity characteristic of the SAN. Short stature homeobox 2 (Shox2) is an early cardiac transcription factor and is crucial in the formation and differentiation of the sinoatrial node (SAN). The present study aimed to improve pacemaker function by overexpression of Shox2 in canine mesenchymal stem cells (cMSCs) to induce a phenotype similar to native pacemaker cells. To achieve this objective, the cMSCs were transfected with lentiviral pLentis‑mShox2‑red fluorescent protein, and then co‑cultured with rat neonatal cardiomyocytes (RNCMs) in vitro for 5-7 days. The feasibility of regulating the differentiation of cMSCs into pacemaker‑like cells by Shox2 overexpression was investigated. Reverse transcription-quantitative polymerase chain reaction and western blotting showed that Shox2‑transfected cMSCs expressed high levels of T box 3, hyperpolarization-activated cyclic nucleotide‑gated cation channel and Connexin 45 genes, which participate in SAN development, and low levels of working myocardium genes, Nkx2.5 and Connexin 43. In addition, Shox2‑transfected cMSCs were able to pace RNCMs with a rate faster than the control cells. In conclusion, these data indicate that overexpression of Shox2 in cMSCs can greatly enhance the pacemaker phenotype in a co-culture model in vitro.

Figure 1

Characterization of cMSCs. (A) Laser confocal microscopy of cMSCs after transfection. cMSCs were transfected with pLentis-mShox2-RFP, as evidenced by the expression of Shox2. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole as a control. (B) Fluorescence images of 1:4 cMSCs: RNCMs co-cultured on day 5 after plating. cMSCs were randomly distributed in culture. Scale bar, 50 µm. (C) Shox2-transfected cMSCs expressing Cx45 were co-cultured with RNCMs. None of the control cells displayed this positive expression. Scale bar, 20 µm. cMSC, canine mesenchymal stem cells; NRMCs, rat neonatal cardiomyocytes; Shox2, short stature homeobox 2; Cx45, Connexin 45; RFP, red fluorescent protein.

Figure 2

Shox2, Tbx3, HCN4, Cx45, Nkx2.5 and Cx43 gene expression was examined using reverse transcription-quantitative polymerase chain reaction. Similar results were obtained in three independent experiments. Data are presented as the mean ± standard error of the mean. ^*P<0.05 vs. control. Shox2, Short stature homeobox 2; Tbx3, T box 3; HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel; Cx45, connexin 45; Cx43, Connexin 43; RFP, red fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RNCMs, rat neonatal cardiomyocytes.

Figure 3

Shox2, Tbx3, HCN4, Cx45, Nkx2.5 and Cx43 protein expression were examined using western blotting. Similar results were obtained in three independent experiments. Shox2, Short stature homeobox 2; Tbx3, T box 3; HCN4, hyperpolarization-activated cyclic nucleotide-gated cation channel; Cx45, connexin 45; Cx43, Connexin 43; RFP, red fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RNCMs, rat neonatal cardiomyocytes.