Transcription factor Tbx18 induces the differentiation of c-kit+ canine mesenchymal stem cells (cMSCs) into SAN-like pacemaker cells in a co-culture model in vitro

Abstract

Bone mesenchymal stem cells (MSCs), as well as cardiomyocytes, are derived from early mesoderm, becoming committed to their fate under the influence of different differentiation factors. We examined whether the overexpression of Tbx18 can induce the differentiation of c-kit⁺ cMSCs into a phenotype similar to that of native pacemaker cells and whether these transfected cells can couple to adjacent atrial cells with functional consequences. The c-kit⁺ cMSCs were first sorted, then transfected with different lentiviral vectors. Tbx18-c-kit⁺ cMSCs represented the experimental group, while EYFP-c-kit⁺ cMSCs and canine sinoatrial node (SAN) cells were used as controls. Within days of transfection, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel HCN4 protein and gap junction protein Connexin 45 (Cx45) expression in Tbx18-c-kit⁺ cMSCs were 12-fold and 5.6-fold higher, respectively, than that in EYFP-c-kit⁺ cMSCs. After co-culture with canine atrial cells in vitro for three days, the funny currents (I_f) were recorded in the Tbx18-c-kit⁺ cMSCs, but not in EYFP-c-kit⁺ cMSCs. The trend of these I_f currents was highly similar to that of SAN cells, although the current density was smaller. The Tbx18-EYFP-c-kit⁺ cMSCs showed responsiveness to β-adrenergic stimulation, and the intracellular cyclic adenosine monophosphate (cAMP) level was higher than that in EYFP-c-kit⁺ cMSCs. The Tbx18-EYFP-c-kit⁺ cMSCs delivered fluorescent dye to neighboring atrial cells via gap junctions, thus these cell pairs could communicate as a pacemaker unit. We propose that the overexpression of Tbx18 in c-kit⁺ cMSCs induces their differentiation to SAN-like pacemaker cells.

Keywords: HCN4; Tbx18; c-kit+ mesenchymal stem cells; co-culture; gap junction.

Figure 1

Sorting and identification of c-kit⁺ cMSCs. (A/P1) Drawing an appropriate gate to exclude the dead or clustered cells. (A/P2) Gate was drawn using negative control. (B/P2) After sorting, the percentage of c-kitpositive cells was 91.25%. (C-E) Next, flow cytometry analysis of the gated c-kit⁺ cells revealed the high expression of cMSC markers CD29 (higher than 99%, n = 3), CD44 (higher than 99%, n = 3) and the absence of CD45 (lower than 2.55%, n = 3).

Figure 2

Verification of hTbx18 overexpression in Tbx18-EYFP-c-kit⁺ cMSCs. Laser confocal microscopy of c-kit⁺ cMSCs after transfection. The c-kit⁺ cMSCs were transfected with pLV-hTbx18-EYFP (A) and pLV-EYFP (B), as evidenced by the expression of yellow fluorescence. Scale bar: 75 μm. (C) Reverse transcription (RT)-polymerase chain reaction (PCR) further confirmed hTbx18 overexpression in Tbx18-EYFP-c-kit⁺ cMSCs. EYFP-c-kit⁺ cMSCs were used as a control. Lane M is a DL 500 ladder marker. The length of the amplicons for lane Tbx18 was 251 bps.

Figure 3

Functional expression of I_f in Tbx18-EYFP-c-kit⁺ cMSCs. (A) Voltage pulse protocol to elicit currents and evaluate activation properties of I_f. (B) Representative I_f traces (top-to-bottom) recorded from a Tbx18-EYFP-c-kit⁺ cMSC before, during and after 5 mmol/l Cs⁺. (C) EYFP-c-kit⁺ cMSCs did not record any currents. (D) Current voltage (I-V) relationship of I_f in Tbx18-EYFP-c-kit⁺ cMSCs and canine SAN cells. Data are presented as the means ± SEM. *P < 0.05 vs. SAN; **P < 0.01 vs. SAN. (E) Normalized tail currents of Tbx18-EYFP-c-kit⁺ cMSCs, which were compared with SAN cells used to construct the I_f activation curves. Solid lines are the Boltzmann equation fit to the data. Data are presented as the means ± SEM. (F) Voltage pulse protocol to measure deactivation properties of I_f. (G) Typical tail current traces of I_f in Tbx18-EYFP-c-kit⁺ cMSCs. (H) I-V relationships of the fully activated I_f in Tbx18-EYFP-c-kit⁺ cMSCs and SAN cells. Solid lines are the linear fit to the different current traces under corresponding test voltage. Data are presented as the means ± SEM. (I) Time constants of (de)activation in Tbx18-EYFP-c-kit⁺ cMSCs. Solid line is currents elicited from activation (A) and deactivation (F) voltage pulse protocols, fit to the equation τ = 1/[A1 × exp (-V/B1) + A2 × exp (V/B2)], where τ is the activation or deactivation time constant, and A1, A2, B1, and B2 are evaluated fitting parameters [58]. (J, K) Effects of isoproterenol on the activation of I_f in Tbx18-EYFP-c-kit⁺ cMSCs. (J) Characteristic I_f traces elicited by a step pulse to -160 mV from holding potential of -30 mV, followed by a step to +20 mV in the absence and presence of 3 μmol/L isoproterenol (ISO). (K) Shifts of I_f activation by isoproterenol. Data arepresented as the means ± SEM.

Figure 4

Tbx18-c-kit⁺ cMSCs recapitulate the critical features of genuine SAN pacemakers. A. Intracellular cAMP level of Tbx18--c-kit⁺ cMSCs was significantly higher compared to EYFP--c-kit⁺ cMSCs (n = 3), reproducing the higher intracellular cAMP level observed in SAN cells (n = 3). Data are presented as the means ± SEM. **P < 0.01 vs. EYFP--c-kit⁺ cMSCs; ***P < 0.001 vs. EYFP--c-kit⁺ cMSCs. B. Relative mRNA levels of Cx45, Kir2.1, PLB, α-actinin comparing Tbx18--c-kit⁺ cMSCs and SAN cells normalized to EYFP--c-kit⁺ cMSCs, respectively. Tbx18--c-kit⁺ cMSCs show similar pattern of normalized transcript levels of SAN cells. Similar results were obtained in 3 independent experiments, each experiment was performed with cells isolated from ≥ 3 wells from a 6-well plate. Data are presented as the means ± SEM. *P < 0.05 vs. EYFP--c-kit⁺ cMSCs or SAN cells; **P < 0.01 vs. EYFP--c-kit⁺ cMSCs; ***P < 0.001 vs. EYFP--c-kit⁺ cMSCs. C. HCN4, Cx45, PLB, p-PLB, α-actinin protein expression levels were detected by using western blot analysis. Similar results were obtained in 3 independent experiments, and each experiment was performed with cells isolated from the 6 wells of a 6-well plate. Data are presented as the means ± SEM. ***P < 0.001 vs. EYFP--c-kit⁺ cMSCs or SAN cells. D. Cx45 immunofluorescence image (Cx45-red, nuclei-blue) of SAN cells (upper, left), Tbx18-c-kit⁺ cMSCs (upper, right) and EYFP--c-kit⁺ cMSCs (bottom). Scale bar: 25 μm.

Figure 5

Dye diffused into neighboring atrial cells. (A) The 0.1% Lucifer yellow dye was loaded into Tbx18-EYFP--c-kit⁺ cMSCs (fluorescent cell, I_f currents expression, n = 6), (B) a SAN cell (I_f currents expression, n = 3) and (C) an EYFP--c-kit⁺ cMSC (fluorescent cell, n = 6) respectively. After the 5-min dye transfer, (A) two adjacent atrial cells are displayed, (B) one adjacent cell was displayed, and (C) the dye did not diffuse into any adjacent atrial cells. Scale bar = 20 μm.