Adipose‑derived stem cells overexpressing SK4 calcium‑activated potassium channel generate biological pacemakers

Figure 1. Expression of SK4 after transduction. (A) Transduction rate of different MOI values detected by flow cytometric analysis. The SK4 transduction efficiency was >70% at MOI≥100. (B) SK4 mRNA was stably expressed in ADSCs at 2 and 7 days. (C and D) Western blot assays revealed significantly increased SK4 expression compared with the GFP group. *P<0.05. SK4, intermediate-conductance Ca2+-activated potassium channel; MOI, multiplicity of infection; ADSCs, adipose-derived stem cells.

Figure 2. Expression of pluripotent markers Oct-4 and Sox-2 and transcription factors Tbx18 and Shox2 following SK4 adenovirus transduction with different MOI values. (A and B) Relative Oct-4 and Sox-2 mRNA levels were detected by RT-qPCR. (C and D) Relative Tbx18and Shox2 mRNA levels were detected by RT-qPCR. *P<0.05. SK4, intermediate-conductance Ca2+-activated potassium channel; MOI, multiplicity of infection; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 3. Expression of myocardial-specific markers after transduction. (A-C) Expression of α-actinin detected using immunofluorescence assay with different MOI values. Scale bar, 100 µm. (D) Quantitative analysis demonstrated that the α-actinin positivity rate in the SK4 group was significantly higher at MOI=150 compared with MOI=100. (E) Expression of cTnI mRNA after transduction with different MOI values. (F) The cell numbers were significantly reduced at 1-5 days after SK4 transduction with MOI=150, and were increased at 3 days after GFP transduction, SK4 transduction with MOI=100, and in the control group. *P<0.05. SK4, intermediate-conductance Ca2+-activated potassium channel; MOI, multiplicity of infection; cTnI, cardiac troponin I.

Figure 4. Expression of HCN4 and pacemaker current If after transduction. (A and B) Western blot analysis of HCN4 expression in the GFP and SK4 groups with MOI=100 and MOI=150 (*P<0.05). (C) If current detected by the patch clamp technique and blockade with CsCl (2 mM/l). (D) Mean current-voltage association of the If current (n=8). SK4, intermediate-conductance Ca2+-activated potassium channel; MOI, multiplicity of infection; HCN4, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4.

Figure 5. Effects of SK4 on the expression of p-ERK 1/2, p-JNK, and p-p38MAPK and total protein detected by western blot analysis. (A-D) The levels of p-ERK 1/2 and p-p38 MAPK increased significantly after SK4 transduction. *P<0.05. SK4, intermediate-conductance Ca2+-activated potassium channel; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Figure 6. ADSCs transduced with SK4 generated biological pacemakers in ex vivo hearts. (A) After AVB model establishment, the electrocardio-graphic morphology was the same as the spontaneous rhythm in the SK4 group following stimulation of the cell injection site, whereas the GFP group exhibited the opposite morphology after stimulation. (B) Heart rate of the SK4 and GFP groups after AVB model establishment in ex vivo hearts (n=8/group) (*P<0.05). (C) Immunofluorescence detected that transduced ADSCs were successfully implanted and expressed HCN4 in the SK4 group. Scale bar, 100 µm. SK4, intermediate-conductance Ca2+-activated potassium channel; ADSCs, adipose-derived stem cells; AVB, atrioventricular block; HCN4, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4.