GW24-e3884 Canine bone marrow mesenchymal stromal cells modified with Shox2 gene rebuild biological pacemakers in vitro

Objectives Heart development is a strictly regulated process, and many gene families contribute to regulating genetic network to achieve this program. The homeobox genes are such a family encodes transcription factors, and it contains two closely related members, Shox and Shox2. During the embryonic cardiac development, Shox2 is restrictedly expressed in the sinoatrial node (SAN) and the sinus valves. Analyses of Shox2 knockout mouse models have demonstrated that Shox2 plays a crucial role during the SAN formation and the pacemaking system development. This study objective was to examine the ability of canine mesenchymal stromal cells (cMSCs) transfected with the mouse Shox2 gene to deliver a biological pacemaker. Methods We used lentivirus vectorto integrate the mouse Shox2 gene into the genome of cMSCs and were able to achieve stable expression of the Shox2 gene permanently. The cMSCs were randomised into two groups: the Shox2 group transfected with LV-Shox2- EGFP-cMSCs, and the control group transfected with LV-EGFP-cMSCs. Then all the positively transduced cMSCs were cultured with the new born rat myocardial cell lysate. After co-cultivation for seven days, we investigated the morphological changes of these transfected cMSCs. At the same time, the expression of cardiomyogenic markers Shox2 and HCN4 were determined by quantitative RT-PCR, Western Blot and immunofluorescence staining. In addition, the kinetics characters of I f like current channels were detected by whole-cellpatch clamp. Results Shox2 and HCN4 proteins were highly expressed in experimental group compared with the control group. Ahyperpolarization-activated inward current was recorded by whole-cell patchclamp in cMSCs transfected with Shox2 gene. The I f current recorded from the experimental group was sensitive to extracellular Cs + . Meanwhile, this current couldn’t be observed in control cells under the same conditions. Conclusions These results demonstrate that cMSCs modified by successful transfection with Shox2 can differentiate so as to develop spontaneous mechanical activity in vitro. Shox2 plays an essential role in the pacemaker development through the promotion of HCN4.