In situ investigation of allografted mouse HCN4 gene–transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices

Abstract

BackgroundRecently, proof-of-concept experiments have shown that genetically modified bone marrow mesenchymal stromal cells (MSCs) carrying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were able to express the funny current (If) in vitro, which played a key role in the process of pacemaker generation for heart rate, and were capable of pacemaker function after transplantation into the host heart. Nevertheless, because of the lack of direct experimental access to the implanted cells in situ, the changes in electrophysiological characteristics and the mechanisms underlying the pacemaker function of engrafted HCN gene–transfected MSCs in vivo remain unclear. Methods and ResultsOn the basis of the improved preparation of ventricular slices, we successfully performed an in situ investigation of allografted mouse HCN4 gene (mHCN4)-transfected rat MSCs (rMSCs) with the use of patch-clamp recording in ventricular slices. We demonstrate that allografted mHCN4-transfected rMSCs survived in the host heart for >4 weeks; that they expressed If, which is generated by the mHCN4 channel, with a similar amplitude but greater negative activation compared with parallel cells cultured in vitro; that they did not express optical action potentials or depolarization-activated inward sodium or calcium currents; and that they exhibited a low incidence of gap-junctional coupling with host cardiomyocytes. ConclusionsThis study provides direct experimental access to investigate MSCs after transplantation into the host heart. We propose that mHCN4-transfected rMSCs survived in the host heart with altered electrophysiological characteristics of If and were accompanied by a low efficiency of connexin 43 expression at 4 weeks after transplantation, which may affect its pacemaker function in vivo.