Triptolide improves early survival of mesenchymal stem cells transplanted into rat myocardium.

OBJECTIVE: To investigate whether triptolide can prolong the survival of rat mesenchymal stem cells (MSCs) transfected with the mouse hyperpolarization-activated cyclic nucleotide-gated channel 4 (mHCN4) gene in the myocardium. METHODS: Grafted cell survival was determined using a sex-mismatched cell transplantation model and analysis of Y chromosome-specific Sry gene expression from hearts harvested at different time points after cell transplantation. ELISA and RT-PCR were used to measure protein and mRNA levels, respectively, of nuclear factor (NF)-κB, IL-1ß, IL-6 and TNF-α. RESULTS: Donor cell numbers decreased over time. Pretreatment with triptolide improved graft survival both 24 (29.3 ± 0.9%) and 72 h (17.5 ± 1.2%) after transplantation of MSCs and resulted in a 2.5-fold increase in the total cell number 72 h after cell transplantation. The mRNA expression and protein content of NF-κB, IL-1ß, IL-6 and TNF-α were significantly reduced in the triptolide-treated group compared with the control groups. In addition, triptolide downregulated Bax but upregulated Bcl-2 in the injected region. CONCLUSIONS: Transient treatment with triptolide may significantly improve the early survival of MSCs in vivo. The mechanism underlying this effect involves attenuating the inflammatory response via inhibition of the NF-κB signaling pathway.

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.

BACKGROUND: Recently, 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 RESULTS: On 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. CONCLUSIONS: This 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.

The integration and functional evaluation of rabbit pacing cells transplanted into the left ventricular free wall.

To evaluate the feasibility of cell transplantation to treat bradyarrhythmia, we analyzed the in vivo integration and pacing function after transplantation of mHCN4-modified rabbit bone marrow mesenchymal stem cells (MSCs) into the rabbit left ventricle free wall epicardium. In our investigation, we injected MSCs transduced with or without mHCN4 into the rabbit left ventricle free wall epicardium. Chemical ablation of the sinoatrial node was performed and bilateral vagus nerves were sequentially stimulated to observe premature left ventricular contraction or left ventricular rhythm. We found that the mHCN4-transduced MSC group had a significantly higher ventricular rate and a shorter QRS duration than that of the control and EGFP group. Furthermore, the mHCN4-transduced MSCs, but not the control cells, gradually adapted long-spindle morphology and became indistinguishable from adjacent ventricle myocytes. The modified MSCs showed pacing function approximately 1 week after transplantation and persisted at least 4 weeks after transplantation. In conclusion, a bradyarrhythmia model can be successfully established by chemical ablation of the sinoatrial node and sequential bilateral vagus nerve stimulation. The mHCN4-modified rabbit MSCs displayed evident dynamic morphology changes after being transplanted into rabbit left ventricle free wall epicardium. Our studies may provide a promising strategy of using modified stem cell transplantation to treat bradyarrhythmia.

Mild electrical pulse current stimulation upregulates S100A4 and promotes cardiogenesis in MSC and cardiac myocytes coculture monolayer.

Parietal endoderm-secreted S100A4 promotes early cardiomyogenesis in embryoid bodies [1]. After an acute ischemic event, S100A4 protein appears in cardiac myocytes only in the border zone in rat and human hearts [2]. In wound research, a large outward current of 4 µA/cm(2) was always measured at the wound edges of rat cornea and human skin [3]. We hypothesize that a special electrical circumstance at the border zone may contribute to the phenomenon. An electric stimulation system was designed to give the cells electric pulse current stimulation (EPCS), the feature of the signal is pulse polarity altered one after another, rectangular 2 ms, 2 Hz, 40 µA. This intensity of stimulation is proved to be safe to cardiac myocytes (both in structure and beating behavior compared with the cardiac myocytes which do not receive stimulation) and MSCs (in cell vitality, proliferation, cell cycle, and gap junction generation potential) through our previous work. Canine MSCs are capable of generating voltage-sensitive Ca(2+) channel and Na(+) channels and generating the Ca(2+) handling system during differentiation. We found that CD44 was reduced in the MSCs monolayer treated with EPCS, compared with non-stimulated MSCs; and EPCS MSCs (3 h/day, 6 h/day, 5 days) showed an 14.04 ± 3.44 and 14.55 ± 3.97 % reduction in CD44, compared with the cotemporary MSCs; these reveal that CD44 reduction amplitude is not correlated with time for EPCS disposure and CD29 (integrin ß1) expression is not affected by EPCS exposure. EPCS was given to the MSCs and cardiac myocytes coculture monolayer (ratio 3:1) for different time (1, 3, and 6 h/day) for 4 days to see the biological effects. Gap junction protein and troponin T show an increase after EPCS. We found that the gap junction protein Cx43 increased with treating time-in the EPCS group, it exhibited 1.5 and 1.7 fold in the 3 h/day group and 6 h/day group (P < 0.01), and troponin T exhibited to about 3.6 and 4.4 fold in the 3 h/day group (P < 0.01) and 6 h/day group (P < 0.05). Since coculture was used as stimuli, immunofluorescence was used to visualize the changes during EPCS for the purpose of elucidating the impact of EPCS on cardiac myocytes and MSCs. We found that after 5 days exposure, EPCS can enhance the expression of S100A4, which is 2.33 fold in cardiac myocytes (P < 0.01) and 1.99 fold in MSCs (P < 0.01) in gray value. A significant increasing expression of the myocyte enhancer factor (MEF) and GATA4 is detected in neonatal rat cardiac myocytes (P < 0.01) compared with cotemporary coculture monolayer in the control group. Also, EPCS can trigger the assembly of MEF2c in the nuclei. In addition, more cardiac myocytes were found to have two nuclei. But MSCs fail to active MEF2C transcriptional factor like that in cardiac myocytes after EPCS exposure. The elevation of MEF2 in both cytoplasm and nuclei of cardiac myocytes can always make a clear distinction of the cardiac myocytes and MSCs in coculture. Some factors show strong upregulation tendency with EPCS in both cardiac myocytes and MSCs-these include the troponin T (P < 0.01) and Cx43 (P < 0.05) in cardiac myocytes, and troponin T (P < 0.01) and Cx43 (P < 0.01) in MSCs. Collagen I expression is not affected with EPCS. In conclusion, mild EPCS can upregulate the secretion of S100A4 in both cardiac myocytes and MSCs, which is a factor supporting the cardiomyogenesis and angiogenesis; it further triggers the development of neonatal rat cardiac myocytes through upregulation of MEF2C and GATA4, the number of cardiac myocytes with two nuclei increases with EPCS, but this phenomenon does not appear in MSCs. Despite this, Cx43 and troponin T in both cardiac myocytes and MSCs are very sensitive to EPCS. EPCS can act as an effective and multi-targeted physical intervention method in cardiomyogenesis.