mHCN4 genetically modified canine mesenchymal stem cells provide biological pacemaking function in complete dogs with atrioventricular block.

BACKGROUND: The study was undertaken to assess the properties of mouse HCN4 (mHCN4)-modified canine mesenchymal stem cells (cMSCs) in dogs with experimentally induced complete atrioventricular (AV) block and electronic pacing. METHODS: Complete AV block was induced in adult dogs who had undergone implantation of backup electronic pacemakers. cMSCs were transfected with mHCN4 genes. Evidence of successful IHCN4 expression was provided by patch-clamp detection. mHCN4-cMSCs or normal cMSCs were injected subepicardially into the left ventricular anterior wall of the dogs. Cardiac parameters were monitored for 6 weeks. Heart rate variability (HRV) was evaluated using quantitative Poincaré plots of R-RN against R-RN+1 intervals. cMSCs survival and expression of HCN4 in vivo were examined by histological studies and Western blot. RESULTS: In 2 weeks, the maximum heart rate and the number of impulses generated from the injection sites were much higher in dogs injected with HCN4-modified MSCs than in control dogs. Basal heart rate increased in the HCN4 group and became fully stabilized by Week 4, evidenced by markedly reduced numbers of electronic pacemaker beats. At Week 2, HRV during exercise was significantly higher in HCN4 dogs than in controls as shown by descriptors of both instantaneous (SD1) and longer term (SD2) beat-to-beat deviations (P < 0.05). Hematoxylin-eosin staining and Western blot proved that cMSCs survive and express HCN4 protein in situ in heart of HCN4 dog. CONCLUSION: Transplantation of mHCN4-modified cMSCs provided a stable biological pacemaking function that allowed an appropriate chronotropic response to physical exercise for up to 6 weeks.

Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4 gene transfer create cardiac pacemakers.

BACKGROUND AIMS: The study objective was to test the ability of canine mesenchymal stromal cells (cMSC) transfected with the mouse hyperpolarization-activated cyclic nucleotide-gated channel 4 (mHCN4) gene to deliver a biologic pacemaker to the canine heart. METHODS AND RESULTS: cMSC that were transfected by lentiviral vector with the cardiac pacemaker gene mHCN4 expressed high levels of Cs(+) -sensitive current (26.4 ± 1.8pA/pF at -140 mV; (n = 17) and were activated in the diastolic potential range with a reversal potential of -29.7 ± 2.5 mV (n = 14), confirming that the expressed current was Funny current (I(f))-like. Next, 3 × 10(6) cMSC transfected with either control plasmid or the mHCN4 gene construct were injected subepicardially into the canine right ventricular wall in situ. During sinus arrest, all control hearts had spontaneous atrioventricular node rhythms [rate = 21 ± 5 beats per minute (b.p.m.)]. In the mHCN4 group, six of eight animals developed spontaneous ventricular rhythms of right-sided origin (rate = 45 ± 9b.p.m.; P < 0.01). Moreover, immunohistochemical analysis of the injected regions demonstrated neither apoptosis nor cellular or humoral rejection at 2 weeks. CONCLUSIONS: These results demonstrate that genetically modified cMSC can express functional HCN4 channels in vitro and in vivo and represent a novel delivery system for pacemaker genes into the heart.

Study on the inward currents of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells / 第三军医大学学报

Objective To observe the electrophysiological characteristics of the inward current of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells (MSCs). Methods Bone marrow specimens was extracted from SD rats aged 1 month. MSCs were isolated by gradient centrifugation and obtained by adherence culture. The second-passage MSCs were treated with 10 ?mol/L 5-azacytidine and incubated for 24 h. After 4 weeks, cardiomyocyte-like cells were identified by immunocytochemical staining technique, then used to detect the inward current with the whole-cell patch-clamp technique, which results were compared with the normal cardiomyocytes and the MSCs without induction. Results Some cardiomyocyte-like cells were stained positive for troponin T, and the quickly activated and inactivated inward currents were elicited 4 weeks after 5-aza cytidine treatment. The inward currents of cardiomyocyte-like cells recorded in the same square pulse were weaker than that of the normal cardiomyocytes, and the I-V curve of cardiomyocyte-like cells was right-shifted. Conclusion After treated with 5-azacytidine, MSCs differentiate into cardiomyocyte-like cells which express the excitable inward current.