【Abstract】 Objective To investigate the expression of connexin 40 (Cx40) and hyperpolarization-activated cycl icnucleotide-gated cation channel 4 (HCN4) in rat bone marrow mesenchymal stem cells (BMSCs) cocultured with the sinoatrialnode (SAN) tissues in vitro, so as to evaluate the possibil ity of BMSCs differentiation into SAN cells. Methods BMSCs wereisolated from Sprague Dawley rats (aged 4-6 weeks, male or female) by the adhesive method and cultured; BMSCs at the 3rdpassage were marked with carboxyfluorescein succinimidyl ester, and then were incubated on 6-well culture plate; cell climingsl ices were prepared at the same time. SAN tissue was taken and cut into 0.3 cm × 0.3 cm mass, and then placed into 4℃ PBSsolution. The SAN tissue mass was cocultured with marked BMSCs at the 3rd passage for 3 weeks as the experimental group, andBMSCs at 3rd passage were cultured alone for 1 week as the control group. At 1, 2, and 3 weeks after coculture, the mean integratedabsorbance (MIA) values of Cx40 and HCN4 were measured by Image pro plus 5.0 through the method of immunohistochemistry,and the mRNA expressions of Cx40 and HCN4 were identified by real-time fluorescent quantitative PCR. Results TheMIA values of Cx40 and HCN4 in the experimental group were higher than that in the control group, showing significantdifferences (P lt; 0.01). In the experimental group, the expressions of Cx40 and HCN4 increased gradually with time. The longerthe culture time was, the higher the expressions of Cx40 and HCN4 were, showing significant differences (P lt; 0.05). The mRNAexpressions of Cx40 and HCN4 in the experimental group were significantly higher than those in the control group (P lt; 0.01); inthe experimental group, the mRNA expressions of Cx40 and HCN4 increased gradually with time, showing significant differencesbetween different time points (P lt; 0.05). Conclusion The expressions of Cx40 and HCN4 increase obviously after coculturingBMSCs with SAN tissue, indicating that BMSCs could differentiate into SAN cells by coculturing with SAN tissue in vitro.
Objective To study the influence of ischemia-reperfusion on the expression of the hyperpolarization activated cycl icnucleotide gated cation channel 4 (HCN4) and to discuss the mechanism of functional disturbance of sinoatrial node tissue (SANT) after ischemia reperfusion injury (IRI). Methods Eighty five healthy adult rabbits, weighing 2-3 kg, were randomly divided into 3 groups: control group [a suture passed under the root section of right coronary artery (RCA) without l igation, n=5], experimental group A (occluding the root section of RCA for 30 minutes, then loosening the root 2,4, 8 and 16 hours, n=10), experimental group B (occluding the root section of RCA for 1 hour, then loosening the root 2, 4,8 and 16 hours, n=10). At the end of the reperfusion, the SANT was cut off to do histopathological, transmission electronmicroscopical and immunohistochemical examinations and semi-quantitative analysis. Results The result of HE stainingshowed that patho-injure of sinoatrial node cell (SANC) happened in experimental groups A and B after 2 hours of reperfusion, the longer the reperfusion time was, the more serious patho-injure of SANC was after 4 and 8 hours of reperfusion, SANC reached peak of damage after 8 to 16 hours of reperfusion; patho-injure of SANC was more serious in experimental group B than in experimental group A at the same reperfusion time. Immunohistochemical staining showed that the expression of HCN4 located in cellular membrane and cytoplasm in the central area of SANC and gradually decreased from the center to borderl ine. The integral absorbance values of HCN4 expression in the control group (397.40 ± 34.11) was significantly higher than those in the experimental group A (306.20 ± 35.77, 216.60 ± 18.59, 155.40 ± 19.11 and 135.00 ± 12.30) and in the experimental group B (253.70 ± 35.66, 138.70 ± 13.28, 79.10 ± 9.60 and 69.20 ± 8.42) after 2, 4, 8 and 16 hours of reperfusion (P lt; 0.05). With reperfusion time, the expression of HCN4 of SANC decreased, which was lowest after 8 hours of reperfusion; showing significant difference among 2, 4 and 8 hours after reperfusion (P lt; 0.05) and no significant difference between 8 and 16 hours after reperfusion (P gt; 0.05). At the same reperfusion time, the expression of HCN4 was higher in the experimental group A than in the experimental group B. The result of transmission electron microscope showed that ultramicrostructure of SANC was damaged after reperfusion in experimental groups A and B. The longer the reperfusion time was, the more serious ultramicrostructure damage of SANC was, and reached the peak of damage after 8 hours of reperfusion. Ultramicrostructure of SANC was not different between 8 and 16 hours of reperfusion. At the same reperfusion time, the ultramicrostructure damage of SANC was moreserious in experimental group B than in experimental group A. Conclusion IRI is harmful to the morphous and structure ofSANC, and effects the expression of HCN4 of SANC, which is concerned with functional disturbance and arrhythmia.
Objective To locate sinoatrial node (SAN) in suckl ing pigs, to develop a rel iable method for isolation, purification and cultivation of SAN cells and to observe the compatibil ity of SAN cells and Col I fiber scaffold. Methods Five newborn purebred ChangBaiShan suckl ing pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, wereused. Multi-channels electrophysiological recorder was appl ied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 × 105 cells/mL) were co-cultured with prewetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM). Results The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% ± 2.9% in the purified cultured SAN cells, to 44.7% ± 2.3% (P lt; 0.01), and the proportion of irregular cells increased from 7.0% ± 1.7% in the purified cultrued SAN cells to 36.1% ± 2.6% (P lt; 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% ± 2.1% and 19.2% ± 2.5%, respectively (P gt; 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobate adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia. Conclusion With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a rel iable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibil ity.