6 months (n=12), control group, without AF (n=8). Atrial muscle samples of the left and right atrial appendage were cut off during heart valve replacement. Expressions of Cx40 and Cx43 were detected by immunoblotting and immunohistologic analyses. Results ①No obvious change of Cx43 and Cx40 in the left and right atrium of each patient in each group was measured. ②Compared with that in the control group, the level of Cx40 was decreased signficantly in GroupⅠ (P

6 months; Control group (n=8) without AF. Atrial muscle samples of the left and right atrial appendage were cut off during heart valve replacement. Morphological changes were observed by light microscopy and electron microscopy. Results ① The incidence rates of Aschoff body were 37.5% (3/8), 46% (5/11) , and 50% (6/12) in Group Ⅰ, Group Ⅱ, and the control group. There was no difference in Aschoff body between the left and right atrial myocytes. ② There was no difference in myocytic degeneration, interstitial hyperplasia, and lymphocytes infiltration between the left and right atrial myocytes in each group. ③ Myolysis, accumulation of glycogen, and fibrosis were detected by electron microscopy in the 3 groups. Changes of myolysis and fibrosis were more obvious in patients with AF than patients without AF. Conclusion There are similar pathological changes in the left and right atria in rheumatic heart disease patients with or without AF. Fibrosis of atrial myocytes may be involved in the occurrence and maintenance of AF.

Objective To investigate the effect of calcium/calmodulin-dependent protein kinaseⅡ (CaMK[KG-*6]Ⅱ) inhibitor,KN93 on calcium overloading of atrial muscle cells in neonatal rats and detect the expression of CaMK[KG-*6]Ⅱ. Methods The atrial muscle cells from neonatal rats were primarily cultured for 96 h and then divided into 6 group,control,calcium overloading group,KN93 group (0.5 ?mol/L),low-,moderate-and high-dose of KN93+ calcium overloading group. A model of calcium overloading for atrial muscle cells was established by using calcium ionophore (ionomycin,1.0 ?mol/L). For the later 3 groups,KN93 at doses of 0.25,0.5 and 1.0 ?mol/L was added into the culture medium for 30 min followed by 1.0 ?mol/L ionomycin treatment for another 30 min. The identification of ?-actin was performed by immunofluorescence staining. In the present of Fluo-3/AM (an indicator of calcium),intracellular calcium and the expression of CaMK[KG-*6]Ⅱ were detected under the intervention of KN93 with laser cofocal microscopy and Western blotting respectively. Results More than 90% of cultured cells were positive to ?-actin antibody. Compared with the control group,the fluorescence intensity of intracellular Ca2+ was increased significantly by ionomycin (660.16?108.47 vs 376.12?57.57,P

Objective To develop new methods to cultivate, retrieve and purify mouse mesenchymal stem cells (mMSCs). Methods Bone marrow was collected from 2-month-old Kunming mice by flushing femurs and tibias with complete medium of DMEM-LG. Cells were plated in a Petri dish. After 24 hours, non-adherent cells were removed by two to three washes with PBS, adherent cells were further cultured in complete medium and retrieved by trypsinisation with 0.25% trypsin for 5 min at 37 ℃. The treated adherent cells were cultivated with 3?dilution for further generations. CD11b-negative cells were retrieved from the collected adherent cells of 3rd generation by using immunomagnetic microbeads, and continued to be cultured in complete medium. After the cultured cells were retrieved, their morphology and their ability of osteoblastic differentiation and adipocytic differentiation were examined. Results Most of mMSCs from 1st generation were of shuttle shape, some of irregular shape. After treatment with magnetic microbeads and several generations, mMSCs were of spindle, star and irregular shape. These cells were of rich cytoplasma, clear nucleolus, and grew in parallel or vortex. The cultured adherent cells from the first and subsequent generations had plenty of CD11b-positive blooding-making cells. After 20-day osteoblastic induction, mMSCs differentiated into bone cells, which showed orange phosphate in extracellular matrix by Alizarin red S staining. mMSCs could differentiate into lipocytes. The size of cells increased along with fat-developing induction period. These cells showed many orange fatty follicles with O Red Oil dyeing. Conclusion Pure mMSCs can not be retrieved by either adhering method or generation cultivation method separately. The combined methods of adhering, immunomagnetic microbeads, and serial subcultivation is effective in vitro in retrieve mMSCs.

AIM: To study influence of ischemia-reperfusion(IR) on apoptosis and expression of apoptosis-related genes Fas-L, Bax and Bcl-2 of sinoatrial node(SAN) cells in rabbits in vivo. METHODS:Ninety healthy adult rabbits were divided randomly into control group, ischemia groups (I 10 min , I 30 min , I 60 min and I 120 min ) and IR groups (I 10 min R 4h , I 30 min R 4h , I 60 min R 4h and I 120 min R 4h ). IR injury model of SAN was established by occluding and loosening the start section of right coronary artery. The apoptosis of SAN cells was detected by TUNEL staining. The expression of Fas-L, Bax and Bcl-2 of SAN cells was detected by immunohistochemistry. RESULTS:①No obvious apoptosis of SAN cells was observed in control group, I 10 min and I 30 min groups. Apoptosis of different degrees in SAN cells were found in 68.3%(41/60) rabbits in I 60 min , I 120 min and 4 subgroups of IR. ②The highest expression of Fas-L and Bax was observed in I 120 min group and that of Bcl-2 was in I 60 min group. ③The highest expression of Fas-L and Bax was observed in I 60 min R 4h group. The peak level of Bcl-2 was observed in I 30 min R 4h group. ④The expression of Fas-L and Bax was significant higher in IR group than that in ischemic group at the same time point. CONCLUSION:Ischemia and IR induced apoptosis of SAN cells in rabbit in vivo . Fas-L、Bax、Bcl-2 may participate in the regulation of apoptosis and the injury during IR aggravates the apoptosis of SAN cells.

Objective To compare the effects of different ischemic preconditioning (IP) protocols on the viability of primary cultured sinoatrial node cells from neonatal rats for investigating the protective effect of IP. Methods The cells were randomized into eleven groups: control, ischemia/reperfusion (I/R), IP1 (preconditioned with ischemia/reperfusion for 5 min), IP2 (preconditioned with 2 cycles of ischemia/ reperfusion for 5 min), IP3 (preconditioned with 3 cycles of ischemia/reperfusion for 5 min), IP4 (preconditioned with ischemia/reperfusion for 10 min), IP5 (preconditioned with 2 cycles of ischemia/reperfusion for 10 min), IP6 (preconditioned with 3 cycles of ischemia/reperfusion for 10 min), IP7 (preconditioned with ischemia/reperfusion for 20 min), IP8 (preconditioned with 2 cycles of ischemia/reperfusion for 20 min), and IP9 (preconditioned with 3 cycles of ischemia/reperfusion for 20 min). PI positive staining rate and changes of D 490 after 3 h ischemia/4 h reperfusion were determined by PI staining and MTT chromatometry. Results ① D 490 value in sinoatrial node cells in each experimental group was significantly lower than that in the control group, but that in IP2, IP3, IP4, IP5, and IP7 groups was significantly higher than that in I/R group (P

Objective To study the effects of mimic ischemic preconditioning (IP) on i and I-LCa of sinoatrial node cells and explore the protective effects of IP. Methods Cells were randomized to three groups: control, ischemia/reperfusion (I/R), IP. After labeled with fura4, fluorescence intensity of i was studied with laser confocal microscopy and I-LCa was recorded by whole-cell patch clamp technology. Results IP significantly reduced the fluorescence intensity of i and enhanced the peak of I-LCa as compared with I/R, shifted the I-V curve to more negative value. Conclusion IP can reduce the overload of i caused by I/R and increase I-LCa weakened by I/R.

AIM: To study the effects of ischemia/reperfusion (I/R) on primary cultured sinoatrial node (SAN) cells and the influence of pinacidil (a K_(ATP) channel activator). METHODS: The SAN cells were isolated from newborn rats and purified. The 48 h cultured cells were cultivated in following mediums: simulated reperfusion solution as normal control, simulated ischemia/reperfusion solution (I/R), Pinacidil+I/R (P+I/R), 5-HD+P+I/R and 5-HD+I/R. Spontaneous action potentials were recorded by ruptured-patch whole-cell technique in current clamp ((I=0)) and the maximum diastolic potential (MDP), upstroke velocity (UV), action potential overshoot (APO), interbeat interval (IBI) and action potential durations at 50% repolarization (APD_(50)) were measured. RESULTS: Compared with control group, simulated ischemia/reperfusion shorten APD_(50), reduced UV, MDP and APO. Exposed to pinacidil, MDP of cells in I/R groups was hyperpolarized; IBI, UV and APO were increased; APD_(50) was shorten. 5-HD couldn't block the effects of pinacdil on APD_(50), IBI and MDP, but reversed its actions on increasing UV and APO. CONCLUSIONS: Pinacidil made changes of AP in I/R group by opening different K_(ATP) channels of SAN cells. The role of this changes on protection in SAN cells during ischemia/reperfusion requires further investigation. [

AIM: To evaluate the effect of hypoxic preconditioning (HP) on the cytoskeleton of sinoatrial node cells primarily cultured from neonatal rat. METHODS: Cells were randomized to three groups of control, hypoxia/reoxygenation ( H/R) and HP. F-actin, vinculin, ?-tubulin and desmin were studied with laser confocal microscopy after labled with different immune fluorescent agents. RESULTS: Compared with control, although both H/R and HP reduced the fluorescence intensity of these four cytoskeletal proteins remarkably (P

Objective To compare the effects of stimulated ischemic preconditioning (IP) and ischemia/reperfusion (I/R) on the hyperpolarization-activated current (I_ f ) of sinoatrial node cells and explore the mechanisms of electrophysiological protection of IP. Methods The sinoatrial node cells cultured for 2 d were randomized to three groups: ① Control in which the cells were cultured at 37 ℃ in the mimic reperfusion solution, meanwhile the mixture of 95% O_ 2 and 5% N_ 2 was ventilated; ② I/R in which the cells were cultured in the mimic ischemic solution and simultaneously the mixture of 95% O_ 2 and 5% N_ 2 was ventilated for 3 h, then cultured in the mimic reperfusion solution and simultaneously the mixture of 95% O_ 2 and 5% N_ 2 was ventilated for 4 h; ③ IP in which the cells underwent ischemia for 10 min and reperfusion for 10 min, repeated once. Then the cells were treated as the same as the I/R group. After 4-hour reperfusion, I_ f was recorded by whole-cell patch clamp technology. Results I/R significantly enhanced the current density of I_ f , shifted the current activation curve to more positive value by changing the half-activation voltage from (-98.9?2.4) mV to (-85.2?2.5) mV (P