基于三维图像处理的心肌细胞中Ca2+浓度研究

心肌细胞中Ca2+浓度的空间和时间分布,对于建立心肌细胞节律运动电路模型提供离子特性参数有很大帮助。提出了一种幼鼠心肌细胞中Ca2+浓度分布分析的新方法。对激光共聚焦显微镜（LSCM）采集的幼鼠心肌细胞序列图像进行图像处理、三维重建、细胞分割、距离变换,从细胞膜向内进行分层剥离,得到心肌细胞中Ca2+浓度的空间和时间分布。该方法极大地方便生物研究和医学诊断,基于三维距离变换研究Ca2+浓度时间空间分布的方法也可运用于类似的生物医学研究。     

巨噬细胞移动抑制因子通过调控自噬抗H9c2心肌细胞缺氧/复氧损伤

目的:研究巨噬细胞移动抑制因子（macrophage migration inhibitory factor,MIF）对H9c2心肌细胞缺氧/复氧（hypoxia/reoxygenation,H/R）过程中自噬的影响，并探讨其分子作用机制。方法:利用MIF特异性siRNA瞬时转染H9c2心肌细胞，建立H9c2心肌细胞H/R损伤模型，给予自噬经典抑制剂3-甲基嘌呤（3-MA），蛋白免疫印迹（Western blot）检测MIF、LC3、Cleaved caspase-3以及mTOR蛋白的表达。 结果:干扰MIF后可抑制H/R诱导的心肌细胞自噬；自噬抑制剂3-MA抑制H/R诱导的心肌细胞自噬，减少细胞凋亡的发生；沉默MIF后可增加H/R过程中p-mTOR的表达。 结论:MIF可通过抑制自噬减少H9c2心肌细胞H/R损伤，其作用机制可能与激活mTOR有关。     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

刺五加叶皂苷对心肌ATP 敏感性钾通道的作用

目的: 研究刺五加叶皂苷(ASS) 对心肌线粒体ATP 敏感性钾通道(mito K_ATP) 和细胞膜ATP 敏感性钾通道(sarcol K_ATP) 的作用, 探讨ASS 对缺血心肌保护作用的机制。方法: 用酶解法获取兔心室肌细胞, 激光扫描共聚焦显微镜观察ASS 对mito K_ATP 的作用, 全细胞膜片钳技术观察ASS 对sarcol K_ATP的作用。结果: 对照组观察10 min 线粒体荧光强度无明显变化。ASS 30、100 和300 mg·L^-1组均可见用药后线粒体荧光强度明显增加, 分别增加(14.8±3.6) %、(30.4±4.3) % 和(38.4±5.7) %。3 μmol·L^-1格列本脲不影响线粒体荧光强度, 但可以阻断ASS 对线粒体荧光强度的作用。而对照组、ASS 10、100 和300 μmol·L^-1组的I_K-ATP 峰值无明显差异。结论: ASS 对mito K_ATP 有开放作用, 而对sarcolK_ATP没有作用。ASS 通过开放mito K_ATP 产生心肌保护作用。     

非酶糖化抑制剂对糖尿病大鼠心肌细胞凋亡作用的研究

目的: 观察非酶糖化抑制剂-氨基胍(AG) 对糖尿病大鼠心肌细胞凋亡的影响。方法: 54 只SD大鼠分为12 周对照组(8 只), 24 周对照组(10 只);糖尿病12 周组(8 只), 糖尿病24 周组(10 只);AG治疗12 周组(8 只), AG 治疗24 周组(10 只), 观察糖尿病大鼠心肌病变过程中(12 周及24 周) 心脏肥厚、心功能指标及心肌细胞凋亡的改变。结果: 链尿佐菌素(STZ) 诱导的糖尿病大鼠12 周时出现心功能异常并可见凋亡的心肌细胞, 随心衰的加重, 24 周时凋亡细胞数目明显增多。透射电镜检查, 发现糖尿病大鼠左室心肌组织中可见凋亡的心肌细胞(具有凋亡形态学特征) 。AG 治疗组大鼠心功能和心肌超微结构明显改善, 心肌细胞凋亡数目减少。结论: 心肌细胞凋亡在糖尿病心肌病发生发展过程中起着重要作用, AG 可有效减少心肌细胞凋亡, 改善心肌病理形态学异常。     

Taxol对缺氧培养乳鼠心肌细胞Cx43蛋白表达及分布的影响

目的: 观察微管稳定剂Taxol对缺氧导致的培养乳鼠心肌细胞Cx43表达和分布的影响。方法: 差速贴壁梯度离心法分离培养乳大鼠心肌细胞,取培养 4 d 细胞缺氧 120 min,分别以不同浓度的Taxol干预;免疫印迹法检测聚合态微管蛋白含量、心肌细胞Cx43的蛋白表达,免疫荧光染色后激光共聚焦显微镜观察Cx43的分布。结果: 正常培养心肌细胞Cx43分布在核膜和细胞的闰盘处,缺氧 120 min 可导致心肌细胞微管解聚,Cx43蛋白表达降低,在心肌细胞间连接处分布规律散失,核膜Cx43分布减弱或消失,而均匀分布在细胞膜上;在低剂量的Taxol作用下,心肌细胞微管解聚状态缓解,Cx43蛋白表达和分布异常明显改善;随着Taxol剂量增加,这种改善作用更明显,呈现剂量依赖性。结论: 缺氧引起乳鼠心肌细胞微管解聚,Cx43蛋白表达降低分布紊乱,微管稳定剂Taxol可以显著地保护缺氧导致的心肌Cx43异常,具有潜在的抗缺血性心律失常价值。     

Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

The cardiac Mg²⁺-sensitive, TRPM6, and TRPM7-like channels remain undefined, especially with the uncertainty regarding TRPM6 expression in cardiomyocytes. Additionally, their contribution to the cardiac action potential (AP) profile is unclear. Immunofluorescence assays showed the expression of the TRPM6 and TRPM7 proteins in isolated pig atrial and ventricular cardiomyocytes, of which the expression was modulated by incubation in extracellular divalent cation-free conditions. In patch clamp studies of cells dialyzed with solutions containing zero intracellular Mg²⁺ concentration ([Mg²⁺]_i) to activate the Mg²⁺-sensitive channels, raising extracellular [Mg²⁺] ([Mg²⁺]_o) from the 0.9-mM baseline to 7.2 mM prolonged the AP duration (APD). In contrast, no such effect was observed in cells dialyzed with physiological [Mg²⁺]_i. Under voltage clamp, in cells dialyzed with zero [Mg²⁺]_i, depolarizing ramps induced an outward-rectifying current, which was suppressed by raising [Mg²⁺]_o and was absent in cells dialyzed with physiological [Mg²⁺]_i. In cells dialyzed with physiological [Mg²⁺]_i, raising [Mg²⁺]_o decreased the L-type Ca²⁺ current and the total delayed-rectifier current but had no effect on the APD. These results suggest a co-expression of the TRPM6 and TRPM7 proteins in cardiomyocytes, which are therefore the molecular candidates for the native cardiac Mg²⁺-sensitive channels, and also suggest that the cardiac Mg²⁺-sensitive current shortens the APD, with potential implications in arrhythmogenesis.     

Pathogenic Mechanisms of Hypertrophic Cardiomyopathy beyond Sarcomere Dysfunction

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, affecting 1 in 500 people in the general population. Although characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, and cardiac fibrosis, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset, and complications. While HCM is generally accepted as a disease of the sarcomere, variable penetrance in families with identical genetic mutations challenges the monogenic origin of HCM and instead implies a multifactorial cause. Furthermore, large-scale genome sequencing studies revealed that many genes previously reported as causative of HCM in fact have little or no evidence of disease association. These findings thus call for a re-evaluation of the sarcomere-centered view of HCM pathogenesis. Here, we summarize our current understanding of sarcomere-independent mechanisms of cardiomyocyte hypertrophy, highlight the role of extracellular signals in cardiac fibrosis, and propose an alternative but integrated model of HCM pathogenesis.     

培养乳鼠心肌细胞时钟基因RT-PCR 检测方法的建立

目的 建立检测培养乳鼠心肌细胞中时钟基因的RT-PCR 方法。 方法 根据GeneBank 基因库大鼠时钟基因bmal1、per2 和dbp 全序列设计合成了3 对寡聚核苷酸引物。以离体培养的乳鼠心肌细胞中提取RNA 为模板, 筛选了(PCR) 最佳反应条件, 并进行了特异性检验。 结果 在20 μl 体积中, PCR 最佳系统组成分别为:dbp:Taq 酶、dNTP 和Mg²⁺的用量分别为0.5 U、0.006 μmol 和0.03 μmol;最佳退火温度为58 ℃, 循环30 次;bmal1:Taq 酶、dNTP 和Mg²⁺ 的用量分别为0.5 U、0.006 μmol 和0.035 μmol;最佳退火温度为57 ℃, 循环30 次;per2:Taq 酶、dNTP 和Mg²⁺的用量分别为0.5 U、0.006 μmol 和0.05 μmol;最佳退火温度为58 ℃, 循环32 次。 结论 成功建立RT-PCR 检测大鼠离体培养心肌细胞时钟基因的方法。     

Calcium waves initiating from the anomalous subdiffusive calcium sparks

The objective of the study is to investigate the propagation of Ca²⁺ waves in full-width cardiac myocytes and carry out sensitivity analysis to study the effects of various physiological parameters on global Ca²⁺ waves. Based on the anomalous subdiffusion of Ca²⁺ sparks, a mathematical model was proposed to characterize the Ca²⁺ waves. The computed results were in agreement with the experimental measurements using confocal microscopy. This model includes variables of current through the Ca²⁺ release unit (CRU; I_CRU), duration of current flow through CRU (T_open), Ca²⁺ sensitivity parameter (K), the longitudinal and transverse spatial separation of CRUs (l_x and l_y, where x denotes longitudinal direction (x-axis) and y denotes transverse direction (y-axis)) and Ca²⁺ diffusion coefficients (D_x, D_y). The spatio-temporal mechanism of the anomalous Ca²⁺ sparks led to results that were different from those based on Fick''s law. The major findings were reported as: I_CRU affected the dynamic properties of Ca²⁺ waves more significantly than T_open; the effect of K on the properties of Ca²⁺ waves was negligible; l_y affected the amplitude significantly, but l_x affected the longitudinal velocity significantly; in turn, the limitation and significance of the study are discussed.