不同运动负荷小鼠心肌活细胞游离钙动态变化的LSCM研究

目的 :探讨运动与心脏重塑的生物学机制中心肌细胞内游离钙的动态变化。方法 :采用激光扫描共聚焦显微技术 ,对不同运动负荷条件下小鼠心肌活细胞内游离钙浓度的变化进行观察、记录和分析 ,采用多道生理记录仪经颈动脉插管 ,对不同运动负荷条件下小鼠心肌收缩功能进行记录与分析。结果 :与对照组比较 ,有氧运动组心脏发生肥大 ,左室内压力二阶导数增加了 47 1 7%(P <0 0 0 1 ) ,表明心肌收缩功能增强 ,静息期心肌细胞内游离 [Ca2 + ]i 基值无显著性变化 ,峰值显著性增加了 1 5 7 2 % (P <0 0 0 1 ) ,达峰时间延长了 3 8 5 % (P <0 0 0 1 ) ;大强度疲劳组心肌肥大 ,但心肌收缩功能降低 (-3 5 2 4% ,P <0 0 0 1 ) ,静息期心肌细胞内游离 [Ca2 + ]i 基值和峰值分别显著增加了 41 9% (P <0 0 0 1 )和 2 0 6 3 % (P <0 0 0 1 ) ,达峰时间延长了 74 8% (P <0 0 0 1 )。有氧运动组与大强度疲劳组比较 ,心肌细胞内游离 [Ca2 + ]i 峰值和达峰时间均有显著性差异 (P <0 0 0 1 )。结论 :不同运动负荷影响心肌细胞内游离 [Ca2 + ]i 的变化 ,大强度疲劳训练可导致心肌细胞内游离[Ca2 + ]i 和静息态基值大幅度升高 。

运动性肥大心肌活细胞胞浆游离Ca^(2+)动态变化的激光共聚焦研究以及局部IGF-Ⅰ和AngⅡ的变化

目的 :研究运动与心脏重塑过程中心肌胞浆游离Ca2 +([Ca2 +]c)动态变化的生物学机制。方法 :采用激光扫描共聚焦显微技术 (LSCM)对STDInCa2 +荧光试剂负载的运动肥大心肌活细胞 [Ca2 +]c 动态变化进行研究 ,采用放射免疫测定运动小鼠心肌局部IGF -Ⅰ和AngⅡ含量。结果 :运动肥大心肌 [Ca2 +]c 变化表现为基值稳态和峰值显著升高 ,达峰时间延长 (P <0 0 0 1)。心肌局部IGF -Ⅰ和AngⅡ显著升高 (P <0 0 0 1)。结论 :运动性心肌肥大与 [Ca2 +]c 变化、机械信号、IGF -Ⅰ和AngⅡ关系密切 ,机械信号、IGF -Ⅰ和AngⅡ可能是引起 [Ca2 +]c 显著升高 ,增强心肌收缩能力的胞外刺激因素。

运动心脏重塑过程中细胞凋亡现象的活细胞观察

为了探讨运动心脏重塑过程中细胞凋亡现象，采用流式细胞技术对不同强度耐力训练及力竭运动后大鼠心肌细胞凋亡情况进行了活细胞观察。结果显示：１运动训练可使大鼠心肌细胞凋亡增加，且与运动强度有关，提示运动性心肌肥大的重塑发生过程有心肌细胞凋亡参与；２反复大强度训练和力竭运动后大鼠心肌细胞凋亡现象的显著增加，可能是运动性心肌微损伤的病理机制之一；３系统训练对心肌细胞凋亡的发生未见明显的保护作用。

The role and modulation of autophagy in experimental models of myocardial ischemia-reperfusion injury

A physiological sequence called autophagy qualitatively determines cellular viability by removing protein aggregates and damaged cyto-plasmic constituents, and contributes significantly to the degree of myocardial ischemia-reperfusion （I/R） injury. This tightly orchestrated cata-bolic cellular‘housekeeping’ process provides cells with a new source of energy to adapt to stressful conditions. This process was first described as a pro-survival mechanism, but increasing evidence suggests that it can also lead to the demise of the cell. Autophagy has been implicated in the pathogenesis of multiple cardiac conditions including myocardial I/R injury. However, a debate persists as to whether autophagy acts as a protec-tive mechanism or contributes to the injurious effects of I/R injury in the heart. This controversy may stem from several factors including the va-riability in the experimental models and species, and the methodology used to assess autophagy. This review provides updated knowledge on the modulation and role of autophagy in isolated cardiac cells subjected to I/R, and the growing interest towards manipulating autophagy to increase the survival of cardiac myocytes under conditions of stress-most notably being I/R injury. Perturbation of this evolutionarily conserved intracellular cleansing autophagy mechanism, by targeted modulation through, among others, mammalian target of rapamycin （mTOR） inhibitors, adenosine monophosphate-activated protein kinase （AMPK） modulators, calcium lowering agents, resveratrol, longevinex, sirtuin activators, the proapoptotic gene Bnip3, IP3 and lysosome inhibitors, may confer resistance to heart cells against I/R induced cell death. Thus, therapeutic ma-nipulation of autophagy in the challenged myocardium may benefit post-infarction cardiac healing and remodeling.

Regulation of swelling-activated chloride channels in embryonic chick heart cells

Swelling-activated Cl- currents, I(ci,swell), were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitor of protein tyrosine kinase (PTK), suppressed I(ci,swell), Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl~ current similar to that in hyposmotic solution, whereas hyposmotic shock did not elicit I(ci,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Con-focal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasin D (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(ci,Sweii) was not elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Ci,swell), The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(ci,sweii) appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl- channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for regulation of I(Ci,swell) in embryonic chick heart cells as compared with often controversial results reported in different cell types.

Cardioprotective effects of Chinese herbal medicine Braintone on myocardial infarction in rats

Recently, we reported the unique effects of cerebral protection of the Chinese herbal medicine-Braintone （a formulation containing Radix Rhodiola, Folium Ginkgo, Radix Notoginseng and Rhizoma Ligustici Chuanxiong）, also known as Remembrance. In the present study, we tested the hypothesis that Braintone may extend cardioprotective effects on ischemic myocardium in Wistar rats. Animal model was created by ligating of left descending coronary artery. Mortality rate and infarct volume were assessed. In addition, capillary density, antioxidant enzymes, apoptosis modulators and VEGF, eNOS mRNA expression level were investigated to reveal the underlying mechanisms. Treatment with Braintone reduced mortality rate from 41.7% to 22.2% associate with notable diminished infarct volume （30.4%±9.0% vs 18.0%±3.0%）. Braintone also acted as antioxidant agent for preserving the activities of catalase （13.07±0.48 U vs 9.71±0.44 U in vehicle, P〈0.01）. Furthermore, Braintone dramatically boosted the expression levels of anti-apoptotic genes Bcl-2 and Bcl-xl （1.43-, 2.30-fold, P〈0.01） as compared to vehicle group and significantly down-regulated the expression level ofpro-apoptotic gene Bax （0.84-fold, P〈0.01） while slightly inhibited Caspase-9 and Caspase-3 signals. Moreover, higher mRNA expression levels of VEGF and eNOS were observed in Braintone group consisting with a remarkable raise of capillary density （46.0±13.3 vs 27.4±12.6, P〈0.01） in myocardium. The findings indicated that Braintone markedly attenuate myocardial damage induced by ischemic insults in vivo. Braintone may confer cardioprotection via scavenging free radicals, inhibiting cardiomyocytes apoptosis and promoting angiogenesis in ischemic region.

Isoflurane induces expression of vascular endothelial growth factor through activating protein kinase C in myocardial cells

Objective： Vascular endothelial growth factor （VEGF） plays important roles in establishing collateral circulation of ischemic myocardium. This study aimed to investigate the effect of isoflurane on VEGF expression and the potential intracellular signal transduction pathway in cultured rat myocardial cells in order to further reveal the molecular mechanism of myocardial preservation of isoflurane. Methods： Primary myocardial cells of Sprague-Dawley rats were isolated and cultured. They were divided randomly into control group, isoflurane group, protein kinase C （PKC） inhibitor group and PKC inhibitor＋isoflurane group where cells were respectively incubated without any treatment, treated by 0.5, 1.0 and 1.5 minimum alveolar concentration （MAC） of isoflurane for 6 hours, by PKC inhibitor calphostin C at a final concentration of 50 nmol/L and by 50 nmol/L calphosfin C＋ 1.0 MAC isoflurane for 6 hours. VEGF expression was detected by enzyme-linked immunosorbent assay （ELISA） and the expression levels of PKC isoforms were determined by Western immunoblotting method. Results： Isoflurane increased the VEGF expression in myocardial cells in a dose-dependent way. VEGF levels were significantly higher in 1.0 and 1.5 MAC isoflurane groups than in the control group （both P〈0.01）. The effect of isoflurane on upregulating VEGF expression was blocked by PKC inhibitor calphostin C （P〈0.01）, but calphostin C did not alter VEGF expression （P〉0.05）. Isoflurane induced the activation and translocation of PKC Immunoblotting analysis revealed that the immunoreactivity of PKC ε increased significantly in the membrane fractions and deceased significantly in the kytoplasm fractions for cells treated with 1.0 MAC isoflurane as compared with the untreated cells, but not of PKC a, PKCα and PKCζ （P〈0.01）. Conclusion： Isoflurane induces myocardial cells to release VEGF through activating PKCε from the endochylema to the cytomembrane, suggesting a possible novel mechanism of isoflurane protecting myocardial cells.