缺氧／复氧诱导培养人肥大心肌细胞凋亡模型的建立

目的　建立缺氧 /复氧诱导体外培养的人肥大心肌细胞模型并观察凋亡情况。方法　用 0 .2 %胰蛋白酶 (trypsin)和0 .1 %胶原酶 (collagenase)进行消化 ,用差速粘附贴壁法、Brdu加入和人为破坏成纤维细胞法纯化心肌细胞 ,用IMDM (Iscove′s改良的Dulbecco′smedium )培养基培养 ,4～ 6d后将体外培养的人肥大心肌细胞置于 92 %氮气及 5 %二氧化碳孵箱中 ,6、1 2、2 4、4 8h缺氧后再恢复正常条件培养 4h ,造成缺氧 /复氧损伤所致细胞凋亡模型 ,分别用倒置显微镜、电镜、TUNEL法技术检测凋亡发生的情况。结果　心肌细胞经历缺氧 /复氧损伤后 ,倒置显微镜、电镜、TUNEL染色均观察到凋亡阳性细胞。TUNEL法定量检测 ,心肌细胞缺氧培养 6、1 2、2 4、4 8h后复氧 4h ,其凋亡率分别为 :(4 .5± 1 .5 ) %、(1 0 .1± 2 .0 ) %、(1 7.3± 2 .3) %和 (1 8.4± 1 .9) %。结论　缺氧 /复氧一定时间可以诱导体外培养的人肥大心肌细胞凋亡 ,心肌细胞凋亡率随着缺氧时间的延长而增高     

Src family tyrosine kinases inhibit single L-type: Ca2+ channel activity in human atrial myocytes

OBJECTIVE: Tyrosine kinases (TKs) are important regulators of the L-type Ca(2+) channel (LTCC) current in various cell types. However, there are no data addressing the role of TKs in the control of single LTCC activity in human atrial cardiac myocytes, where changes in LTCC gating properties have been described in a number of disease states. METHODS AND RESULTS: Single LTCC activity was recorded in isolated human atrial myocytes. The broad-spectrum TK inhibitor genistein and the Src family-selective TK inhibitor PP1 significantly enhanced single LTCC ensemble average current, availability, and open probability; the latter was due to significant increases of mean open time and mode 2 gating. Conversely, the tyrosine phosphatase inhibitor bisperoxo-phenanthroline-vanadate inhibited single LTCC activity, indicating that LTCC gating properties in human atrial myocytes are controlled by TKs and tyrosine phosphatases in a reciprocal fashion. The effects of genistein on single LTCC activity were not affected by stimulation (8Br-cAMP) or inhibition (Rp-8-CPT-cAMPS) of protein kinase A (PKA) or by inhibition of serine/threonine phosphatases types I and IIa (okadaic acid), indicating that TKs inhibit LTCC gating in human atrial myocytes independent of PKA and phosphatases types I and IIa. However, inhibition of protein kinase C (PKC) by staurosporine or bisindolylmaleimide reversed the stimulatory effects of genistein on single LTCC gating properties, indicating that PKC is required for the inhibitory effect of TKs on single LTCC activity. CONCLUSION: Src family TKs inhibit single LTCC activity in human atrial myocytes via PKC-dependent, but PKA and phosphatase types I and IIa-independent, molecular pathways.     

诱导多能性干细胞与心肌细胞的融合细胞遗传表型特征研究

目的体外构建诱导多能性干细胞(induced pluripotent stem cells,i PSCs)与心肌细胞(cardiomyocytes,CMs)的融合细胞,探讨其细胞遗传学以及定向分化等生物学特点。方法 i PSCs与原代心肌细胞通过聚乙二醇(PEG-4000)诱导融合,检测融合细胞染色体数分布情况,融合细胞碱性磷酸酶染色,鉴定亲本细胞特异性基因和蛋白在融合细胞中的表达情况,以及当融合细胞失去亲本心肌细胞表型时,其向心肌细胞分化的能力。结果融合细胞表现为i PSCs样的特点;融合细胞主要表现为四倍体或者近似四倍体的核型(超过75%的融合细胞染色体数目在76~80条之间);碱性磷酸酶的阳性率低于同时间点的i PSCs;干细胞特异性基因Oct-4和Nanog的表达量在融合细胞与i PSCs中没有差异,而心肌细胞特异性基因α-MHC和β-MHC在融合细胞中的表达量明显低于心肌细胞;Oct-4蛋白在不同代数的融合细胞中均有表达,而随着时间的延长,c Tn T蛋白逐渐呈阴性表达;失去亲本心肌细胞表型的融合细胞能够向心肌细胞分化,且分化率高于同时间点的i PSCs。结论 i PSCs与原代心肌细胞的融合细胞,初期表现出双向重建,P5代后完全表现出干细胞显型,并且能向心肌细胞分化。     

体外模拟心肌微环境下骨髓间充质干细胞向心肌样细胞分化的实验研究

目的 :探讨粒细胞集落刺激因子 （G CSF）和干细胞因子 （SCF）预处理对骨髓间充质干细胞 （MSCs）向心肌细胞分化的影响。方法 :MSCs均采用全骨髓法提取 ,通过换液传代逐渐纯化细胞 ,取传 4代的MSCs,在共培养前用DAPI标记 ,心肌细胞在培养的第 3d与MSCs共培养。对照组 :未施加任何干预的MSCs与心肌细胞共培养 ;实验组 :联合使用G CSF和SCF对大鼠在体动员 ,提取其MSCs与心肌细胞共培养。共培养前测定心肌细胞的活力 ,在共培养第 3、4、5d用免疫荧光标记肌钙蛋白T ,分析DAPI MSCs向心肌细胞分化的百分率。结果 :在共培养的第3、4、5d ,G CSF和SCF处理的DAPI MSCs的分化率分别为 （1.2 4± 0 .16 ） %、（3.4 6± 0 .2 1） %、（7.2 1± 0 .11） % ,均显著高于对照组的 0、（1.2 7± 0 .13） %、（1.32± 0 .2 5 ） %。结论 ::G CSF和SCF对MSCs具有促分化作用。     

Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium

Transplantation of neonatal cardiomyocytes is a novel approach for the treatment of heart failure and myocardial infarction, but quantitative information on long-term cell survival and development is limited. Male donor cardiomyocytes were isolated from neonatal Fischer 344 rats (1-2 days), purified, and injected into the left ventricular wall of female syngeneic adult rats. One hour to 12 weeks later, genomic DNA was isolated from recipient hearts. The amount of male DNA per sample was determined by quantitative real-time TaqMan PCR of the male-specific Sry gene. Transplanted cell survival was 57 +/- 9% at 0-1 h, 24 +/- 6% at 24 h, 28 +/- 11% at 7 days, 27 +/- 3% at 14 days, 23 +/- 8% at 4 weeks and 15 +/- 3% at 12 weeks. The caspase inhibitor AcYVADcmk failed to improve transplanted cell survival at 24 h, suggesting that apoptosis did not play a major role in cell loss. Histology revealed that transplanted cells became more elongated over time, developed cross-striations, and that their nuclei increased in size. However, at 12 weeks, transplanted cells and their nuclei were still smaller than those of host myocardium. We established a quantitative survival profile for neonatal cardiomyocytes transplanted into normal adult myocardium. There was significant loss of cells within 24 h, but 15% of transplanted cells survived 12 weeks. Those cells that did survive underwent differentiation and developed visible sarcomeres, suggesting a potential contribution toward ventricular function.     

Inhibition of K+ currents by homocysteine in rat ventricular myocytes

INTRODUCTION: Clinical evidence suggests that increased blood levels of homocysteine may be an independent risk factor for the development of cardiovascular disease, but the functional effects of this sulfhydryl amino acid on the myocardium are poorly understood. The present study was conducted to determine the direct effects of homocysteine on the electrophysiologic properties of the heart. METHODS AND RESULTS: Whole-cell voltage-clamp recordings were made in ventricular myocytes isolated from normal rat hearts to analyze the Ca2+-independent, transient outward K+ current (I(to)), a major repolarizing current in these cells. Maximum I(to) density (measured at +60 mV) was decreased approximately 47% from baseline in the presence of 500 microM homocysteine (P < 0.05), but the amount of block varied in a frequency- and voltage-dependent manner. Decreased I(to) density was not accompanied by significant changes in voltage- or time-dependent properties of the current, nor was it affected by pretreating myocytes with the protein kinase inhibitor staurosporine. Because a portion of total extracellular homocysteine is oxidized, we examined the response to homocystine, the oxidized form of homocysteine. In myocytes superfused with 500 microM homocystine, maximum I(to) density was decreased by approximately 40% from baseline (P < 0.05). In contrast, the thiolactone form of homocysteine did not alter I(to) amplitude. CONCLUSION: These data suggest that homocysteine and its oxidized form homocystine acutely inhibit I(to) channels in ventricular myocytes by mechanisms involving the free thiol or disulfide moieties of these compounds. High homocysteine or homocystine levels may contribute to abnormal repolarization and arrhythmogenic conditions in the intact heart.     

Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2⁺/CD13⁺/KDR⁺/PDGFRα⁺ cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2⁺ cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2⁺/CD13⁺/KDR⁺/PDGFRα⁺ cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.     

Differential effects ofd, l-Sotalol andd-Sotalol on isoproterenol-increased delayed rectifier outward potassium current in guinea pig single ventricular myocytes

Summary The aim of this study was to compare the effects ofd, l-Sotalol andd-Sotalol on the delayed rectifier K⁺ outward current in the presence of isoproterenol at different concentrations. Time-dependent delayed rectifier K⁺ outward currents were measured in isolated guinea pig single myocytes using the whole-cell configuration of the patch-clamp technique. Currents were measured in response to 300 ms depolarizing pulses from a holding potential of −40 mV in three experimental protocols [control, isoproterenol (10^-9mollL-10^-6 mollL), and isoproterenol (10^-9mollL-10^-6mollL) plus eitherd, l-Sotalol (10^-4 mollL) ord-Sotalol (10^-4 mollL)]. I_k tail currents were measured upon repolarization to −40 mV. It was found that Ik was significantly amplified in the presence of isoproterenol (10^-9 mollL-10^-6 mollL) plusd-Sotalol. At 10^-8 mollL isoproterenol, I_k was increased by 92. 7%± 17. 1% (P<0. 05) and 54. 3 %±13. 4% afterd-Sotalol addition (P<0. 05). In contrast,d, l-Sotalol completely conteracted the increase of I_k by isoproterenol (<10^-8 mollL), and compared to control, Ik was decreased by 35. 6%± 8. 1 % at 10^-8 mollL isoproterenol plusd, l-Sotalol (P<0. 05). It is concluded that the β-adrenergic blocking property ofd, l-Sotalol but not that ofd-Sotalol maintains the delayed rectifier K⁺ outward current blockade in the presence of isoproterenol in guinea pig myocytes. This might contribute to a superior antiarrhythmic efficacy as compared tod-Sotalol.     

Myocardial ultrastructure of rats differing in resistance to hypoxia,after acute and periodic exposure to it

Research Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 8, pp. 148–151, August, 1991.