二氮嗪预处理对成年大鼠心肌异丙肾上腺素损伤的保护作用及其与剂量的关系

目的 探讨二氮嗪(DZ)预处理对大鼠心肌异丙肾上腺素(ISO)损伤的保护作用及其与剂量的关系.方法 采用成年大鼠心肌ISO损伤模型,观察不同浓度的DZ预处理对心肌细胞ISO损伤后的肌酸激酶(CK)和肌钙蛋白Ⅰ(cardiac troponin Ⅰ,cTnⅠ)释放的影响.结果 DZ预处理培养大鼠心肌细胞后,对ISO损伤有保护作用,能减少心肌细胞的损伤.在5~20mg/kg浓度内,随着DZ剂量的增加,其预处理心肌细胞的保护效应也相应增加.结论 在5~20mg/kg浓度范围内,DZ能剂量依赖地产生预处理心肌保护效应.     

Sphingosine modulation of cAMP levels and beating rate in rat heart

Sphingolipids, especially as elements of the sphingomyelin signal transduction cycle, are thought to play a significant role as second messengers and modulators of events in heart muscle cells. A possible modulatory role of sphingosine in signal transduction in the beta-adrenergic pathway in the heart was examined. Neonatal rat cardiomyocytes were incubated with sphingosine and/or other agents after which cAMP levels and contraction rates were measured. Heart rate in anaesthetized rats was also measured before and after sphingosine injection in the jugular vein. Sphingosine caused a decrease in basal cAMP levels and diminished isoproterenol-induced increase in cAMP levels. These changes were dose- and time-dependent and showed a significant negative effect on signal transmission in the beta-adrenergic pathway in cardiomyocytes. Increase in cAMP intracellular levels by forskolin, which activates adenylcyclase, was not inhibited by sphingosine. A phosphodiesterase inhibitor was used in all experiments in which cAMP was measured excluding effects on cAMP breakdown. It was also demonstrated that sphingosine caused reduction in the beating rate of cultured cardiomyocytes and a dose-dependent reduction in heart rate of anaesthetized rats. The sphingosine-induced inhibition of bradycardic response of anaesthetized rats reached a maximum about 5-10 min after the onset of sphingosine administration and returned to normal within 60 min. Sphingosine may modulate the signal transmission of the beta-adrenoceptor pathway upstream of adenylcyclase in rat cardiomyocytes. This may contribute to the sphingosine-induced decrease in heart rate of rats in vivo.     

低氧对小鼠胚胎干细胞诱导分化成心肌细胞的影响

目的探讨在胚胎干细胞诱导分化过程的不同阶段进行低氧处理对分化心肌细胞的影响。方法建立胚胎干细胞向心肌细胞诱导分化的实验方法,在分化过程的不同阶段采取低氧（氧浓度为4%）处理,并设立常氧组（约为20%）作为对照,用免疫荧光鉴定分化后的心肌细胞,以流式细胞术检测低氧对分化心肌细胞的增殖、分化、凋亡的影响。结果分化细胞经免疫荧光检测显示,部分细胞呈α辅肌动蛋白阳性（红色）、心肌肌钙蛋白I阳性（绿色）;分化细胞经流式检测显示,与常氧组相比,悬滴低氧组α-actinin阳性细胞和cTnI阳性细胞的比率分别提高了12.55%（P〈0.01）和6.11%（P〈0.05）,悬浮低氧组凋亡比率与常氧组相比明显提高（P〈0.01）,悬滴低氧组处于S期的细胞比率与常氧组相比明显降低（P〈0.01）。结论在胚胎干细胞向心肌细胞的定向诱导分化过程中,采用悬滴阶段低氧处理,使心肌特异性蛋白阳性细胞比率提高,细胞凋亡率稳定,细胞增殖能力因细胞分化成熟而有所降低,为低氧处理的最佳方案。     

黄芪注射液与5-氮杂胞苷体外诱导大鼠骨髓间充质干细胞分化为心肌细胞的研究

目的研究黄芪注射液与5-氮杂胞苷（5-Aza）对大鼠骨髓间充质干细胞（mesenchymal stem cells,MSCs）分化为心肌细胞的影响。方法在无菌条件下从成年大鼠胫骨骨髓分离出MSCs。以1：3的比例传代,取第3代的细胞．接种后随机将MSCs分为黄芪注射液诱导分化组、5-Aza诱导分化组、黄芪注射液预处理后5-Aza诱导分化组。分别以黄芪注射液、5-Aza及黄芪注射液＋5-Aza进行诱导,用相差显微镜观察各纽细胞形态变化;免疫细胞化学鉴定MSCs表面标志物及心肌特异性肌钙蛋白I。结果各组诱导后细胞形态发生改变,细胞之问形成连接,排列方向趋于一致,免疫组化检测结果显示肌钙蛋白I（eTnI）表达均阳性。黄芪注射液组与5-Aza组相比其诱导率无明显区别．但黄芪注射液＋5一Aza组阳性细胞比率均高于5-Aza组及黄芪注射液组．差异具有统计学意义（P〈0．05）。结论黄芪注射液可体外诱导MSCs分化为心肌样细胞,采用黄芪注射液预处理后5-Aza诱导分化的方法优于单纯采用黄芪注射液或5-Aza谤导法。     

吡格列酮对过氧化氢诱导的乳鼠心肌细胞凋亡的抑制作用

目的观察吡格列酮对乳鼠心肌细胞凋亡的抑制作用及其可能作用机制。方法采用培养的新生大鼠乳鼠心肌细胞制备H2O2200μmol·L-1氧化损伤模型。实验分为正常对照组、模型组、吡格列酮组(0.1,1及10μmol·L-1)、吡格列酮(10μmol.L-1)+GW9662(10μmol·L-1)组、维拉帕米1μmol.L-1组。MTT法测心肌细胞的活力;采用硫代巴比妥酸比色法、黄嘌呤氧化酶法分别测定心肌细胞中脂质过氧化产物丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;流式细胞仪检测心肌细胞的凋亡率;采用Till阳离子测定系统,以Fura-2/AM为荧光探针,观察心肌细胞[Ca2+]i瞬间变化。结果H2O2200μmo.lL-1作用于心肌细胞12h能引起心肌细胞凋亡。吡格列酮浓度依赖性地增加受损心肌细胞的活力及SOD活性,降低MDA含量。吡格列酮10μmol·L-1抑制作用最明显,其与维拉帕米1μmol·L-1有相似的抑制作用,二者都可以减少心肌细胞的凋亡率,降低由H2O2诱导的升高的[Ca2+]i静息水平及频率。过氧化物酶体增殖物激活受体PPARγ受体特异性拮抗剂GW966210μmol.L-1能拮抗吡格列酮的部分抑制作用,但不影响吡格列酮对[Ca2+]i的瞬变作用。结论吡格列酮可以抑制H2O2诱导的乳鼠心肌细胞凋亡,其作用机制可能与其抗脂质氧化和减少细胞内钙超载有关,其抑制作用部分是通过PPARγ受体介导的。     

Adenovirus-mediated FKBP12.6 overexpression induces hypertrophy and apoptosis in cultured neonatal cardiomyocytes

1. Cardiac ryanodine RyR2 receptors regulate Ca(2+) release from the sarcoplasmic reticulum (SR). FK506 binding protein (FKBP) 12.6 prevents aberrant SR Ca(2+) leakage during diastole, thereby maintaining the integrity of RyR2 function. Previous studies have focused mainly on FKBP12.6 deficiency and so the pathophysiological consequences of FKBP12.6 overexpression remain unclear. Herein, we investigate the effect of FKBP12.6 overexpression on cardiac hypertrophic and apoptotic signalling. 2. Human FKBP12.6 cDNA was cloned into pAdTrack-CMV and the resulting plasmid, along with a control empty plasmid, were transfected into bacteria. The resulting virus, namely Ad-FKBP12.6 containing green fluorescent protein, was propagated and purified. Neonatal rat cardiomyocytes were infected with this virus. Protein and DNA synthesis were measured by [(3)H]-leucine and [(3)H]-thymidine incorporation, respectively. Expression of p38 mitogen-activated protein kinase (MAPK), phosphorylated extracellular signal-regulated kinase 1 or 2 (p-ERK1/2) and Bax were examined by western blotting. 3. Compared with control cells, cardiomyocytes that overexpressed FKBP12.6 became hypertrophic and hyperplastic, with increased levels of both p38 MAPK and p-ERK1/2. At the same time, overexpression of FKBP12.6 induced apoptosis of cardiomyocytes, as determined by both Bax protein expression and DNA fragmentation. Rapamycin treatment downregulated the expression of p-ERK1/2, p38 MAPK and Bax in stimulated cardiomyocytes, with or without FKBP12.6 overexpression, and enhanced protein synthesis, but had no effect on DNA synthesis in cardiomyocytes. 4. In conclusion, FKBP12.6 overexpression may participate in pathophysiological processes through both hypertrophic and apoptotic signalling pathways, leading to cardiomyocyte damage and death.     

Creatine kinase inhibitor iodoacetamide antagonizes calcium-stimulated inotropy in cardiomyocytes

1. Inhibition of creatine kinase is known to suppress cardiac contractile reserve in intact hearts, although the underlying mechanism has not been elucidated. 2. The present study was designed to examine whether cardiac depression induced by creatine kinase inhibition was due to action at the level of the essential contractile element, namely cardiomyocytes. Adult rat cardiomyocytes were perfused with the creatine kinase inhibitor iodoacetamide (90 micromol/L) for 90 min. Mechanical and intracellular Ca(2+) properties were evaluated using edge-detection and fluorescence microscopy, respectively. Myocytes were superfused with normal (1.3 mmol/L) or high (3.3 mmol/L) extracellular Ca(2+) contractile buffer. Mechanical function was examined, including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time to 90% PS (TPS(90)), time to 90% relengthening (TR(90)) and integration of shortening/relengthening (normalized to PS). Intracellular Ca(2+) transients were evaluated using the following indices: resting and rise of fura-2 fluorescence intensity (Delta FFI) and intracellular Ca(2+) decay time constant. 3. The results indicate that elevated extracellular Ca(2+) stimulated cardiomyocyte positive inotrope, manifested as increased PS, +/-dL/dt, area of shortening, resting FFI and Delta FFI associated with a shortened TR(90) and intracellular Ca(2+) decay time constant. High extracellular Ca(2+) did not affect TPS(90) and area of relengthening. Iodoacetamide ablated high Ca(2+)-induced increases in PS, +/-dL/dt, area of shortening, resting FFI, Delta FFI and shortened TR(90) and intracellular Ca(2+) decay time constant. Iodoacetamide itself significantly enhanced the area of relengthening and TR(90) without affecting other indices. 4. Collectively, these data demonstrate that inhibition of creatine kinase blunts high extracellular Ca(2+)-induced increases in cardiomyocyte contractile response (i.e. cardiac contractile reserve).     

Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation

Mesenchymal stem cells (MSCs) are considered to be a source of stem cells in tissue regeneration and therapeutics, due to their ability to undergo proliferation and differentiation. Complications associated with bone marrow-derived MSCs has prompted researchers to explore alternative sources of MSCs. The human umbilical cord is one such source; it is easily available and its collection is non-invasive. The sources of MSCs are non-controversial and thus they are not subjected to ethical constraints, as in the case of embryonic stem cells. MSCs are multipotent stem cells and has the ability to differentiate into various cell types of the mesodermal lineage. The aim of this study was to establish a reproducible method for the isolation of MSCs from human umbilical cord, as the few methods published till date gave inconsistent results and had a mixed population of contaminating endothelial cells. In our isolation strategy, we isolated a pure population of MSCs from Wharton's jelly of the human umbilical cord, which is very rich in collagen, and we used a high concentration of collagenase enzyme in the isolation of MSCs. Extensive phenotypic characterization analysis of these cells, using flow cytometry and antibody staining methods, have shown that we were able to isolate a pure population of the mesenchymal lineage cells that is devoid of haematopoietic and endothelial cell contaminants. When these MSCs were subjected to cardiomyocyte differentiation, we observed a change in the morphological characteristics, which was accompanied by the formation of myotube structures and spontaneous beating after 21 days.     

Anticancer prodrugs of butyric acid and formaldehyde protect against doxorubicin-induced cardiotoxicity

Formaldehyde has been previously shown to play a dominant role in promoting synergy between doxorubicin (Dox) and formaldehyde-releasing butyric acid (BA) prodrugs in killing cancer cells. In this work, we report that these prodrugs also protect neonatal rat cardiomyocytes and adult mice against toxicity elicited by Dox. In cardiomyocytes treated with Dox, the formaldehyde releasing prodrugs butyroyloxymethyl diethylphosphate (AN-7) and butyroyloxymethyl butyrate (AN-1), but not the corresponding acetaldehyde-releasing butyroyloxydiethyl phosphate (AN-88) or butyroyloxyethyl butyrate (AN-11), reduced lactate dehydrogenase leakage, prevented loss of mitochondrial membrane potential (DeltaPsim) and attenuated upregulation of the proapoptotic gene Bax. In Dox-treated mice, AN-7 but not AN-88 attenuated weight-loss and mortality, and increase in serum lactate dehydrogenase. These findings show that BA prodrugs that release formaldehyde and augment Dox anticancer activity also protect against Dox cardiotoxicity. Based on these observations, clinical applications of these prodrugs for patients treated with Dox warrant further investigation.