Decreased intercellular coupling improves the function of cardiac pacemakers derived from mouse embryonic stem cells

The aim of this study was to determine if embryonic stem cell derived cardiomyocyte aggregates (ESdCs) can act as pacemakers in spontaneously active cardiomyocyte preparations when their connexin isoform expression is tuned toward a more sinus nodal phenotype. Using microelectrode array recordings (MEAs), we demonstrate that mouse ESdCs establish electrical coupling with spontaneously active cardiomyocyte preparations (HL-1 monolayer) and obtain pacemaker dominance. WT- and Cx43(−/−)-ESdCs comparably established intercellular coupling with cardiac host tissue (Cx43(−/−): 86% vs. WT: 91%). Although both aggregates had a 100% success rate in pacing quiescent cardiac preparations, Cx43(−/−)-ESdCs had an increased likelihood of gaining pacemaker dominance (Cx43(−/−): 40% vs. WT: 13%) in spontaneously active preparations. No differences in size, beating frequency, V_m, or differentiation were detected between WT- and Cx43(−/−)-ESdCs but the intercellular coupling resistance in Cx43(−/−)-ESdCs was significantly increased (Cx43(−/−): 1.2nS vs. WT: 14.8nS). Lack of Cx43 prolonged the time until Cx43(−/−)-ESdCs established frequency synchronization with the host tissue. It further hampered the excitation spread from the cardiomyocyte preparation into the ESdC. However rectifying excitation spread in these co-cultures could not be unequivocally identified. In summary, ESdCs can function as dominant biological pacemakers and Cx43 expression is not a prerequisite for their electrical integration. Maintenance of pacemaker dominance depends critically on the pacemaker's gap junction expression benefiting those with increased intercellular coupling resistances. Our results provide important insight into the design of biological pacemakers that will benefit the use of cardiomyocytes for cell replacement therapy.     

Flexible electrical recording from cells using nanowire transistor arrays

Semiconductor nanowires (NWs) have unique electronic properties and sizes comparable with biological structures involved in cellular communication, thus making them promising nanostructures for establishing active interfaces with biological systems. We report a flexible approach to interface NW field-effect transistors (NWFETs) with cells and demonstrate this for silicon NWFET arrays coupled to embryonic chicken cardiomyocytes. Cardiomyocyte cells were cultured on thin, optically transparent polydimethylsiloxane (PDMS) sheets and then brought into contact with Si-NWFET arrays fabricated on standard substrates. NWFET conductance signals recorded from cardiomyocytes exhibited excellent signal-to-noise ratios with values routinely >5 and signal amplitudes that were tuned by varying device sensitivity through changes in water gate–voltage potential, V_g. Signals recorded from cardiomyocytes for V_g from −0.5 to +0.1 V exhibited amplitude variations from 31 to 7 nS whereas the calibrated voltage remained constant, indicating a robust NWFET/cell interface. In addition, signals recorded as a function of increasing/decreasing displacement of the PDMS/cell support to the device chip showed a reversible >2× increase in signal amplitude (calibrated voltage) from 31 nS (1.0 mV) to 72 nS (2.3 mV). Studies with the displacement close to but below the point of cell disruption yielded calibrated signal amplitudes as large as 10.5 ± 0.2 mV. Last, multiplexed recording of signals from NWFET arrays interfaced to cardiomyocyte monolayers enabled temporal shifts and signal propagation to be determined with good spatial and temporal resolution. Our modular approach simplifies the process of interfacing cardiomyocytes and other cells to high-performance Si-NWFETs, thus increasing the experimental versatility of NWFET arrays and enabling device registration at the subcellular level.     

缺氧对H9C2心肌细胞膜葡萄糖转运蛋白表达量的影响

目的观察缺氧对H9C2心肌细胞膜葡萄糖转运蛋白（GLUT1和GLUT4）表达量的影响。方法将H9C2心肌细胞随机分为对照组、胰岛素组、叠氮钠模拟的缺氧组，提取各实验组细胞膜后，用蛋白质印记杂交法测定膜组份中GLUTs的表达量。结果同对照组相比，胰岛素组和缺氧组细胞膜GLUTs表达量均明显增加，且胰岛素组对细胞膜GLUTs表达量的影响高于缺氧组（P〈0．05）。结论缺氧诱导H9C2心肌细胞膜GLUTs表达量增加，此种增加至少部分程度上介导了缺氧状态下H9C2心肌葡萄糖转运活动。     

同种异体心肌细胞移植对大鼠梗死心肌修复作用的研究

目的观察同种异体心肌细胞移植对心肌梗死大鼠心肌的修复作用和心功能的影响。方法选取110只雄性SD大鼠用于制作心肌梗死模型,术后2周共存活53只,死亡57只。从53只建模成功的SD大鼠中随机选取30只分为3组（每组10只）：心肌细胞移植组（A组）、培养液基质注射组（B组）和单纯心肌梗死组（C组）。A组将原代培养的乳鼠心肌细胞移植到心肌梗死区和梗死边缘区;B组采用与A组同样方法在心肌梗死区和梗死边缘区注射细胞培养液基质;C组不做任何处理。2周后观察A组移植细胞存活情况。采用Langendorff装置检测各组左心室收缩压（LVSP）、左心室舒张末压（LVEDP）、左心室内压最大上升速率（＋dp/dtmax）、左心室内压最大下降速率（-dp/dtmax）和左心室做功（LVW）。结果A组与B组及C组相比,LVSP分别从（47.9±4.6）mmHg和（47.6±3.9）mmHg升高到（52.0±3.0）mmHg（P〈0.05或P〈0.01）;LVEDP分别从（18.8±2.7）mmHg和（24.7±5.8）mmHg降低到（15.8±1.5）mmHg（P〈0.05或P〈0.01）;＋dp/dtmax分别从（1 900±198）mmHg/s和（1 737±347）mmHg/s升高到（2 260±337）mmHg/s（P〈0.05）。3组间-dp/dtmax和LVW比较差异无显著性（P〉0.05）。A组大鼠的心肌中均发现移入的乳鼠心肌细胞,经5-溴脱氧尿核苷（BrdU）标记的免疫组化染色和抗心肌肌钙蛋白I抗体（anti-cTn I）标记的免疫组化染色证明移入的细胞是心肌细胞。结论异体移植的乳鼠心肌细胞可在心肌梗死区存活,并能改善心功能,尤其是改善左心室收缩功能。     

尼可地尔对培养乳鼠心肌细胞的保护实验

目的 :通过建立心肌细胞缺氧再复氧模型 ,观察尼可地尔对其保护作用 ,揭示保护机制 .方法 :培养心肌细胞分4组 ,Ⅰ :对照K H液 ;Ⅱ :St.Thomas Ⅱ液 ;Ⅲ :1.6mmol·L-1尼可地尔液 ;Ⅳ :1.6mmol·L-1尼可地尔 +2 0mmol·L-1牛磺酸 .分别于处理前后台盼蓝染色测定心肌细胞存活率、原子光谱法测定钙含量、自动生化仪测定乳酸脱氢酶 (LDH)释放量、比色法测定超氧化物歧化酶 (SOD)活性、丙二醛(MDA)含量 .结果 :处理前各组心肌细胞指标无差异 ,Ⅰ组细胞存活率缺氧后明显下降 ,复氧后进一步下降 ,钙含量增加 ,LDH释放量增加 ,SOD活性下降 ,MDA含量升高 .Ⅱ和Ⅲ组各指标在复氧后较Ⅰ组有明显改善 ,Ⅲ组保护作用更明显 ,与Ⅱ组比较具有统计学差异 .Ⅳ组可进一步增加尼可地尔的保护效果 .结论 :超极化停跳液心肌保护效果明显 ,临床应用前景好 .牛磺酸可进一步增加超极化停搏的保护作用     

Na/Ca交换在心肌细胞Ca~(2+)平衡和CICR过程中的作用

Ca~（2+）平衡是维持心肌细胞正常电生理活动的重要前提。在各种机制中,肌质网Ca-ATPase与肌膜Na/Ca交换蛋白对于维持胞内Ca~（2+）的平衡发挥主要作用。其中肌膜Na/Ca交换蛋白是Ca~（2+）排出胞外的主要途径,并通过调节细胞内静息状态下[Ca~（2+）]调节肌质网的[Ca（2+）]含量,从而调节心肌细胞的收缩力。少量Ca（2+）内流入胞后可触发肌质网释放大量Ca（2+）（CICR）。已证实动作电位峰电位及平台期有Ca（2+）通过Na/Ca内流,这种除极化诱导的Ca（2+）经Na/Ca内流可能是触发CICR的主要因素。总之Na/Ca交换蛋白在兴奋-收缩耦联中的作用需要重新加以评价。     

Morphological manifestations of heart remodeling in anthracycline-induced dilated cardiomyopathy

The morphogenesis of anthracycline-induced dilated cardiomyopathy was studied after single sublethal dose of doxorubicin. Cardiomyocyte depopulation (up to 27%) and decrease in their regeneratory plastic reactions were the main mechanisms of cardiac failure development after anthracycline (doxorubicin) treatment, determining the type of heart remodeling by the dilatation variant. Cardiomyocyte elimination and atrophy during the development of anthracycline-induced regeneratory plastic cardiac insufficiency were paralleled by hypertrophy of remaining cardiomyocytes and diffuse and small focal sclerosis of the myocardium, which could be regarded as a correlated compensatory reaction of the connective tissue to the decrease in the number of muscle fibers.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 12, pp. 684–689, December, 2004This revised version was published online in April 2005 with a corrected cover date.     

Cardiac hypertrophy: a matter of translation

1. Left ventricular hypertrophy (LVH) of the heart is an adaptive response to sustained increases in blood pressure and hormone imbalances. Left ventricular hypertrophy is associated with programmed responses at the molecular and biochemical level in different subsets of cardiac cells, including the cardiac muscle cells (cardiomyocytes), fibroblasts, conductive tissue and coronary vasculature. 2. Regardless of the initiating cause, the actual increase in chamber enlargement is, in each case, due to an increase in size of a pre-existing cardiomyocyte population, with little or no change in their number; a process referred to as cellular hypertrophy. 3. An accelerated rate of global protein synthesis is the primary mechanism by which protein accumulation increases during cardiomyocyte hypertrophy. In turn, increased rates of synthesis are a result of increased translational rates of existing ribosomes (translational efficiency) and/or synthesis and recruitment of additional ribosomes (translational capacity). 4. The present review examines the relative importance of translational capacity and translational efficiency in the response of myocytes to acute and chronic demands for increased protein synthesis and the role of these mechanisms in the development of LVH.     

Dystrophin and the Cardiomyocyte Membrane Cytoskeleton in the Healthy and Failing Heart

The cardiomyocyte membrane cytoskeleton consists of the costameric proteins that mediate force transduction from the cell to the extracellular matrix, and a sub-membrane network composed of dystrophin and associated proteins. Studies of the precise cellular distribution of dystrophin and of the consequences of genetic mutations leading to abnormal expression of the dystrophin molecule, as occurs in Duchenne and Becker's muscular dystrophies, highlight potential functional roles of this sub-membrane protein complex in cardiomyocytes. Detailed investigation of dystrophin distribution using the complementary cell imaging techniques of immunoconfocal microscopy and freeze-fracture cytochemistry at the electron-microscopical level show that, in contrast to rat cardiomyocytes, the dystrophin network in human cardiomyocytes is locally enriched at costameres. Thus located, the dystrophin network appears to have a mechanical role, involving stabilization of the peripheral plasma membrane during the repetitive distortion associated with cardiac contraction and, in the human myocyte, contributing to lateral force-transduction. Evidence from animal models of muscular dystrophy and from investigation of the interactions of the sub-membrane cytoskeleton with other membrane-associated proteins including ion channels, receptors and enzymes, further suggests a role for dystrophin in organization and regulation of membrane domains. The relative preservation of the membrane cytoskeleton in non-dystrophic dilated cardiomyopathy and in ischemic cardiomyopathy, conditions in which the myocyte contractile apparatus and internal desmin-based cytoskeleton are commonly disrupted, emphasizes the vital role of the membrane cytoskeleton in cell survival. Continued cardiomyocyte survival despite loss of contractile protein organization has implications in the potential for reversibility of left ventricular remodeling that can be achieved in the clinical setting.     

心肌细胞培养环境对骨髓间充质干细胞定向分化成心肌样细胞有促进作用

目的:研究心肌细胞培养微环境对骨髓间充质干细胞(MSCs)定向分化成心肌样细胞的影响.方法:取SD大鼠骨髓,通过贴壁法进行骨髓间充质干细胞培养,经多次传代后获得较纯的MSCs.用5-氮胞苷(5-aza)进行诱导分化,实验组为在玻片上的5-aza干预的MSCs移入到心肌细胞培养环境中;对照组为5-aza干预的MSCs.显微镜下观察两组形态变化,并通过免疫组化鉴别心肌心肌特异蛋白Desmin和肌钙蛋白Ⅰ(cTnI)表达及其出现时间的早晚.结果:对照组MSCs诱导后10天可见细胞由原来扁平多角形变成长梭形,15天后部分细胞体明显增粗,可见到有些细胞间有融合样情况发生,20天左右类似肌管样细胞出现.而实验组在17天左右可以看到类似心肌管样细胞.免疫组化表明实验组的阳性结果出现的时间比对照组要早.结论:心肌细胞培养环境对骨髓间充质干细胞定向分化成心肌样细胞有促进作用.