PDF(Pubmed)
Abstract：
Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7 days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5 Hz, 1 Hz, and 2 Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24 h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7 d. Pacing rates above 1 Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7 d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1 d and 7 d, while Ca2+ transient duration was prolonged after 7 d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7 d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.
摘要：
简介：最近的进展使跳动的人心肌切片的器官型培养能够稳定数周。然而,人体心肌样本很少见,表现出高度的变异性，经常起源于患病的心脏。因此，有必要适应动物心肌的长期切片培养。当应用于动物心脏切片时，在健康或转基因心肌的研究将是可能的。我们介绍了兔子心脏切片的培养，在微观结构上类似于人类心脏，电生理学和兴奋-收缩耦合。方法：使用振动切片机切开新西兰白兔的左心室心肌，并在仿生室中培养长达7天（d）。在培养开始期间添加了机电解偶联剂2,3-丁二酮单肟（BDM）和细胞松弛素D（CytoD），并定量了对心肌细胞存活的影响。我们调查了起搏率(0.5Hz，1Hz,和2赫兹）和荷尔蒙补充剂（皮质醇，T3，儿茶酚胺）在生理血浆浓度下。使用BSA缓冲T3。连续记录收缩力。测量葡萄糖消耗和乳酸生产。记录全切片Ca2+瞬变和动作电位。通过共聚焦显微镜和图像分析研究了培养对微观结构的影响。结果：人心肌培养方案在24小时内导致兔切片持续挛缩和心肌细胞死亡，这可以通过瞬时应用BDM和CytoD的组合来预防。皮质醇稳定了培养物中的收缩幅度和动力学。T3和儿茶酚胺能刺激没有进一步改善稳定性。T3和更高的起搏速率增加了代谢率和乳酸产生。T3在7d内稳定了对β-肾上腺素能刺激的反应。超过1Hz的起搏速率导致收缩力明显下降。图像分析显示7d后心肌细胞体积分数或纤维化指标无变化。1d和7d后，Ca2瞬时振幅和对异丙肾上腺素的反应性相当，培养7d后Ca2+瞬时持续时间延长。结论:建立了兔心肌培养的工作流程,该研究强调了糖皮质激素信号在维持组织功能和延长培养时间方面的重要性。此外，BDM和CytoD似乎在组织培养的起始阶段保护免受组织损伤。
