Data_Sheet_1_Long-Term Cultivation of Human Atrial Myocardium.PDF

Organotypic culture of human ventricular myocardium is emerging in basic and translational cardiac research. However, few institutions have access to human ventricular tissue, whereas atrial tissue is more commonly available and important for studying atrial physiology. This study presents a method for long-term cultivation of beating human atrial myocardium. After written informed consent, tissues from the right-atrial appendage were obtained from patients with sinus rhythm undergoing open heart surgery with cardiopulmonary bypass. Trabeculae (pectinate muscles) prepared from the samples were installed into cultivation chambers at 37°C with a diastolic preload of 500 μN. After 2 days with 0.5 Hz pacing, stimulation frequency was set to 1 Hz. Contractile force was monitored continuously. Beta-adrenergic response, refractory period (RP) and maximum captured frequency (fmax) were assessed periodically. After cultivation, viability and electromechanical function were investigated, as well as the expression of several genes important for intracellular Ca2+ cycling and electrophysiology. Tissue microstructure was analyzed by confocal microscopy. We cultivated 19 constantly beating trabeculae from 8 patient samples for 12 days and 4 trabeculae from 3 specimen for 21 days. Functional parameters were compared directly after installation (0 d) with those after 12 d in culture. Contraction force was 384 ± 69 μN at 0 d and 255 ± 90 μN at 12 d (p = 0.8, n = 22), RP 480 ± 97 ms and 408 ± 78 ms (p = 0.3, n = 9), fmax 3.0 ± 0.5 Hz and 3.8 ± 0.5 Hz (p = 0.18, n = 9), respectively. Application of 100 nM isoprenaline to 11 trabeculae at 7 d increased contraction force from 168 ± 35 μN to 361 ± 60 μN (p < 0.01), fmax from 6.4 ± 0.6 Hz to 8.5 ± 0.4 Hz (p < 0.01) and lowered RP from 319 ± 22 ms to 223 ± 15 ms. CACNA1c (L-type Ca2+ channel subunit) and GJA1 (connexin-43) mRNA expressions were not significantly altered at 12 d vs 0 d, while ATP2A (SERCA) and KCNJ4 (Kir2.3) were downregulated, and KCNJ2 (Kir2.1) was upregulated. Simultaneous Ca2+ imaging and force recording showed preserved excitation-contraction coupling in cultivated trabeculae. Confocal microscopy indicated preserved cardiomyocyte structure, unaltered amounts of extracellular matrix and gap junctions. MTT assays confirmed viability at 12 d. We established a workflow that allows for stable cultivation and functional analysis of beating human atrial myocardium for up to 3 weeks. This method may lead to novel insights into the physiology and pathophysiology of human atrial myocardium.

人类心室心肌细胞的原位培养技术在基础和转化型心脏研究中逐渐崭露头角。然而，由于人类心室组织获取难度较大，仅有少数研究机构能够获得，相比之下，心房组织更为常见，且对于研究心房生理学至关重要。本研究提出了一种长期培养跳动的人类心房心肌细胞的方法。在获得书面知情同意后，从接受心脏直视手术并采用心肺转流的患者右侧心耳瓣中获取组织。从样本中制备的乳头肌（梳状肌）被置于37°C的培养室中，静息前负荷为500 μN。经过2天的0.5 Hz节律刺激后，将刺激频率调整为1 Hz。持续监测收缩力。定期评估β-肾上腺素能反应、不应期（RP）和最大捕获频率（fmax）。培养后，研究了生存能力和电机械功能，以及与细胞内Ca2+循环和电生理学相关的多种基因的表达。通过共聚焦显微镜对组织微结构进行了分析。我们从8个患者样本中培养了19个持续跳动的乳头肌，持续培养12天，以及从3个标本中培养了4个乳头肌，持续培养21天。直接在安装后（0天）与12天培养后的功能参数进行了比较。在0天时，收缩力为384 ± 69 μN，而在12天时为255 ± 90 μN（p = 0.8，n = 22），RP分别为480 ± 97 ms和408 ± 78 ms（p = 0.3，n = 9），fmax分别为3.0 ± 0.5 Hz和3.8 ± 0.5 Hz（p = 0.18，n = 9）。在第7天对11个乳头肌应用100 nM异丙肾上腺素后，收缩力从168 ± 35 μN增加到361 ± 60 μN（p < 0.01），fmax从6.4 ± 0.6 Hz增加到8.5 ± 0.4 Hz（p < 0.01），而RP从319 ± 22 ms降低到223 ± 15 ms。与0天相比，在第12天CACNA1c（L型Ca2+通道亚单位）和GJA1（连接蛋白-43）mRNA表达没有显著改变，而ATP2A（SERCA）和KCNJ4（Kir2.3）下调，KCNJ2（Kir2.1）上调。同时进行的Ca2+成像和力量记录显示，培养的乳头肌中兴奋-收缩偶联得到保留。共聚焦显微镜表明，心肌细胞的结构得到保留，细胞外基质和缝隙连接的数量未发生变化。MTT试验在12天时证实了生存能力。我们建立了一套工作流程，允许对跳动的人类心房心肌细胞进行长达3周的培养和功能分析。该方法可能为揭示人类心房心肌细胞的生理学和病理生理学提供新的见解。