Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, RC Leiden, The Netherlands.
Department of Cell Biology and Genetics, Center for Anti-ageing and Regenerative Medicine, Shenzhen Key Laboratory for Anti-ageing and Regenerative Medicine, Shenzhen University Medical School, Shenzhen University, Nanhai Ave 3688, Shenzhen, China.
Cardiovasc Res. 2018 Dec 1;114(14):1848-1859. doi: 10.1093/cvr/cvy134.
The generation of homogeneous cardiomyocyte populations from fresh tissue or stem cells is laborious and costly. A potential solution to this problem would be to establish lines of immortalized cardiomyocytes. However, as proliferation and (terminal) differentiation of cardiomyocytes are mutually exclusive processes, their permanent immortalization causes loss of electrical and mechanical functions. We therefore aimed at developing conditionally immortalized atrial myocyte (iAM) lines allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition.
Freshly isolated neonatal rat atrial cardiomyocytes (AMs) were transduced with a lentiviral vector conferring doxycycline (dox)-controlled expression of simian virus 40 large T antigen. Under proliferative conditions (i.e. in the presence of dox), the resulting cells lost most cardiomyocyte traits and doubled every 38 h. Under differentiation conditions (i.e. in the absence of dox), the cells stopped dividing and spontaneously reacquired a phenotype very similar to that of primary AMs (pAMs) in gene expression profile, sarcomeric organization, contractile behaviour, electrical properties, and response to ion channel-modulating compounds (as assessed by patch-clamp and optical voltage mapping). Moreover, differentiated iAMs had much narrower action potentials and propagated them at >10-fold higher speeds than the widely used murine atrial HL-1 cells. High-frequency electrical stimulation of confluent monolayers of differentiated iAMs resulted in re-entrant conduction resembling atrial fibrillation, which could be prevented by tertiapin treatment, just like in monolayers of pAMs.
Through controlled expansion and differentiation of AMs, large numbers of functional cardiomyocytes were generated with properties superior to the differentiated progeny of existing cardiomyocyte lines. iAMs provide an attractive new model system for studying cardiomyocyte proliferation, differentiation, metabolism, and (electro)physiology as well as to investigate cardiac diseases and drug responses, without using animals.
从新鲜组织或干细胞中生成均质的心肌细胞群体既费力又昂贵。解决此问题的一种潜在方法是建立永生化的心肌细胞系。然而，由于心肌细胞的增殖和（终末）分化是相互排斥的过程，因此它们的永久永生化会导致丧失电和机械功能。因此，我们的目标是开发条件性永生化的心房肌细胞（iAM）系，通过培养基成分的单一成分变化在增殖和收缩表型之间进行切换。
用赋予四环素（dox）控制的猴病毒 40 大 T 抗原表达的慢病毒载体转导新鲜分离的新生大鼠心房心肌细胞（AMs）。在增殖条件下（即在存在 dox 的情况下），所得细胞失去了大多数心肌细胞特征，每隔 38 小时倍增一次。在分化条件下（即在不存在 dox 的情况下），细胞停止分裂并自发重新获得与原代 AMs（pAMs）在基因表达谱、肌节组织、收缩行为、电特性和对离子通道调节化合物的反应（通过膜片钳和光学电压映射评估）非常相似的表型。此外，分化的 iAMs 的动作电位更窄，传播速度比广泛使用的鼠心房 HL-1 细胞快 10 倍以上。对分化的 iAMs 的单层培养物进行高频电刺激会导致类似于心房颤动的折返传导，该传导可以通过 tertiapin 治疗来预防，就像 pAMs 的单层一样。
通过 AMs 的受控扩增和分化，生成了具有优于现有心肌细胞系分化后代的性能的大量功能性心肌细胞。iAMs 为研究心肌细胞增殖、分化、代谢和（电）生理学以及研究心脏疾病和药物反应提供了一个有吸引力的新模型系统，而无需使用动物。
