Keeping the beat. Focus on "enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro".
Keeping the beat. Focus on "enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro".
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DOI：
10.1152/ajpcell.00310.2012
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Lynch,RonaldM
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作者：
Konhilas,JohnP;Behunin,SamanthaM;Lynch,RonaldM
尽管对心血管疾病（CVD）发展的细胞和分子机制进行了20-30年的广泛研究，CVD仍然是男性和女性死亡的主要原因（25%的全因死亡率）。然而，CVD治疗的进步，加上改善的专业CVD护理，导致更多的CVD患者进展为充血性心力衰竭（CHF）。事实上，CHF的发病率范围为0.1%至0.5%（每年400，000例新发CHF患者），发达国家80岁或以上患者的发病率高达10%（5）。这后一点清楚地表明，CVD是一种渐进的，不断演变的条件，其病理轨迹在很大程度上取决于疾病的病因，治疗策略，以及环境和遗传因素的相互作用。因此，阐明潜在的细胞和分子机制如何与临床后遗症同时发生变化，就等同于确定和开发适当的治疗方案。这种类型的CVD研究的成功在很大程度上取决于建立用于研究心血管组织和细胞类型（特别是心脏细胞）的纵向体内和体外范例的能力。可以说，到目前为止，只有动物模型（大型和小型;手术和转基因）是CVD研究所需的纵向和转化研究类型的最合适模型。尽管如此，许多研究人员已经成功地利用从整个心脏中分离的心肌细胞群来研究与心脏生物学的分子，细胞和生理机制相关的特定问题，这些问题无法使用动物模型实现（9）。目前，新生大鼠心肌细胞（NRCM）的原代细胞培养物是体外模型的选择。然而，使用NRCM的主要限制是在NRCM由于去分化和细胞骨架重塑而失去其收缩和心肌细胞表型之前的有限时间范围（通常为2-3周）。与之前的研究一致，最近的一项分析发现，成年小鼠和大鼠心肌由约56%的收缩性肌细胞、27%的成纤维细胞、7%的内皮细胞和10%的血管平滑肌细胞组成（2）。增殖的非心肌细胞是群体去分化的主要原因（1，8）。因此，研究人员采用了几种技术来最大限度地减少酶消化完整心脏后的细胞异质性，例如预铺板以去除粘附的非心肌细胞和/或Percoll梯度分离出低密度的非心肌细胞。然而，这些富集策略仍然不足以维持具有收缩表型的NRCM的长期培养。(4)他们能够利用功能性的多巴胺特异性荧光染料、四甲基罗丹明甲酯高氯酸盐（TMRM）和荧光激活细胞分选（FACS）的效用，分离富含多巴胺的胚胎干细胞衍生的心肌细胞的高纯度培养物（4）。这种方法的优点是无需遗传操作即可实现分离，因此有可能应用于包括大鼠和人类在内的多种物种。
DESPITE 20–30 YEARS of extensive research into the cellular and molecular mechanisms underlying the development of cardiovascular disease (CVD), CVD remains the leading cause of death (25% all-cause mortality) in men and women. Nevertheless, advances in CVD therapeutics, coupled with improved specialty CVD care, have resulted in more CVD patients progressing to congestive heart failure (CHF). In fact, CHF has reached an incidence that ranges from 0.1% to 0.5%(400,000 new CHF patients per year) and up to 10% of patients 80 years of age or older in developed countries (5). This latter point clearly illustrates that CVD is a progressive, continuously evolving condition whose pathological trajectory depends largely on the interaction of disease etiology, treatment strategies, and both environmental and genetic factors. Consequently, elucidation of how the underlying cellular and molecular mechanisms are changing concurrent with the clinical sequelae becomes tantamount to the identification and development of appropriate treatment regimens. Success for this type of CVD research largely depends on the ability to establish longitudinal in vivo and in vitro paradigms for studying the cardiovascular tissues and cell types, in particular the cardiac cells. It can be argued that, up to this point, only animal models (large and small; surgical and transgenic) are the most suitable models for the type of longitudinal and translational research required for the study of CVD. Still, many investigators have successfully utilized myocardial cell populations isolated from whole hearts to study specific issues related to the molecular, cellular, and physiological mechanisms of cardiac biology that cannot be achieved using an animal model (9). Currently, primary cell cultures of neonatal rat cardiomyocytes (NRCMs) are the in vitro model of choice. However, the major limitation to the use of NRCMs is the restricted time frame (usually 2–3 wk) before NRCMs lose their contractile and myocardial cell phenotype due to dedifferentiation and cytoskeletal remodeling. Consistent with previous studies, a recent analysis identified that the adult murine and rat myocardium is composed of approximately 56% contractile myocytes, 27% fibroblasts, 7% endothelial cells, and 10% vascular smooth muscle cells (2). The proliferating non-myocardial cells are the primary cause of dedifferentiation of the population (1, 8). Therefore, investigators have employed several techniques to minimize the cellular heterogeneity following enzymatic digestion of the intact heart, such as preplating to remove adherent non-myocardial cells and/or Percoll gradients to separate out lower-density, nonmyocardial cells. Still, these enrichment strategies remain insufficient for maintaining long-term culture of NRCMs with a contractile phenotype.Applying an alternative approach, Hattori et al.(4) were able to exploit the utility of a functional mitochondria-specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM) and fluorescence-activated cell sorting (FACS), to isolate high-purity cultures of mitochondria-rich embryonic stem cell-derived cardiomyocytes (4). The advantage of this approach is that the isolation is achieved without genetic manipulation and therefore has the potential to be applied to several species including rat and human.