In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter.
In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter.
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通过用GCaMP3报告物测量心脏比钙流量体外评估心脏重编程。
DOI：
10.3791/62643
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发表时间：
2022-02-22
期刊：
影响因子：
--
通讯作者：
Wang Z
中科院分区：
文献类型：
--
作者：
Li Z;Liu L;Wang Z
心脏重编程已成为修复受损心脏的潜在有前途的疗法。通过引入多种转录因子，包括Mef 2c、Gata 4、Tbx 5（MGT），成纤维细胞可以重编程为诱导的心肌细胞（iCM）。这些iCM在梗死心脏中原位生成时，与周围心肌进行电气和机械整合，导致疤痕尺寸减小和心脏功能改善。由于iCM的重编程效率、纯度和质量相对较低，iCM的表征仍然是一个挑战。目前在该领域使用的方法，包括流式细胞术，免疫细胞化学和qPCR，主要集中在心脏特异性基因和蛋白质的表达，而不是iCM的功能成熟。由动作电位触发，心肌细胞中电压门控钙通道的开放导致钙快速流入细胞。因此，定量钙内流率是评价心肌细胞功能的一种很有前途的方法。在此，该方案介绍了通过钙（Ca 2+）通量评价iCM功能的方法。通过将Tg（Myh 6-cre）1 Jcnt/J（以下称为Myh 6-Cre）与Gt（ROSA）26 Sortm 38（CAG-GCaMP 3）Hze/J（以下称为Rosa 26 A-Flox-Stop-Flox-GCaMP 3）小鼠杂交，建立αMHC-Cre/Rosa 26 A-Flox-Stop-Flox-GCaMP 3小鼠品系。从P0-P2新生小鼠分离并体外培养新生心脏成纤维细胞（NCF），并将MGT的多顺反子结构引入NCF，这导致其重编程为iCM。因为只有成功重编程的iCM才会表达GCaMP 3报告基因，所以iCM的功能成熟可以通过荧光显微镜下的Ca 2+通量来视觉评估。与未重编程的NCF相比，NCF-iCM显示出显著的钙瞬时流动和自发收缩，与CM相似。该方案详细描述了小鼠品系建立、新生小鼠心脏的分离和选择、NCF分离、用于心脏重编程的逆转录病毒的生产、iCM诱导、使用我们的报告细胞系评估iCM Ca 2+通量以及相关的统计分析和数据呈现。预期本文所述的方法将提供一个有价值的平台，以评估iCM的功能成熟，用于心脏重编程研究。
Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.
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影响因子：
64.8
作者：
Qian, Li;Huang, Yu;Srivastava, Deepak
通讯作者：
Srivastava, Deepak
影响因子：
11.1
作者：
Tian, Shuo;Lei, Ienglam;Wang, Zhong
通讯作者：
Wang, Zhong
通过对心脏转录因子的非肌细胞进行重编程通过重编程。
影响因子：
64.8
作者：
Song, Kunhua;Nam, Young-Jae;Luo, Xiang;Qi, Xiaoxia;Tan, Wei;Huang, Guo N.;Acharya, Asha;Smith, Christopher L.;Tallquist, Michelle D.;Neilson, Eric G.;Hill, Joseph A.;Bassel-Duby, Rhonda;Olson, Eric N.
通讯作者：
Olson, Eric N.
DOI：
10.3791/53426
发表时间：
2015-11-13
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Wang L;Liu Z;Yin C;Zhou Y;Liu J;Qian L
通讯作者：
Qian L
影响因子：
82.9
作者：
Ikeda K;Kang Q;Yoneshiro T;Camporez JP;Maki H;Homma M;Shinoda K;Chen Y;Lu X;Maretich P;Tajima K;Ajuwon KM;Soga T;Kajimura S
通讯作者：
Kajimura S