Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.
Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.
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DOI：
10.1161/circresaha.116.310283
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发表时间：
2017-06-09
影响因子：
20.1
通讯作者：
Pu WT
中科院分区：
文献类型：
--
作者：
Guo Y;VanDusen NJ;Zhang L;Gu W;Sethi I;Guatimosim S;Ma Q;Jardin BD;Ai Y;Zhang D;Chen B;Guo A;Yuan GC;Song LS;Pu WT
心肌细胞（CM）的功能丧失研究目前受到限制，需要适当的条件敲除等位基因。调节CM成熟的因素知之甚少。先前关于CM成熟的研究被整个器官基因失活引起的心功能障碍所混淆。开发一种新的技术平台，以快速表征出生后小鼠CM中的细胞自主基因功能，并将其应用于鉴定调节T-小管（成熟心肌细胞的标志）的基因。我们开发了CASAAV（基于CRISPR/Cas9-AAV 9的体细胞诱变），这是一种平台，其中AAV 9将靶向感兴趣基因的串联向导RNA和心肌肌钙蛋白T启动子（cTNT）驱动的Cre递送至RosaCas 9 GFP/Cas9 GFP新生小鼠。当针对嗜连接蛋白-2（Jph 2），一个先前参与T-小管成熟的基因时，我们实现了有效，快速和CM特异性的JPH 2消耗。高剂量AAV 9在64%的CM中消融JPH 2并引起致死性心力衰竭，而低剂量AAV 9在22%的CM中消融JPH 2并保留正常的心脏功能。在保存心脏功能的背景下，缺乏JPH 2的CM发育出几乎形态正常的T-小管，表明JPH 2在T-小管成熟中不具有主要的细胞自主作用。然而，在具有严重功能障碍的心脏中，AAV转导的和未转导的CM都表现出T-小管破坏，这在转导的亚组中更严重。这些数据表明，心脏功能障碍破坏T-小管结构，JPH 2在这种情况下保护T-小管。然后，我们使用CASAAV筛选8个额外的基因，用于T小管形成中所需的细胞自主作用。我们确定兰尼碱受体2（RYR 2）作为一种新的，细胞自主需要的T-小管成熟因子。CASAAV是研究细胞自主基因功能的有力工具。遗传镶嵌对于精确定义细胞自主基因功能是非常宝贵的。JPH 2在正常的T-小管成熟中具有次要作用，但需要稳定衰竭心脏中的T-小管。RYR 2是一种新的T-小管成熟因子。
Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Prior studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate T-tubules, a hallmark of mature cardiac myocytes. We developed CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis), a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin T promoter (cTNT)-driven Cre to RosaCas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both AAV-transduced and non-transduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure, and that JPH2 protects T-tubules in this context. We then used CASAAV to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified ryanodine receptor 2 (RYR2) as a novel, cell-autonomously required T-tubule maturation factor. CASAAV is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.