Journal article
Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo
Circulation research, Vol.120(12), pp.1874-1888
06/09/2017
DOI: 10.1161/CIRCRESAHA.116.310283
PMCID: PMC5466492
PMID: 28356340
Abstract
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. Previous 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 transverse tubules (T-tubules), a hallmark of mature CMs.
We developed 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-driven Cre to
neonatal mice. When directed against junctophilin-2 (
), 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 adeno-associated virus-transduced and nontransduced 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 CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor.
CRISPR/Cas9/AAV9-based somatic mutagenesis 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.
Details
- Title: Subtitle
- Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo
- Creators
- Yuxuan Guo - Boston Children's HospitalNathan J VanDusen - Boston Children's HospitalLina Zhang - Shanghai University of Traditional Chinese MedicineWeiliang Gu - Shanghai University of Traditional Chinese MedicineIsha Sethi - Dana-Farber Cancer InstituteSilvia Guatimosim - Universidade Federal de Minas GeraisQing Ma - Boston Children's HospitalBlake D Jardin - Boston Children's HospitalYulan Ai - Boston Children's HospitalDonghui Zhang - Boston Children's HospitalBiyi Chen - Roy J. and Lucille A. Carver College of MedicineAng Guo - Roy J. and Lucille A. Carver College of MedicineGuo-Cheng Yuan - Dana-Farber Cancer InstituteLong-Sheng Song - Roy J. and Lucille A. Carver College of MedicineWilliam T Pu - Boston Children's Hospital
- Resource Type
- Journal article
- Publication Details
- Circulation research, Vol.120(12), pp.1874-1888
- DOI
- 10.1161/CIRCRESAHA.116.310283
- PMID
- 28356340
- PMCID
- PMC5466492
- ISSN
- 0009-7330
- eISSN
- 1524-4571
- Grant note
- UM1 HL098166 / NHLBI NIH HHS U01 HL131003 / NHLBI NIH HHS F32 HL134235 / NHLBI NIH HHS R01 HL090905 / NHLBI NIH HHS R01 HL130346 / NHLBI NIH HHS R01 HL116461 / NHLBI NIH HHS T32 HL007572 / NHLBI NIH HHS
- Language
- English
- Date published
- 06/09/2017
- Academic Unit
- Cardiovascular Medicine; Fraternal Order of Eagles Diabetes Research Center; Biochemistry and Molecular Biology; Internal Medicine
- Record Identifier
- 9984288732202771
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