title: Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  RESULTS
  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 toRosaCas9GFP/Cas9GFPneonatal 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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Guo
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: VanDusen
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Lina
authors.initials: L
authors.lastname: Zhang
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Weiliang
authors.initials: W
authors.lastname: Gu
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Isha
authors.initials: I
authors.lastname: Sethi
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Silvia
authors.initials: S
authors.lastname: Guatimosim
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Qing
authors.initials: Q
authors.lastname: Ma
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Blake D
authors.initials: BD
authors.lastname: Jardin
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Yulan
authors.initials: Y
authors.lastname: Ai
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Donghui
authors.initials: D
authors.lastname: Zhang
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Biyi
authors.initials: B
authors.lastname: Chen
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Ang
authors.initials: A
authors.lastname: Guo
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Guo-Cheng
authors.initials: GC
authors.lastname: Yuan
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.).
authors.forename: Long-Sheng
authors.initials: LS
authors.lastname: Song
authors.affiliation: From the Cardiology, Boston Children's Hospital, MA (Y.G., N.J.V., Q.M., B.D.J., Y.A., D.Z., W.T.P.); Institute of Basic Medicine (L.Z.) and Pharmacology, School of Pharmacy (W.G.), Shanghai University of Traditional Chinese Medicine, China; Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA (I.S., G.-C.Y.); Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (S.G.); Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City (B.C., A.G., L.-S.S.); Veterans Affairs Medical Center, Iowa City (L.-S.S.); and Harvard Stem Cell Institute, Cambridge, MA (W.T.P.). wpu@pulab.org.
authors.forename: William T
authors.initials: WT
authors.lastname: Pu
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000502:physiology*; D048708:Cell Growth Processes / Q000502:physiology*; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000172:deficiency / Q000235:genetics; D032383:Myocytes, Cardiac / Q000502:physiology*
chemical_list: D008565:Membrane Proteins; D009124:Muscle Proteins; C000589042:JP2 protein, mouse
keywords: alleles; gene silencing; heart failure; maturation; mutagenesis; transverse tubule