Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo

Abstract

Rationale 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. 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 T-tubules, a hallmark of mature cardiac myocytes. Methods and Results 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. Conclusions 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.

Considerations for Cardiac CRISPR

“…Cardiac-restricted expression of Cas9 was induced using mice in which a cassette encoding Cas9 linked to a GFP reporter was expressed from the Rosa26 locus, crossed to αMHC-Cre mice. Consistent with other models that have expressed Cas9 in either a broad or tissue restricted manner 5, 6, 9 , no changes in baseline cardiac function were observed following Cas9 overexpression, suggesting that Cas9 alone has no adverse impact on the heart. To test the utility of these mice for gene editing, the authors chose to target Tbx20 , which is essential for cardiac function, and Sav1 , a component of the Hippo signaling pathway, which governs cardiac growth.…”

“…First, one of the major hurdles is the use of AAV as a viral delivery vector for sgRNA components, as the efficiency of gene editing depends on viral titer, as well as levels of expression of Cas9 and sgRNAs 6 . As a result, strategies to enhance AAV penetrance in cardiac tissue, particularly in adult heart, could prove especially critical in generating more widespread infection of cardiomyocytes.…”

CASAAV Technology to Examine Regulators of Heart Failure

“…As proof of concept, the authors 12 chose JPH2 as target protein based on its association with dyad microdomain and t-tubule organization. 3, 4, 13 It was first confirmed that CASAAV system conducts efficient and specific gene silencing in postnatal cardiomyocytes, consistent with an earlier report.…”

“…Early diagnosis and thus interventions earlier in the disease process should allow clinicians the ability to reverse disease progression. The technical platform developed by Guo et al 12 offers a new method for scientists to identify the gene expression changes which are upstream triggers of cardiomyocyte dysfunction. In the present study, JPH2 deletion had minimal impact on t-tubule organization and function, but may exacerbate and manifest the failing phenotype.…”

“…In the present study, JPH2 deletion had minimal impact on t-tubule organization and function, but may exacerbate and manifest the failing phenotype. 12 It is possible that other proteins such as cBIN1 may serve as potential upstream regulators of t-tubules, together with RyR2. The technology introduced by Guo et al 12 is an important new tool to help advance our understanding of heart failure progression.…”