Cardiovascular Research. 2022 May 16
Yuxuan Guo, Yangpo Cao, Blake D. Jardin, Xiaoran Zhang, Pingzhu Zhou, Silvia Guatimosim, Junsen Lin, Zhan Chen, Yueyang Zhang, Neil Mazumdar, Fujian Lu, Qing Ma, Yao-Wei Lu, Mingming Zhao, Da-Zhi Wang, Erdan Dong, William T. Pu
Products used in the paper Details Operation
AAV vector packaging We thank PackGene Biotech for assistance in AAV production. Request Quote

Research Field: (AAV)-delivered CRISPR/Cas9-mediated somatic mutagenesis (CASAAV) technique; Cardivascular research

Keywords: Cardiomyocyte maturation, Ryanodine receptor 2, ER stress

AAV Serotype: AAV9

Dose: To rule out the expression of truncated RYR2 proteins, we injected high-dose (3.6×10^9 vg/g) AAV-sgRNA[Ryr2]-cTNT-Cre (AAV-sgRNA[Ryr2]; KO) or AAV-cTNT-Cre (AAV-Cre; control) into Post-natal Day 1 (P1) mice and collected ventricular tissues at P7 for western blot analysis.

Animal or cell line strain: mice


Aims: Calcium-handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the pre-dominant calcium channel that releases calcium from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) to activate cardiomyocyte contraction. Although RYR2 was previously implied as a key regulator of cardiomyocyte maturation, the mechanisms remain unclear. The aim of this study is to solve this problem.

Methods and results: We performed Cas9/AAV9-mediated somatic mutagenesis to knockout RYR2 specifically in cardiomyocytes in mice. We conducted a genetic mosaic analysis to dissect the cell-autonomous function of RYR2 during cardiomyocyte maturation. We found that RYR2 depletion triggered ultrastructural and transcriptomic defects relevant to cardiomyocyte maturation. These phenotypes were associated with the drastic activation of ER stress pathways. The ER stress alleviator tauroursodeoxycholic acid partially rescued the defects in RYR2-depleted cardiomyocytes. Overexpression of ATF4, a key ER stress transcription factor, recapitulated defects in RYR2-depleted cells. Integrative analysis of RNA-Seq and bioChIP-Seq data revealed that protein biosynthesis-related genes are the major direct downstream targets of ATF4.

Conclusion: RYR2-regulated ER homeostasis is essential for cardiomyocyte maturation. Severe ER stress perturbs cardiomyocyte maturation primarily through ATF4 activation. The major downstream effector genes of ATF4 are related to protein biosynthesis.

Popular Services

AAV Packaging Service

AAV Analytical Service

Vector Design