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Ryanodine receptor 2 (RYR2) dysfunction activates the unfolded protein response and perturbs cardiomyocyte maturation.

AIMS
Calcium handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the predominant 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 (CASAAV) 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 (TUDCA) 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.


CONCLUSIONS
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.


TRANSLATIONAL PERSPECTIVE
Dysfunctional calcium handling is a major factor contributing to cardiac pathogenesis, but the molecular mechanisms remain unclear. This study uncovered RYR2 as a new regulator of ER stress and cardiomyocyte maturation, providing significant insights to guide the development of therapeutic approaches to control cardiac pathogenesis. Because cardiomyocyte maturation is a major bottleneck in translational medicine using stem cell-derived cardiomyocytes, this study also pointed out RYR2 and ER homeostasis as potential targets to manipulate the maturity of stem cell-derived cardiomyocytes.