Abstract 288: Rnd3/RhoE Regulates Cardiac Ryanodine Receptor Type 2 Stability

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Rationale: Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration and proliferation. The biological function of Rnd3 in the heart remains unexplored.

Objective: To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism.

Methods and Results: By loss-of-function approaches, we discovered a new role in which Rnd3 stabilizes the ryanodine receptor type 2 (RyR2) Ca2+ release channel. Genetic deletion of Rnd3 in mice resulted in embryonic lethality with heart failure and arrhythmia. Both Rnd3-/- embryonic and Rnd3+/- adult cardiomyocytes showed severe Ca2+ leakage. Single channel assessment showed the destabilized RyR2 channel, and this irregular spontaneous Ca2+ release was curtailed by protein kinase A (PKA) inhibitor treatment. Further studies found that RyR2 protein was hyperphosphorylated by PKA in the mutant heart. Remarkable increases in the PKA activity along with elevated cyclic adenosine monophosphate levels were detected in vivo in Rnd3-null embryos and in vitro in neonatal rat cardiomyocytes and non-cardiac cell lines with Rnd3 knockdown. Moreover, we found increasing β2-adrenergic receptor (β2AR) protein levels, but no correlated mRNA changes in both the Rnd3-null heart and non-cardiac cells with Rnd3 knockdown. Immunoprecipitation analysis demonstrated that Rnd3 and β2AR physically interacted. Multiple post-translational modification analyses of β2AR revealed that downregulation of Rnd3 attenuated β2AR protein lysosomal targeting and ubiquitination, which in turn resulted in the elevation of β2AR protein levels contributing to the activation of PKA signaling. Rnd3 deficiency had no effects on the hydroxylation- and sumoylation-mediated β2AR protein degradation.

Conclusion: Rnd3 is a unique stabilizer of RyR2 that impacts intracellular Ca2+ handling in the heart.

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