Targeting RyR2 with a phosphorylation site–specific nanobody reverses dysfunction of failing cardiomyocytes in rats

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca 2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2‐targeting phosphorylation site—specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2‐specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain—only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno‐associated virus (AAV)9—mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca 2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA‐mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.—Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C, Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site–specific nanobody reverses dysfunction of failing cardiomyocytes in rats. FASEB J. 33, 7467–7478 (2019). www.fasebj.org