Abstract 13228: SUMO2 Induces Cardiac Hypertrophy in Vivo and in Vitro , Independent of Sumoylation

Abstract

Introduction: The calcineurin (Cn)-NFAT signaling axis is a pivotal pathway in cardiac physiology and pathophysiology including remodeling and pathological hypertrophy. Methods and results: We have previously shown that SUMO2 (S2) is a strong activator of Cn-NFAT signaling and, furthermore, is a direct interaction partner of Cn. S2 is sufficient and necessary to induce cardiomyocyte hypertrophy in NRVCMs. Interestingly, a sumoylation-deficient mutant of S2, S2ΔGG, yielded similar results in every parameter investigated. Overexpression of S2 or S2ΔGG enhances nuclear CnA localization as evidenced by subcellular fractionation as well as immunofluorescence experiments. The direct interaction of S2 and CnA is further supported by proximity-ligation-experiments in NRVCM. Additionally, knockdown of Ubc9, the sole E2-enzyme in the sumoylation cascade, had no significant influence on S2‘s effects, further supporting a sumoylation-independent mechanism. Knockdown of S2 revealed its necessity for agonist induced hypertrophy as shown by an abrogation of phenylephrine induced hypertrophy (median cell size: miRNeg+PE=512 μm 2 , miRS2+PE=519 μm 2 , n>3000 cells) and diminished expression of fetal genes and Rcan-1.4 (miRNeg+PE vs. miR-S2+PE: Nppa : -41%, Nppb : -57%, Rcan1.4 : -80%; p<0.001, n=6). On the basis of these in vitro findings, we designed an AAV9 gene transfer approach for S2 and S2ΔGG in mice, as a proof-of-principle for the in vivo relevance of this mechanism. In line with the proposed mechanism, both S2 constructs induced cardiac hypertrophy with elevated heart weight to body weight ratio (+25%, p<0.05; n=6 mice) and increased lung weight to body weight ratio (+20%, p<0.05) as a sign of heart failure. Consistent with pathological hypertrophy, fractional shortening (FS) and ejection fraction (EF) were reduced (FS, Ctrl: 25%, S2: 19%, S2ΔGG: 20%; p<0.05; EF: Ctrl: 37%, S2: 26%, S2ΔGG: 30%; p<0.01). Furthermore these animals showed elevated Nppb (S2: 2.7-fold, S2ΔGG: 2.4-fold; p<0.01) and Rcan1.4 levels (S2: 4.5-fold, S2ΔGG: 5.5-fold; p<0.01). Conclusions: We propose a mechanism where S2 causes cardiac hypertrophy, independent of sumoylation, by a direct interaction with calcineurin A, facilitating its translocation to the nucleus.