Succinylation of SERCA2a at K352 Promotes Its Ubiquitinoylation and Degradation by Proteasomes in Sepsis-Induced Heart Dysfunction.

Abstract

Background: Intracellular Ca²⁺ cycling governs effective myocardial systolic contraction and diastolic relaxation. SERCA2a (sarco/endoplasmic reticulum Ca²⁺ ATPase type 2a), which plays a crucial role in controlling intracellular Ca²⁺ signaling and myocardial cell function, is downregulated and inactivated during sepsis-induced heart dysfunction. However, the cause of this dysregulation remains unclear. In this study, we investigated the effect of lysine succinylation in lipopolysaccharide-induced septic heart dysfunction through global succinylome analysis of myocardial tissues from septic rats.

Methods: We conducted a succinylome profiling and developed a protein language model-based framework to prioritize succinylation at a functionally important site, and further analysis revealed crosstalk between ubiquitination and succinylation of SERCA2a. The succinylation of SERCA2a in septic rats or lipopolysaccharide-treated cells were detected by co-immunoprecipitation. Thereafter, a desuccinylated SERCA2a^K352R was introduced and its function and stability were determined by Ca²⁺ transient and Western blot, respectively. Meanwhile, the effect on SERCA2a^K352R on heart function was assessed in vivo by echocardiography and hemodynamics.

Results: We identified 10 324 succinylated lysine sites in heart tissues, including 1042 differentially succinylated lysine sites, in response to lipopolysaccharide. SERCA2a was hypersuccinylated in the myocardial tissues of septic rats and lipopolysaccharide-treated cardiomyocytes. Increased ubiquitination level, reduced protein level, and activity of SERCA2a were observed, along with increased succinylation of SERCA2a in vivo and in vitro. K352 was essential for SERCA2a succinylation, which reduced SERCA2a protein level by promoting formation of the K48 ubiquitin chain on SERCA2a and its degradation by proteasomes. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry identified that SIRT2 (sirtuin2), a deacylase, exhibited interaction with SERCA2a. Furthermore, SIRT2 decreased K352 succinylation of SERCA2a, suggesting that SIRT2 may function as a desuccinylase for SERCA2a.

Conclusions: Succinylation of SERCA2a at K352, which was controlled by SIRT2, promotes its ubiquitinoylation and degradation by proteasomes in sepsis-induced heart dysfunction.