Characterization of the cardiac succinylome and its role in ischemia–reperfusion injury

Abstract

Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD+-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5−/− mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5−/− samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5−/− heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5−/− hearts. 16 peptides from 9 proteins were significantly increased in Sirt5−/− by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5−/− (108.4 fold). The data indicate that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5−/− hearts compared to WT littermates (68.5+/− 1.1% Sirt5−/− vs 39.6+/− 6.8% WT) following 20 minutes of ischemia and 90 minutes reperfusion. We further demonstrate that I/R injury in Sirt5−/− heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.

Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function

“…In the present study, we have established that SIRT5 deficiency leads to decreased ECHA activity and deficiency in cardiac energy metabolism, and ultimately cardiomyopathy. Very recently, Boylston et al (39) have showed that Sirt5 KO mice are more susceptible to ischemia-reperfusion injury compared with WT.…”

New and revisited approaches to preserving the reperfused myocardium

“…Post-translational modifications of electron transport complexes and endogenous ROS-scavengers via sirtuins 84 and O-GlcNAc modifications 85 , or succinylation 86 might help in the search for novel pharmacological interventions to treat post-ischaemic injury. Given that the adaptor protein p66 Shc catalyses ROS formation via cytochrome c , several studies have investigated the potential role of p66 Shc as a regulator of myocardial injury in murine models of cardiac ischaemia and reperfusion.…”

Mitochondrial sirtuins in the heart

“…Myocardial infarct size was increased, and recovery of contractile function was impaired in hearts of SIRT5 -/-mice following IR [86]. IR injury was restored to WT levels by inhibition of succinate dehydrogenase, which was hypersuccinylated and probably more active in SIRT5 -/-hearts [13,86].…”

“…Myocardial infarct size was increased, and recovery of contractile function was impaired in hearts of SIRT5 -/-mice following IR [86]. IR injury was restored to WT levels by inhibition of succinate dehydrogenase, which was hypersuccinylated and probably more active in SIRT5 -/-hearts [13,86]. Since succinate accumulates during ischemia and drives mitochondrial ROS generation at the onset of reperfusion by increasing reverse electron transport in the electron transport chain, the authors proposed that inhibition of SDH may restore IR injury to WT levels by reducing succinate-driven ROS production [86,87].…”

“…IR injury was restored to WT levels by inhibition of succinate dehydrogenase, which was hypersuccinylated and probably more active in SIRT5 -/-hearts [13,86]. Since succinate accumulates during ischemia and drives mitochondrial ROS generation at the onset of reperfusion by increasing reverse electron transport in the electron transport chain, the authors proposed that inhibition of SDH may restore IR injury to WT levels by reducing succinate-driven ROS production [86,87]. Interestingly, SIRT5 is downregulated in cardiomyocytes upon oxidative stress, and SIRT5 knockdown results in a marked reduction in cell viability, a significant increase in the number of apoptotic cells, and increased activity of caspase 3 activity [73].…”