Evaluating changes in reversible cysteine oxidation of cardiac proteins as metabolic syndrome develops into cardiovascular disease
Behring, Jessica Belle
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Oxidative stress is commonly associated with diet-induced metabolic syndrome (MetS) and left ventricular cardiac remodeling, but much remains unknown about the role of redox signaling, sensors, and switches in mediating the effects of high fat and sugar intake. In this work, I describe and apply an optimized method for quantifying changes in reversible protein-cysteine oxidation in the heart. This method uses isobaric tagging of cysteine thiols and mass spectrometry in a modified biotin switch on whole tissue lysate. Analyzing the resulting data with systems biology approaches helped delineate redox pathways playing a role in disease development, while cysteine-specificity provided exact targets for mutation-based mechanistic studies. Initial findings in a mouse model for MetS, wherein C57Bl6J mice were fed a high fat/high sucrose diet, identified energy pathways as the primary target of changing reversible cysteine oxidation. In follow-up studies, our collaborators helped validate the pathophysiological role of two particular cysteines in complex II; their early reversible oxidation and later irreversible oxidation contributed to decreased ATP output from cardiac mitochondria. A subsequent, more robust study revealed a weakness in our original method. While investigating the role of hydrogen peroxide-induced oxidative post-translational modifications (OPTMs) in the development of MetS sequelae, analysis of four mouse groups, each with an n=5, revealed that measurements of reversibly oxidized cysteine thiols were highly variable compared to those of all available thiols. Thus, I developed a strategy to address the source of variability and, in the process, improved many additional steps in the switch protocol. Finally, in an effort to clarify the role of the most stable reversible OPTM, glutathionylation (RSSG), we characterized the HFHS diet response in mice engineered to have more or less RSSG via genetic manipulation of glutaredoxin-1 expression. Those with more RSSG suffered worsened cardiac function, making them an ideal model for future studies with the methods optimized in this work. Studying the progression from poor diet to cardiac involvement in these and other mouse models using the methods described herein will aid in the design of diagnostics and targeted therapies against the growing burden of metabolic CVD.