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Redox signaling and cardiac function are tightly linked. Still, it is largely unknown which protein targets are affected by H2O2 in cardiomyocytes that underly the impaired inotropic effects in oxidative stress. Here, we combined a new chemogenetic mouse model (HyPer-DAO mice) and a redox proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we prove that increased endogenous production of H2O2 in cardiomyocytes leads to a reversibly impaired cardiac contractility in vivo. We identified the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch and link its modification to mitochondrial metabolism and glutathione synthesis. Microsecond molecular dynamics simulations combined with experiments using cysteine-gene-edited point mutated cells revealed that IDH3γ Cys148 and 284 are critically involved in H2O2-dependent regulation of IDH3 activity. The oxidative modification of IDH3γ regulates the competition between energy production by TCA cycle flux and cellular anti-oxidant defense by glutathione synthesis.

Genes that are highly conserved in food seeking behaviour, such as protein kinase G (PKG), are of interest because of their potential role in the global obesity epidemic. PKG1α can be activated by binding of cyclic guanosine monophosphate (cGMP) or oxidant-induced interprotein disulfide bond formation between the two subunits of this homodimeric kinase. PKG1α activation by cGMP plays a role in reward and addiction through its actions in the ventral tegmental area (VTA) of the brain. ‘Redox dead’ C42S PKG1α knock-in (KI) mice, which are fully deficient in oxidant-induced disulfide-PKG1α formation, display increased food seeking and reward behaviour compared to wild-type (WT) littermates. Rewarding monoamines such as dopamine, which are released during feeding, are metabolised by monoamine oxidase to generate hydrogen peroxide that was shown to mediate PKG1α oxidation. Indeed, inhibition of monoamine oxidase, which prevents it producing hydrogen peroxide, attenuated PKG1α oxidation and increased sucrose preference in WT, but not KI mice. The deficient reward phenotype of the KI mice was rescued by expressing WT kinase that can form the disulfide state in the VTA using an adeno-associated virus, consistent with PKG1α oxidation providing a break on feeding behaviour. In conclusion, disulfide-PKG1α in VTA neurons acts as a negative regulator of feeding and therefore may provide a novel therapeutic target for obesity. Subject areas: PKG1α, Food-seeking behaviour, Reward, Ventral Tegmental Area

Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wild-type mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output. These responses are typical features of sepsis and their combined effect is a lowering of blood pressure. This hypotension, a hallmark of sepsis, resulted in underperfusion of end organs, resulting in their damage. A central role for PKG Iα oxidative activation in injury is supported by oxidation-resistant Cys42Ser PKG Iα knock-in mice being markedly protected from these clinical indices of injury during sepsis. We conclude that oxidative activation of PKG Iα is a key mediator of hypotension and consequential organ injury during sepsis.