Glutaredoxin-2 and Sirtuin-3 deficiencies impair cardiac mitochondrial energetics but their effects are not additive.

Altered redox biology and oxidative stress have been implicated in the progression of heart failure. Glutaredoxin-2 (GRX2) is a glutathione-dependent oxidoreductase and catalyzes the reversible deglutathionylation of mitochondrial proteins. Sirtuin-3 (SIRT3) is a class III histone deacetylase and regulates lysine acetylation in mitochondria. Both GRX2 and SIRT3 are considered as key in the protection against oxidative damage in the myocardium. Knockout of either contributes to adverse heart pathologies including hypertrophy, hypertension, and cardiac dysfunction. Here, we created and characterized a GRX2 and SIRT3 double-knockout mouse model, hypothesizing that their deletions would have an additive effect on oxidative stress, and exacerbate mitochondrial function and myocardial structural remodeling. Wildtype, single-gene knockout (Sirt3-/-, Grx2-/-), and double-knockout mice (Grx2-/-/Sirt3-/-) were compared in heart weight, histology, mitochondrial respiration and H2O2 production. Overall, the hearts from Grx2-/-/Sirt3-/- mice displayed increased fibrosis and hypertrophy versus wildtype. In the Grx2-/- and the Sirt3-/- we observed changes in mitochondrial oxidative capacity, however this was associated with elevated H2O2 emission only in the Sirt3-/-. Similar changes were observed but not worsened in hearts from Grx2-/-/Sirt3-/- mice, suggesting that these changes were not additive. In human myocardium, using genetic and histopathological data from the human Genotype-Tissue Expression consortium, we confirmed that SIRT3 expression correlates inversely with heart pathology. Altogether, GRX2 and SIRT3 are important in the control of cardiac mitochondrial redox and oxidative processes, but their combined absence does not exacerbate effects, consistent with the overall conclusion that they function together in the complex redox and antioxidant systems in the heart.

Glutaredoxin-2 controls cardiac mitochondrial dynamics and energetics in mice, and protects against human cardiac pathologies.

Glutaredoxin 2 (GRX2), a mitochondrial glutathione-dependent oxidoreductase, is central to glutathione homeostasis and mitochondrial redox, which is crucial in highly metabolic tissues like the heart. Previous research showed that absence of Grx2, leads to impaired mitochondrial complex I function, hypertension and cardiac hypertrophy in mice but the impact on mitochondrial structure and function in intact cardiomyocytes and in humans has not been explored. We hypothesized that Grx2 controls cardiac mitochondrial dynamics and function in cellular and mouse models, and that low expression is associated with human cardiac dysfunction. Here we show that Grx2 absence impairs mitochondrial fusion, ultrastructure and energetics in primary cardiomyocytes and cardiac tissue. Moreover, provision of the glutathione precursor, N-acetylcysteine (NAC) to Grx2-/- mice did not restore glutathione redox or prevent impairments. Using genetic and histopathological data from the human Genotype-Tissue Expression consortium we demonstrate that low GRX2 is associated with fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, GRX2 is important in the control of cardiac mitochondrial structure and function, and protects against human cardiac pathologies.

Glutaredoxin-2 is required to control oxidative phosphorylation in cardiac muscle by mediating deglutathionylation reactions.

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2(-/-)) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2(-/-) hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2(+/-)) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2(+/-) mice were far less hypertensive than Grx2(-/-) mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.

Glutaredoxin-2 is required to control proton leak through uncoupling protein-3.

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2(-/-)) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3(-/-) cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.

Crucial yet divergent roles of mitochondrial redox state in skeletal muscle vs. brown adipose tissue energetics.

Reduced glutathione (GSH) is the major determinant of redox balance in mitochondria and as such is fundamental in the control of cellular bioenergetics. GSH is also the most important nonprotein antioxidant molecule in cells. Surprisingly, the effect of redox environment has never been examined in skeletal muscle and brown adipose tissue (BAT), two tissues that have exceptional dynamic range and that are relevant to the development of obesity and related diseases. Here, we show that the redox environment plays crucial, yet divergent, roles in modulating mitochondrial bioenergetics in skeletal muscle and BAT. Skeletal muscle mitochondria were found to naturally have a highly reduced environment (GSH/GSSG≈46), and this was associated with fairly high (∼40%) rates of state 4 (nonphosphorylating) respiration and decreased reactive oxygen species (ROS) emission. The deglutathionylation of uncoupling protein 3 (UCP3) following an increase in the reductive potential of mitochondria results in a further increase in nonphosphorylating respiration (∼20% in situ). BAT mitochondria were found to have a much more oxidized status (GSH/GSSG≈13) and had basal reactive oxygen species emission that was higher (∼250% increase in ROS generation) than that in skeletal muscle mitochondria. When redox status was subsequently increased (i.e., more reduced), UCP1-mediated uncoupling was more sensitive to GDP inhibition. Surprisingly, BAT was found to be devoid of glutaredoxin-2 (Grx2) expression, while there was abundant expression in skeletal muscle. Taken together, these findings reveal the importance of redox environment in controlling bioenergetic functions in both tissues, and the highly unique characteristics of BAT in this regard.