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The 66-kDa Src homology 2 domain-containing protein (p66Shc) is a master regulator of reactive oxygen species (ROS). It is expressed in many tissues where it contributes to organ dysfunction by promoting oxidative stress. In the vasculature, p66Shc-induced ROS engenders endothelial dysfunction. Here we show that p66Shc is a direct target of the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetylation of p66Shc governs its capacity to induce ROS. Using diabetes as an oxidative stimulus, we demonstrate that p66Shc is acetylated under high glucose conditions and is deacetylated by Sirt1 on lysine 81. High glucose-stimulated lysine acetylation of p66Shc facilitates its phosphorylation on serine 36 and translocation to the mitochondria, where it promotes hydrogen peroxide production. Endothelium-specific transgenic and global knockin mice expressing p66Shc that is not acetylatable on lysine 81 are protected from diabetic oxidative stress and vascular endothelial dysfunction. These findings show that p66Shc is a target of Sirt1, uncover a unique Sirt1-regulated lysine acetylationdependent mechanism that governs the oxidative function of p66Shc, and demonstrate the importance of p66Shc lysine acetylation in vascular oxidative stress and diabetic vascular pathophysiology.p66Shc | sirt1 | lysine acetylation | diabetes | oxidative stress

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes1. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Context: Doxorubicin (Dox) is one of the most active chemotherapeutic agents used to treat various types of cancers. Its clinical utility is compromised due to fatal cardiac toxicity characterized by an irreversible cardiomyopathy. Objective: This study evaluates the cardioprotective potential of naringin (NR) against Dox-induced acute cardiac toxicity in rats. Materials and methods: Male Wistar rats were randomly divided into five groups. NR (50 and 100 mg/kg) was administered intraperitoneally (i.p.) daily from 0 to 14 d. Doxorubicin (15 mg/kg, i.p.) was given as a single dose on the 10th day. On the 14th day, all animals were sacrificed and oxidative stress parameters that include malondialdehyde (MDA), glutathione (GSH) level, superoxide dismutase (SOD), catalase (CAT) activities, and all mitochondrial complexes (I-IV) activities were evaluated along with histopathological studies of the heart. Results: Doxorubicin-induced cardiotoxicity was confirmed by increased (p50.05) MDA, decreased (p50.05) GSH levels, SOD, and CAT activities, mitochondrial complexes (I-IV) activities in the heart tissue. NR (100 mg/kg) showed cardioprotection as evident from significant decreased MDA (p50.001) level, raised (p50.001) GSH level, SOD and CAT activities and increased mitochondrial complexes I (p50.01), II (p50.001), III (p50.001), and IV (p50.05) activities. Further, Dox-induced cardiotoxicity was confirmed by histopathological studies. These obtained results indicated the protective role of NR against Dox-induced cardiac toxicity in rats. Conclusion: NR can be used in combination with Dox due to its high cardioprotective effect against Dox-induced cardiomyopathy.

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Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified ≥1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, β-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.

Highlights d RBM10 interacts with Star-PAP and directs Star-PAP activity toward polyadenylation d RBM10 is enriched in the heart and controls expression of anti-hypertrophy regulators d RBM10 depletion or compromised Star-PAP interaction results in myocyte hypertrophy d RBM10 expression is lost in the in vivo model of hypertrophic heart in Wistar rats