Glutamine protects cardiomyocytes from hypoxia/reoxygenation injury under high glucose conditions through inhibition of the transforming growth factor-ß1-Smad3 pathway.

Activation of transforming growth factor-ß1 (TGF-ß1)-Smad3 pathway aggravates myocardial ischemia/reperfusion injury (IRI). We previously showed that glutamine (Gln) protects cardiomyocytes from hypoxia/reoxygenation (H/R) injury under high glucose (HG) conditions. The aim of this study was to investigate whether Gln exerts its protective effect in H/R via inhibiting TGF-ß1-Smad3 pathway. In vitro, H9c2 rat cardiomyocytes were treated with Gln with HG (33 mM) and/or H/R. We also performed in vivo experiments in which we treated normal and diabetic rats with Gln or solvent control following IRI. We assessed protein levels of TGF-ß1, total Smad3, phosphorylated (p)-Smad3 and cleaved caspase-3 in H9c2 cells and rat myocardium by Western blotting. H9c2 cells treated with HG + H/R exhibited high apoptosis rates, as well as a highly activated TGF-ß1-Smad3 pathway. TGF-ß1 receptor inhibitor (SB431542) or Smad3 inhibitor (SIS3) reduced HG + H/R induced apoptosis. Similarly, Gln supplementation alleviated apoptosis and decreased p-Smad3 levels. However, Gln's protective effect was significantly weakened by TGF-ß1. Diabetic rats treated with Gln had improved hemodynamics, smaller infarct size after IRI, and a significant decrease in TGF-ß1-Smad3 pathway activation. We conclude that Gln inhibits HG + H/R induced activation of the TGF-ß1-Smad3 pathway and decreases cell apoptosis in cardiomyocytes.

Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism.

Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.