Oxidative stress has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). Indeed, the prevalence of AF increases with age as does oxidative stress. However, the mechanisms linking redox state to AF are not well understood. In this study we identify a link between oxidative stress and aberrant intracellular Ca2+ release via the type 2 ryanodine receptor (RyR2) that promotes AF. We show that RyR2 are oxidized in the atria of patients with chronic AF compared with individuals in sinus rhythm. To dissect the molecular mechanism linking RyR2 oxidation to AF we used two murine models harboring RyR2 mutations that cause intracellular Ca2+ leak. Mice with intracellular Ca2+ leak exhibited increased atrial RyR2 oxidation, mitochondrial dysfunction, reactive oxygen species (ROS) production and AF susceptibility. Both genetic inhibition of mitochondrial ROS production and pharmacological treatment of RyR2 leakage prevented AF. Collectively, our results indicate that alterations of RyR2 and mitochondrial ROS generation form a vicious cycle in the development of AF. Targeting this previously unrecognized mechanism could be useful in developing effective interventions to prevent and treat AF.

The incidence of diabetic nephropathy has been increasing. Studies have shown that oxidative stress (due to increased oxidant production and/or decreased antioxidant activity) is a critical underlying mechanism. The principal intracellular reductant is NADPH whose production is mainly dependent on glucose-6-phosphate dehydrogenase (G6PD) activity. Our work in cultured cells previously showed that high glucose caused activation of protein kinase A (PKA) and subsequent phosphorylation and inhibition of G6PD activity and hence decreased NADPH (Zhang Z, Apse K, Pang J, and Stanton RC. J Biol Chem 275:40042-40047, 2000). The purpose of this study was to determine whether these findings occur in diabetic rats (induced by streptozotocin) compared with control. G6PD activity and accordingly NADPH levels and glutathione levels were significantly decreased in diabetic kidneys compared with control kidneys. Lipid peroxidation was significantly increased, which correlated with decreased G6PD activity (r = 0.48). G6PD expression was significantly reduced, which correlated with decreased G6PD activity (r = 0.72). PKA activity and serine phosphorylation of G6PD were significantly increased and were closely correlated with decreased G6PD activity (r = 0.51 for PKA activity; r = 0.93 for serine phosphorylation of G6PD). Insulin treatment and/or correction of hyperglycemia ameliorated the changes caused by diabetes. In conclusion, chronic hyperglycemia caused inhibition of G6PD activity via decreased expression and increased phosphorylation of G6PD, which therefore led to increased oxidative stress.

The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here we report the 2.5 Å crystal structure of a regulatory domain construct (aa 78-355) containing both cGMP binding sites of PKG Iα. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.

Endothelial cells (ECs) are critical mediators of blood pressure (BP) regulation, primarily via the generation and release of vasorelaxants, including nitric oxide (NO). NO is produced in ECs by endothelial NO synthase (eNOS), which is activated by both calcium (Ca 2+ )-dependent and independent pathways. Here, we report that intracellular Ca 2+ release from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca 2+ -dependent eNOS activation. EC-specific type 1 1,4,5-trisphosphate receptor knockout (IP3R1 −/− ) mice are hypertensive and display blunted vasodilation in response to acetylcholine (ACh). Moreover, eNOS activity is reduced in both isolated IP3R1-deficient murine ECs and human ECs following IP3R1 knockdown. IP3R1 is upstream of calcineurin, a Ca 2+ /calmodulinactivated serine/threonine protein phosphatase. We show here that the calcineurin/nuclear factor of activated T cells (NFAT) pathway is less active and eNOS levels are decreased in IP3R1-deficient ECs. Furthermore, the calcineurin inhibitor cyclosporin A, whose use has been associated with the development of hypertension, reduces eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 plays a crucial role in the ECmediated vasorelaxation and the maintenance of normal BP.is a major cause of morbidity and mortality affecting millions of adults worldwide (1, 2). Globally, among the ∼17 million deaths caused by cardiovascular diseases, nearly half can be attributed to complications of HTN (3-9). In addition, vasodilator drugs that activate nitric oxide (NO) production have been used to treat HTN for decades (4, 10). Calcineurin (also known as protein phosphatase 2B), is a Ca 2+ /calmodulin-activated serine/ threonine protein phosphatase. Calcineurin inhibitors are first-line immunosuppressants used in organ transplantation (11,12). However, calcineurin inhibition causes HTN in up to 70% of patients (13), and the exact underlying mechanisms are not fully understood.Vascular endothelial cells (ECs), located at the interface between the vessel wall and blood, release vasorelaxants that influence vascular smooth muscle tone in response to mechanical (e.g., shear stress) and biochemical stimuli (14, 15). These stimuli induce a rapid increase in intracellular Ca 2+ ([Ca 2+ ] i ) in ECs activating Ca 2+ -dependent signaling pathways, resulting in release of endothelium-derived relaxing factors, including NO (14,[16][17][18][19]. Mice lacking endothelial nitric oxide synthase (eNOS) develop severe HTN (20). However, how eNOS is regulated in vivo remains essentially unclear. The modulation of both plasma membrane Ca 2+ entry and endoplasmic reticulum (ER) Ca 2+ release is critical in EC function (21,22). Moreover, recent meta-analysis and genome-wide association studies in hypertensive individuals have linked type 1 1,4,5-trisphosphate receptor (IP3R1), a major [Ca 2+ ] i release channel (23,24), to high blood pressure (BP) (25,26). On these grounds, we sought to inve...