Modulation of atrial fibrillation susceptibility by gp91phox-containing NADPH oxidases

Atrial fibrillation (AF) is the most frequently encountered cardiac rhythm disorder in humans, with affected individuals facing an increased risk of stroke and mortality. Published findings implicate oxidative stress in triggering the new onset of AF and in promoting the atrial electrical and structural changes that drive disease progression. NOX2-containing NADPH oxidases are a significant source of superoxide production in atrial tissues. Previous work showed that a NOX2-mediated increase in superoxide accompanies AF in patients and animal models of tachypacing-induced AF, and further reported a strong independent association between atrial NOX2 activity and post-operative AF in patients undergoing cardiac surgery. However, a direct causal role for NOX2 in AF has not been investigated. This thesis aimed to test the hypothesis that superoxide released specifically from NOX2 directly contributes to the development of AF. To investigate the role of NOX2 in AF, mice with cardiac-specific overexpression of the human NOX2 gene (NOX2-Tg) were characterized. Atrial homogenates from these mice were found to show approximately 3-fold higher expression of NOX2 protein compared with wild-type controls, which was associated with a significant increase in NADPH-stimulated superoxide production. AF susceptibility assessed in vivo by transesophageal atrial burst stimulation was significantly higher in NOX2-Tg mice compared with wild-type, in the absence of significant alterations in cardiac function. However, dietary administration of atorvastatin (30 mg/kg daily for two weeks) did not eliminate AF susceptibility in NOX2-Tg mice, despite preventing the increase in NADPH-stimulated superoxide production in the left and right atria of these animals. Instead, atorvastatin significantly reduced the duration of pacing-induced AF, an effect which was found to be independent of genotype, and thus independent of NOX2 inhibition. Mechanistic studies do not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of di-4-ANEPPS-stained atrial tissue preparations revealed no significant differences in action potential duration or conduction velocity between wild-type and NOX2-Tg mice. Instead, NOX2 overexpression was associated with impaired atrial Ca2+ handling. In left atrial myocytes of NOX2-Tg mice, the RyR2 Ca2+ leak-SR load ratio was lower when compared with wild-type, despite an increase in SR Ca2+ load. In the right atria, the leak-load ratio was similar between NOX2-Tg and wild-type myocytes, but the proportion of myocytes with ‘leaky’ RyR2 channels was greater among NOX2-Tg mice. RyR2 phosphorylation at Ser-2814 and Ser-2808 was not significantly different between WT and NOX2-Tg atrial homogenates, indicating that the changes in diastolic leak are not caused by differences in CaMKII or PKA activity.</p

Modulation of atrial fibrillation susceptibility by gp91phox-containing NADPH oxidases

Atrial fibrillation (AF) is the most frequently encountered cardiac rhythm disorder in humans, with affected individuals facing an increased risk of stroke and mortality. Published findings implicate oxidative stress in triggering the new onset of AF and in promoting the atrial electrical and structural changes that drive disease progression. NOX2-containing NADPH oxidases are a significant source of superoxide production in atrial tissues. Previous work showed that a NOX2-mediated increase in superoxide accompanies AF in patients and animal models of tachypacing-induced AF, and further reported a strong independent association between atrial NOX2 activity and post-operative AF in patients undergoing cardiac surgery. However, a direct causal role for NOX2 in AF has not been investigated. This thesis aimed to test the hypothesis that superoxide released specifically from NOX2 directly contributes to the development of AF. To investigate the role of NOX2 in AF, mice with cardiac-specific overexpression of the human NOX2 gene (NOX2-Tg) were characterized. Atrial homogenates from these mice were found to show approximately 3-fold higher expression of NOX2 protein compared with wild-type controls, which was associated with a significant increase in NADPH-stimulated superoxide production. AF susceptibility assessed in vivo by transesophageal atrial burst stimulation was significantly higher in NOX2-Tg mice compared with wild-type, in the absence of significant alterations in cardiac function. However, dietary administration of atorvastatin (30 mg/kg daily for two weeks) did not eliminate AF susceptibility in NOX2-Tg mice, despite preventing the increase in NADPH-stimulated superoxide production in the left and right atria of these animals. Instead, atorvastatin significantly reduced the duration of pacing-induced AF, an effect which was found to be independent of genotype, and thus independent of NOX2 inhibition. Mechanistic studies do not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of di-4-ANEPPS-stained atrial tissue preparations revealed no significant differences in action potential duration or conduction velocity between wild-type and NOX2-Tg mice. Instead, NOX2 overexpression was associated with impaired atrial Ca2+ handling. In left atrial myocytes of NOX2-Tg mice, the RyR2 Ca2+ leak-SR load ratio was lower when compared with wild-type, despite an increase in SR Ca2+ load. In the right atria, the leak-load ratio was similar between NOX2-Tg and wild-type myocytes, but the proportion of myocytes with âleakyâ RyR2 channels was greater among NOX2-Tg mice. RyR2 phosphorylation at Ser-2814 and Ser-2808 was not significantly different between WT and NOX2-Tg atrial homogenates, indicating that the changes in diastolic leak are not caused by differences in CaMKII or PKA activity.</p

Mechanisms Underlying the Pathogenesis of Atrial Arrhythmias in RGS4-deficient Mice

Atrial arrhythmias are very common clinically relevant conditions that are strongly associated with aging and parasympathetic tone. Additionally, ATP-sensitive K+ (KATP) channel activation has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here investigate whether KATP channel activity is increased under decreased RGS4 activity in a manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-induced re-entrant rotors in the RGS4-deficient mouse model.MAS

RGS4-Deficiency Alters Intracellular Calcium and PKA-Mediated Control of Insulin Secretion in Glucose-Stimulated Beta Islets

A number of diverse G-protein signaling pathways have been shown to regulate insulin secretion from pancreatic β-cells. Accordingly, regulator of G-protein signaling (RGS) proteins have also been implicated in coordinating this process. One such protein, RGS4, is reported to show both positive and negative effects on insulin secretion from β-cells depending on the physiologic context under which it was studied. We here use an RGS4-deficient mouse model to characterize previously unknown G-protein signaling pathways that are regulated by RGS4 during glucose-stimulated insulin secretion from the pancreatic islets. Our data show that loss of RGS4 results in a marked deficiency in glucose-stimulated insulin secretion during both phase I and phase II of insulin release in intact mice and isolated islets. These deficiencies are associated with lower cAMP/PKA activity and a loss of normal calcium surge (phase I) and oscillatory (phase II) kinetics behavior in the RGS4-deficient β-cells, suggesting RGS4 may be important for regulation of both Gαi and Gαq signaling control during glucose-stimulated insulin secretion. Together, these studies add to the known list of G-protein coupled signaling events that are controlled by RGS4 during glucose-stimulated insulin secretion and highlight the importance of maintaining normal levels of RGS4 function in healthy pancreatic tissues

The autonomic nervous system and cardiac GLP-1 receptors control heart rate in mice

Objectives: Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine cells and exerts a broad number of metabolic actions through activation of a single GLP-1 receptor (GLP-1R). The cardiovascular actions of GLP-1 have garnered increasing attention as GLP-1R agonists are used to treat human subjects with diabetes and obesity that may be at increased risk for development of heart disease. Here we studied mechanisms linking GLP-1R activation to control of heart rate (HR) in mice. Methods: The actions of GLP-1R agonists were examined on the control of HR in wild type mice (WT) and in mice with cardiomyocyte-selective disruption of the GLP-1R (Glp1rCM−/−). Complimentary studies examined the effects of GLP-1R agonists in mice co-administered propranolol or atropine. The direct effects of GLP-1R agonism on HR and ventricular developed pressure were examined in isolated perfused mouse hearts ex vivo, and atrial depolarization was quantified in mouse hearts following direct application of liraglutide to perfused atrial preparations ex vivo. Results: Doses of liraglutide and lixisenatide that were equipotent for acute glucose control rapidly increased HR in WT and Glp1rCM−/− mice in vivo. The actions of liraglutide to increase HR were more sustained relative to lixisenatide, and diminished in Glp1rCM−/− mice. The acute chronotropic actions of GLP-1R agonists were attenuated by propranolol but not atropine. Neither native GLP-1 nor lixisenatide increased HR or developed pressure in perfused hearts ex vivo. Moreover, liraglutide had no direct effect on sinoatrial node firing rate in mouse atrial preparations ex vivo. Despite co-localization of HCN4 and GLP-1R in primate hearts, HCN4-directed Cre expression did not attenuate levels of Glp1r mRNA transcripts, but did reduce atrial Gcgr expression in the mouse heart. Conclusions: GLP-1R agonists increase HR through multiple mechanisms, including regulation of autonomic nervous system function, and activation of the atrial GLP-1R. Surprisingly, the isolated atrial GLP-1R does not transduce a direct chronotropic effect following exposure to GLP-1R agonists in the intact heart, or isolated atrium, ex vivo. Hence, cardiac GLP-1R circuits controlling HR require neural inputs and do not function in a heart-autonomous manner