Regulation of Cardiac Connexin43 by Reactive Oxygen Species
thesisposted on 01.07.2016, 00:00 by Cody Rutledge
Sudden cardiac arrest (SCA) refers to the abrupt and unexpected cessation of cardiac mechanical function, often attributable to electrical abnormalities in the heart. SCA is the leading cause of death in the United States, responsible for between 200,000 and 450,000 American deaths yearly. SCA is most often caused by the formation of reentrant arrhythmias in the heart, which are favored by slow electrical conduction velocity. In the ventricles, connexin43 (Cx43) is the most important gap junction protein responsible for maintaining rapid conduction velocity. Cx43 disruption occurs in nearly every cardiac disease. Recent evidence has implicated the tyrosine kinase c-Src as a crucial regulator of Cx43 function. The first part of this thesis investigates the role of c-Src in Cx43 regulation in a mouse model of myocardial infarction (MI). We hypothesized that increased production of reactive oxygen species (ROS), a known consequence of MI, causes c-Src activation, resulting in displacement of Cx43 from the gap junction. Additionally, we evaluated the roles of c-Src inhibitors, including PP1 and AZD0530, for their efficacy in protecting Cx43 expression following MI. The second part of this thesis evaluates the role of ROS in c-Src activation. Transgenic ACE8/8 mice, which feature elevated cardiac-specific renin-angiotensin system signaling, are used to evaluate Cx43 expression and SCA in the presence of various ROS inhibitors and scavengers. Targeting specific sources of oxidative stress, including xanthine oxidase, NAPDH oxidase, uncoupled eNOS, and depleted glutathione, did not affect SCA in this model. Mitochondrial-specific ROS scavengers, however, prevented c-Src activation, protected Cx43 expression, and lowered the incidence of SCA in the ACE8/8 model, suggesting that mitochondrial ROS is responsible for c-Src activation. Part three of this thesis attempts to explain the mechanistic link between mitochondrial ROS production and c-Src activation. One potential mediator is the scaffolding protein caveolin-1 (Cav-1), which has been linked to c-Src activation in a variety of tissue models. Cav-1 binds the inactive form of c-Src, but this binding is disrupted in pathological conditions. We demonstrated decreased Cav-1/c-Src interaction in the ACE8/8 model, which could be reversed with mitochondrial antioxidant therapy. Additionally, we demonstrated that Cav-1 null mice have activated c-Src, decreased Cx43 expression, and increased risk of SCA. Finally, we evaluated Cav-1/c-Src interaction in the MI model and evaluated the role of mitochondrial antioxidant therapy following MI. Collectively, this work supports a novel signaling cascade involving mitochondrial ROS, Cav-1 disruption, and c-Src activation in the regulation of Cx43 in a variety of mouse models. Additionally, it supports the role of ROS scavengers and c-Src inhibitors as potential therapeutic options in the prevention of SCA.