Gap junction channels (GJCh) comprise the pathway for intercellular propagation of the electrical signals responsible for coordinated activation of cardiac contraction; heterogeneity and failure of GJCh function lead to heterogeneous slowing of conduction and consequent micro- or macro-reentry circuits that set the stage for tachycardia and fibrillation in the atria and ventricles. Heterogeneity and failure of GJCh function reflect acute, phosphorylation-dependent regulation of GJCh gating and conductance. This application focuses on the GJCh gating and conductance changes that are antiarrhythmic (preconditioning) and proarrhythmic, contributing to sudden cardiac death. The long-term goals of the project are to: 1) understand the mechanisms underlying phosphorylation-dependent regulation of Cx43 GJCh and hemichannel (HCh) function, and 2) develop from that knowledge peptidomimetics that will target or induce specific functional properties of Cx43 channels that will preserve cardiac rhythmicity and prevent irreversible injury. Two key issues limit our ability to implement Cx43 based therapies. First, we do not understand the consequences of differential Cx43 phosphorylation on GJCh function. Second, the impact of heterogeneous Cx43 phosphorylation on impulse propagation in the heart is unknown. Because these issues cannot be addressed in pairs of adult ventricular myocytes (due to heterogeneity of Cx43 phosphorylation state and the large number of functioning GJChs that preclude study of gating and single channel behavior by the necessary whole-cell voltage clamp methods), we address our aims using site-mutants of Cx43 that mimic the phosphorylation state of Cx43 in normally functioning, preconditioned and injured heart. We express these Cx43 site mutants in Cx-deficient rat insulinoma cells, Cx43-deficient cardiomyocytes, and wild-type cardiomyocytes, where their gating, conductance and antiarrhythmic properties can be studied. The results of our studies will guide design and testing of function- specific, high-affinity peptidomimetics to interfere with or mimic the interactions tht underlie anti-arrhythmic GJCh and HCh function. The proposed combination of molecular, structural and functional approaches can be expected to provide new mechanistic insight on regulation of the dynamic function of Cx43 GJChs as they support the extraordinary dynamic range of cardiac function. In addition, we expect to develop Cx43- and function-specific, experimentally and therapeutically useful tools that will protect coordinated function of the heart despite ongoing disease and pharmacologic therapies.
The heart's ability to dynamically adjust its output to meet the body's demand for blood flow is orchestrated by signaling cascades that modulate the function of numerous proteins including those essential for intercellular communication, the connexins. In heart disease, function of this essential communication pathway is compromised, leading to arrhythmias. The goal of our project is to understand how this communication pathway is regulated by signaling cascades normally active in the heart and those activated by injury and disease such that novel therapeutic tools can be developed that will protect against fatal arrhythmias.
