Gap-Junction Uncoupling Paradoxically Increase Synchronization of Spontaneous Calcium Release in the Intact Heart

Abstract

Intracellular calcium (Ca) dysregulation associated with cardiac disease has been linked to mechanisms of ventricular arrhythmias. We have previously shown that spontaneous calcium release from an aggregate of many cells in situ (an SCR) occurs in a synchronized fashion; however, the mechanism responsible for synchronizing SCR activity in coupled myocardium is unknown. Since others have reported that Ca can diffuse through gap junctions (GJ), we hypothesize that uncoupling cells by blocking GJ will desynchronize, and attenuate SCR activity. Methods: To test this hypothesis high resolution optical mapping of Ca (Indo-1AM) from the anterior surface of the Langendorff perfused guinea pig heart (n=5) was performed in hearts under high Ca conditions ([Ca2+]e=5.5mM), with and without carbonoxolone (CBX, 50μM) to reduce GJ coupling. Endocardial cryoablation were performed to eliminate Purkinje fibers and cytochalasin-D (7μM) was administered to remove motion artifact. Fifteen seconds of rapid pacing (350-160 ms cycle length) followed by a pause was used to induce SCR activity. Results: In all preparations, SCR activity was observed across the entire mapping field before and after CBX. With CBX, the amplitude of SCR activity increased (+14.8%, p < 0.05) and its time to peak occurred earlier (−11.2%, p < 0.01) compared to no CBX. CBX also decreased the range of local SCR time to peaks across the mapping field (−17.2%, p < 0.05), suggesting that uncoupling myocytes synchronizes spontaneous calcium release across cells. There was no statistical difference in the occurrence of triggered activity before and during CBX. Conclusions: These results demonstrate that the occurrence of spontaneous calcium release in tissue (an SCR) does not require Ca diffusion though GJs. In fact, spontaneous calcium release in tissue is paradoxically enhanced during GJ inhibition.