Ryanodine Receptors Outside of Couplons are Involved in Excitation-Contraction Coupling in Rabbit Ventricular Myocytes

Abstract

Current theories of excitation-contraction coupling (ECC) in ventricular myocytes assert that L-type Ca channels interact with clusters of ryanodine-sensitive Ca release channels (RyRs) within couplons. We hypothesized that RyR clusters exist also outside of couplons and contribute to ECC. We investigated this hypothesis by three-dimensional imaging of RyR clusters and sarcolemma of isolated myocytes lying flat (horizontal) and on end (vertical). We deconvolved the image stacks, created reconstructions of cell segments, and identified RyR cluster types. RyR clusters remote to sarcolemma were assumed to be outside of couplons. Similar studies were performed on intact ventricular tissue. Furthermore, we imaged evoked Ca transients and sarcolemma of horizontal cells labeled with fluo-4 and di-8-anepps. Image sequences were acquired using rapid two-dimensional scanning (Zeiss LSM5Live, rates up to 300HZ). The image sequences were corrected for bleaching and cross-talk. In horizontal and vertical isolated cells, RyR clusters appeared to be arranged in sheets in the vicinity of Z-disks. Some RyR clusters were associated with sarcolemma, in particular transverse tubules, and are presumably part of couplons. However, most RyR clusters were not. Examination of cells in intact tissue revealed a smaller number of RyR clusters not associated with sarcolemma than in isolated cells. The density of transverse tubules was higher than in isolated cells. This loss of transverse tubules might be caused by the isolation procedure. Analysis of the rapid image sequences indicated that both types of RyR clusters were activated during an action potential. However, the RyR clusters not associated with sarcolemma were activated with delays of up to 10ms. In conclusion, we demonstrated that RyR clusters outside of couplons are involved in ECC. We suggest that activation of RyR clusters outside of couplons occurs by a common pool mechanism.