Understanding the Heartbeat at the Nanoscale

The heartbeat is generated by the coordinated contraction of individual heart muscle cells. However, despite decades of research, the precise manner by which cellular contraction is triggered remains elusive. Much of this uncertainty stems from the fact that the structures which initiate cellular contraction are truly tiny, measuring only 1/10,000th of the width of a human hair! This project is aimed at investigating the arrangement and function of these units using new microscopy techniques capable of revealing extraordinary 3-dimensional detail. Two ion channels will be specifically examined, which are known to cooperate to release calcium from stores within the cell, signalling contraction. Using heart muscle cells from mice and human cardiac patients we will examine both the precise location and function of these channels. This work will importantly reveal how these two channels "talk" to each other, enabling them to fine-tune the power of the heartbeat. For example, we believe that improved communication during times of need allows the cell, and thus the whole heart, to contract more forcefully. However, in diseases such as heart failure, we expect that that impaired communication between the channels causes a weaker heartbeat. Thus, our project will provide a new, more detailed understanding of how the heartbeat is triggered both in health and disease, and the adaptability of this process.

The heartbeat is generated by the coordinated contraction of cardiac muscle cells. In each cell, contraction is initiated at tiny structures called dyads which release Ca2+. Despite decades of research, the precise arrangement and function of Ca2+ handling proteins within dyads remains unclear. However, our recent data employing 3D, live-cell super-resolution imaging have revealed functional groupings of Ca2+ release channels (Ryanodine Receptors, RyRs) in unprecedented detail. In the present project, we will extend these findings to examine how RyRs are triggered by neighbouring L-type Ca2+ channels (LTCCs). This work will provide quantitative insight into the basic, yet elusive mechanisms which trigger and regulate the heartbeat. The precise 3D arrangement of LTCCs, RyRs, and dyadic membranes will be revealed by combining super-resolution imaging and electron microscopy tomography (CLEM imaging). To this end, a transgenic mouse with fluorescent labels on LTCCs and RyRs will be employed, with cells examined during rest, beta-adrenergic stimulation, and heart failure progression. Dyads will be similarly compared in tissue from explanted healthy and failing human hearts. Live-cell experiments will examine the consequences of dyadic organization for LTCC-RyR crosstalk, with channel localization and activity respectively assessed by super-resolution imaging and local Ca2+ recordings. Acquired data will be subsequently integrated by mathematical modeling. We hypothesize that beta-adrenergic stimulation augments LTCC-RYR crosstalk by recruiting additional channels, both via physical displacement and increased phosphorylation status, representing a new paradigm for understanding the fight-or-flight response. Conversely, we anticipate that loss of LTCC-RyR plasticity and crosstalk reduces contractility during heart failure. These data will provide novel, nanoscale understanding of how the heartbeat is strengthened during times of need, and weakened during disease.