Periodic Reporting for period 4 - CARDYADS (Controlling Cardiomyocyte Dyadic Structure) Reporting period: 2020-01-01 to 2020-12-31 Summary of the context and overall objectives of the project Contraction and relaxation of the heart are dependent on the function of individual muscle cells, called cardiomyocytes. Within these cells are small structures called dyads, which are junctions between two cellular membranes. During the heartbeat, release of calcium occurs at these dyads, which triggers contraction, and relaxation occurs as calcium is removed. Prior to initiating the project, existing data indicated that dyads are broken down during diseases such as heart failure, which reduces the power of the heartbeat. In order to eventually treat these patients, we aimed to precisely understand how dyads work and what regulates their structure. We further sought to examine the specific consequences of changing dyadic structure, and to gain insight into approaches that may repair dyads in disease.Using advanced microscopy, our work during this project has provided significant new knowledge of dyadic structure and function. We have observed the precise locations of different proteins which cycle calcium in the cell, providing information for how these proteins collaborate with each other. We have observed that these proteins are mobile and that they are carefully assembled into functional groups during cardiac development. However, these groupings are degraded during conditions such as heart failure. These changes in protein localization are paralleled by alterations in the membranes where they are located, as more membranes are grown in the developing and compensating heart, but are lost in the failing heart. What regulates the formation and destruction of dyads? Our investigations have shown that the heart’s workload critically regulates the structure and function of dyads, and thus the function of the whole heart. In the developing heart, increasing workload drives the formation of dyads as cardiac function increases. In these hearts, we have specifically identified protein partners that work together to build new dyads. Interestingly, this functional reserve remains present in the healthy adult heart. Indeed, moderate increases in workload allow compensatory increases in dyad formation. However, excessive workload, as occurs during diseases such as heart failure, causes dyads to be broken down, weakening the heartbeat. Importantly, our observations have shown that this degradation of dyadic structure only occurs in forms of heart failure where the contractile power of the heart is reduced. These findings have important clinical implications, as interventions that reduce workload and/or mechanical signaling in cardiomyocytes have potential to treat some but not all forms of heart failure. We look forward to continuing our search for such therapies in our ongoing studies. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the project, our group has made several interesting discoveries. These can be divided into 3 parts, which describe our 3 areas of scientific focus:1. Examining dyadic structure in unprecedented detailWe employed state-of-the-art technologies to understand the 3D structure of dyads. These techniques include super-resolution imaging and electron microscopy, both of which are able to separate objects which are as close together as a few nanometers (billionths of a meter). These studies have illustrated how the membranes are placed within dyads, and the precise 3D locations of different proteins that transport calcium. We have observed how these membranes and proteins are assembled as the heart develops, and how these constituent parts of dyads are degraded during certain types of heart failure.2. Determining the precise consequences of changing dyadic structureIt is critical that we link how the structure of dyads affects their function, and thus the function of the entire heart. Therefore, our work involved measurements of calcium in the cell. Using new technologies, we were able to measure calcium being transported by individual dyads, and understand how the structure of the dyad affects calcium regulation. Our data show that in some types of heart failure (but not others) breakdown of dyads during disease has serious detrimental consequences for how calcium is transported. This results in impaired contraction and relaxation of the cell. Thus, overall dysfunction of the whole heart in these patients can be traced in part to tiny changes in dyadic structure and function within the heart's muscle cells.3. Finding signals that control dyadic structureLittle has been known of the signals that regulate how dyads are put together and kept intact. Our studies in this project showed that high physical stress placed on the walls of the heart is a key trigger for breaking dyads down. This has important implications for heart failure, as the hearts of many of these patients are under significant stress. Our data show that proteins which are necessary for anchoring dyads in place are lost during these high stress conditions, causing dyads to degrade. We have also identified other proteins that act to build new dyads. We hope that by using this knowledge we can build and/or maintain dyads to strengthen the heartbeat in heart failure patients in future therapies. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Heart failure has an enormous socio-economic burden and current treatments are inadequate. Instead of simply treating disease symptoms, we must strive to identify the key underlying events that cause heart failure to progress, and find ways to reverse or prevent them. The results of our study show that changes in cell structures called dyads are an important cause of certain types of heart failure. This work has yielded unprecedented insight into the precise structure and function of dyads, the consequences of changing these structures, and the signals which are important for forming and maintaining them. We have specifically identified that elevated cardiac workload is a key driver of dyadic destruction during heart failure, indicating that future therapies should be aimed at reducing workload and/or inhibiting mechanical signaling in cardiac muscle cells. Differing mechanisms of HFrEF and HFpEF, from Frisk et al., JACC 2021 3D reconstruction of t-tubule and RyR arrangement, from Shen et al, J Physiol, 2019 T-tubules grown in iPSC-derived cardiomyocytes, from Parikh et al, 2017 Regulation of dyadic structure by workload
