Multi-Scale Cardiomyocyte Organisation as a Determinant of Cardiac Function

Abstract

Excitation-contraction (EC) coupling is a critical process underlying cardiac function, and for this to be effective, precise organisation of key proteins and structures is required. Transverse (t)-tubules and the sarcoplasmic reticulum (SR) form a close association known as the junction, where several EC coupling proteins localise, including the SR calcium release channels - the ryanodine receptors (RyR). In cardiomyocytes, formation of both junctions and t-tubules is promoted and maintained by junctophilin-2 (JPH2). Three mechanisms have been proposed through which JPH2 may impact cardiac function: 1) organisation of the junction, 2) t-tubule organisation, and 3) stabilisation of RyR. By investigating these mechanisms, this thesis aimed to examine aspects of the relationship between cardiomyocyte organisation and cardiac function in three studies. The first study aimed to determine the role of JPH2 in maintaining junctional protein and t-tubule organisation. Using JPH2 transgenic mice, super resolution imaging revealed an increased RyR cluster size in JPH2 over- expressing mice, in which smaller calcium sparks are observed, while JPH2 knockdown demonstrated no change to RyR cluster size, despite increased calcium spark size and frequency. Changes in density and co-localisation of junctional proteins provided an explanation for the apparent disparity between RyR cluster size and calcium handling properties. Increased longitudinal branching of the t-tubule network was identified following JPH2 over-expression, which overall, had no detrimental effect on EC coupling. The second study used a novel approach to directly correlate force development and cardiomyocyte structure in trabeculae from failing human hearts. Cardiomyocyte content of the myocardium was identified as a key indicator of contractile performance in these samples. Several changes in the microscopic organisation of cardiomyocytes were also confirmed as occurring in the failing human heart. The final study investigated the development of EC coupling structures, including t-tubules and junctions, in the large mammalian fetal heart. In the first known fluorescent study to examine this, t-tubules (which develop only postnatally in rodents) were identified as developing in utero in the sheep heart, and were associated with early co- localisation of junctional proteins. Combined, these findings support the three proposed mechanisms of JPH2, and provide new insights of implications for EC coupling.