Nanoscale organisation of calcium regulatory proteins in the healthy and failing heart

  • Lauren Toms

Student thesis: Phd


Contraction of cardiac myocytes is driven by a process known as excitation-contraction coupling (ECC). Briefly, this involves influx of calcium (Ca2+) through L-type Ca2+ channels on the sarcolemma, which triggers further release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR). Ca2+ removal is essential for myocyte relaxation. This primarily occurs by the SR Ca2+-ATPase (SERCA), replenishing the intracellular Ca2+ store, and is dependent upon the phosphorylation status of phospholamban (PLN). In the SR, Ca2+ is mainly bound to buffers, which maintains a low concentration of free Ca2+ during diastole. The recent advent of super resolution microscopy and the use of computer models has revealed the importance of the nanoscale organisation of Ca2+ handling proteins to their functioning. Although the underlying mechanism is the same, there are chamber-specific differences in Ca2+ handling which are reflected in the spatiotemporal properties of the systolic Ca2+ transient. In heart failure (HF), ECC is disrupted, resulting in diminished contractility. Therefore, the aims of this study were to determine the nanoscale organisation of Ca2+ handling proteins and if this is (1) chamber-specific, and (2) altered in HF. Left atrial and ventricular myocytes were isolated from control and HF sheep, induced by right ventricular tachypacing. Ca2+ handling proteins were visualised using stochastic optical reconstruction microscopy (STORM), with spatial arrangements determined using custom-written software. Tissue protein levels were quantified by Western blot. Differences were determined if p < 0.05 using linear mixed model analysis or Student’s t-test. Firstly, we found RyR cluster spatial properties to be t-tubule dependent in atrial myocytes, with larger clusters and reduced inter-cluster spacing in the areas lacking t-tubules. These arrangements may aid in the centripetal propagation of the Ca2+ release required. RyR clusters on a t-tubule in the atria did not differ from that in the ventricle. Additionally, higher protein levels of the SR Ca2+ buffer, calsequestrin, was seen in the ventricle, which may aid in priming the junctional SR for a larger release of Ca2+, contributing to the increased systolic Ca2+ transient amplitude. SERCA was found to align predominantly to the z-lines. In the atria, increased levels of SERCA and decreased levels of PLN protein were found in comparison to the ventricle, correlating with the accelerated rate of relaxation in the atria. In the failing left ventricle and atria, RyR protein was reduced, with smaller clusters and increased inter-cluster spacing. This may contribute to a slow and dyssynchronous release of Ca2+ from the SR which may lead to contractile dysfunction. In addition, there was hypophosphorylation of PLN in HF for both chambers, likely contributing to the reduced SERCA activity. In the failing ventricle, more SERCA was present between z-lines compared to control, with the effect of this currently unknown. The findings in this study reveal differences in the nanoscale organisation of Ca2+ handling proteins between the chambers and in HF, which may underlie some of the differences in the spatiotemporal properties of the systolic Ca2+ transient. The latter finding may prove beneficial in the identification of novel therapeutic targets.
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAndy Trafford (Supervisor) & Katharine Dibb (Supervisor)


  • Super resolution
  • Dyad
  • Ryanodine receptor

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