Oral Presentation 2014 International Biophysics Congress

Zooming in on the transverse tubules: dSTORM imaging of human cardiac junctions (#63)

David Crossman 1 , Yu-Feng Hou 1 , Peter Ruygrok 2 , David Baddeley 3 , Christian Soeller 4
  1. The University of Auckland, Auckland, New Zealand
  2. Auckland City Hospital, Auckland, New Zealand
  3. Yale University, New Haven, USA
  4. University of Exeter, Exeter, UK

Transverse tubules (TT) are invaginations of the plasma membrane that allow propagation of the action potential into the interior of the myocyte, triggering Ca2+ induced Ca2+ release from the ryanodine receptor (SR), facilitating a synchronous contraction. A key structure in this signalling cascade is the cardiac junction, which forms a restricted space (~15 nm) between the TT and sarcoplasmic reticulum (SR). Literature evidence suggests changes in the expression of the protein junctophilin (JPH) leads to nano-scale changes in junctional geometry that precipitates loss of contractile function in heart failure. To examine this hypothesis direct stochastic optical reconstruction microscopy (dSTORM) was used to image junctional architecture in the failing human heart. Direct-STORM is a form of localisation microscopy that allows imaging substantially below the traditional optical limit (~250 nm) with a resolution approaching 15 nm. An advantage of this technique is that it utilises conventional fluorescence dyes allowing samples to be first imaged with confocal. Human cardiac tissue labelled for JPH and RyR were first imaged with confocal. Identified regions were then correlatively-imaged with dSTORM. Preliminary analysis of dSTORM images shows that in TT regions 87% ± 1% of RyR label is located within 20 nm of the JPH label in normal myocytes compared to 92 ± 2% in failing myocytes. Likewise at the outer sarcolemma 88 ± 1% of RyR label is located within 20 nm of the JPH label in normal myocytes compared to 83 ± 3% in failing myocytes. Imaging with dSTORM clearly detected the distribution of RyR and JPH but the confocal volume data provided cellular context for the super-resolution data.