Shining light on the molecular scale remodelling in a heart's path to failure

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Myocytes are the mechanical workhorses of the human heart. The uninterrupted contraction andrelaxation of these large cells powers the movement of blood throughout the body over an individual'slifetime. Intricate extensions of the plasma membranes known as t-tubules form thousands of'intracellular synapses' (known as 'dyads') deep within the cell's interior. Calcium signals released ateach dyad are the primary determinants of the cell's contraction. Previous work has reported a seriesof gradual sub-microscopic biochemical and structural changes occurring throughout each myocyte ina wide range of heart diseases. Such remodelling has catastrophic effects on the calcium signals andthe cell's mechanical performance, placing the heart on an irreversible path towards failure. Until now,optical microscopy techniques have lacked the resolving power to fully visualise compact structuressuch as t-tubules and dyads; therefore our understanding of the time-course, the spatial scales andthe protein modifications underpinning the human pathology has been very limited. With a mortalityrate of up to 50% in the first 5 years in such heart failure patients, it is now urgent that we combinestate-of-the-art microscopy, biochemical and cell biology tools to gain a visual understanding of thisremodelling process. Among the currently-popular super-resolution microscopies, the novel DNA-PAINT method is the only technique that allows adequate resolution to fully visualise t-tubules, as wellas individual proteins among the tightly-packed clusters within dyads.The proposed study will bring together a highly interdisciplinary supervision team to test thehypothesis that the unique shape and protein composition of the dyads within the human myocytesundergo biochemical and structural remodelling in response to hormonal stimuli at a spatial scale thathas not been visualised before. The project will consist of three primary aims: (i) to utilise DNA-PAINTimaging to examine the time-course of t-tubule remodelling in myocytes that are freshly isolated fromcardiac tissue biopsies from patients in Leeds General Infirmary, (ii) to perform quantitativemeasurements of the expression and phosphorylation levels of structural proteins of the dyads withthe utility of super-resolution images and quantitative Western Blot protocols and (iii) to develop an exvivo culture assay of stem cell-derived human cardiomyocytes that can facilitate long term (weeksmonths)chemical and structural observations in the presence of cardiotropic hormones typicallyobserved in failing hearts. In supervision, Dr Isuru Jayasinghe (IJ) will lead the DNA-PAINT imaging,image analysis and membrane study; Dr Andrew Smith (AJS) will share clinical knowledge of thebiopsy samples, knowhow of culture of cardiac stem cells and derived myocytes and Prof John Colyer(JC) will provide the quantitative Western Blot tools.The primary endpoints of this study will be (i) an unprecedented visual understanding of thetimescales of the nanoscale membrane and protein remodelling in human myocytes, (ii) the targeteddetection of nanoscale morphologies of remodelled t-tubules and calcium release sites in specificcases of human heart failure and (iii) a novel ex vivo human myocyte assay that enables aquantitative profiling of these changes under a controlled humoral environment that typifies specifichuman heart diseases.