In October 2018 I flew across the world to the laboratory of Dr David Crossman at the University of Auckland in New Zealand. The goal: to study pathological remodelling in human heart biopsies from patients with idiopathic dilated cardiomyopathy (IDCM), using the super-resolution imaging technique expansion microscopy (ExM).
Our interest lies in seeing whether nanodomain remodelling observed in a rat model of heart failure in Leeds, including reorganisation of the internal calcium compartments and functional modification to calcium-handling proteins, is also present in end-stage human heart failure. Understanding the mechanisms of remodelling is one of the first steps towards investigating whether they can be targeted for preventative therapies.
ExM is novel imaging technique, enabling super-resolution imaging by spatially separating fluorophores within a swellable hydrogel. The compatibility of ExM gels with standard microscopes enables greater imaging depth and improved axial resolution over competing super-resolution techniques. ExM therefore provides a practical tool to observe remodelling within dyadic calcium release clusters. I was responsible for starting ExM experiments from scratch in a new lab across the world, requiring efficient independent work to obtain meaningful data in the space of just 4 weeks.
It was fantastic to take this journey and work in a laboratory that is home to a strong consortium of leading cardiovascular researchers. In my final week I gave a 30-minute seminar, in which I presented work to the physiology department and the wider bio-imaging facility. This allowed me to reach an international audience and receive valuable feedback on the progression of my research.
Many thanks to the MRC and DiMeN flexible fund grant which made this trip possible, and special thanks to David Crossman for welcoming me into his lab.
Background: Cardiac arrhythmias are a leading cause of mortality and long-term morbidity worldwide. Over 25% of these originate in the heart’s electrically-autonomous (pacemaking) cells without a detectable heart attack. Inappropriate release of calcium from internal compartments (e.g. endoplasmic reticulum) into the cytoplasm of pacemaker cells is central to this pathology. Recent reports suggest that a small group of intracellular calcium channels, ryanodine receptor type-3 (RyR3) – historically associated with neuronal signalling – can interact with other calcium handling proteins to trigger arrhythmogenic calcium signals. The molecular mechanism and locations of RyR3 and its partners seeding these arrhythmias are unknown.
Objectives: (i) To locate RyR3 and its partner proteins which trigger arrhythmogenic calcium signals in pacemaker cells at nanometre precision (ii) To use pharmacological inhibitors tested previously on neuronal cells for perturbing the calcium signals inappropriately triggered by RyR3.
Timeliness: A number of current anti-arrhythmic therapies target other calcium handling proteins and involve significant risks. Identification of RyR3 and partners as a trigger mechanism for arrhythmias in pacemaker cells would be crucial to developing alternative and potentially safer therapies.
Novelty: RyR3, until now, has not been linked with heart disease. This project will utilise the latest super-resolution microscopy tools like Expansion Microscopy and DNA-PAINT to pinpoint the epicentres of arrhythmogenic calcium signals at an unprecedented spatial resolution.
Approach: You will exploit the leading-edge super-resolution microscopy tools (DNA-PAINT and Expansion Microscopy) pioneered by the Nanoscale Microscopy Group in Leeds (website: https://musclesuperres.com/ ), to visually examine how RyR3s are organised in the pacemaking (sinoatrial node) cells of healthy hearts. You will then examine a rat model prone to arrhythmias, comparing how RyR3, IP3-receptors and SERCA proteins are re-arranged in the nanometre-scale during the pathology. You will complete at least two secondments with the industrial partner Badrilla Ltd (website: https://badrilla.com/) to design, manufacture and test bespoke antibody labels which will allow you to map the arrhythmogenic calcium signals against the underpinning RyR3 and partner protein layouts. For this, you will harness a novel correlative microscopy protocol developed by the primary supervisor for overlaying calcium and DNA-PAINT images. Using pharmacological inhibitors of IP3R and SERCA established for studying dorsal root ganglion neurons, you will modulate the calcium handling of RyR3 and partners to understand how the signals can be reverted towards a healthy phenotype.
Key outcomes will include mechanistic schemes of the molecular-scale factors in RyR3-mediated calcium signals seeding arrhythmias and a set of pharmacological strategies to impede them.
Research environment: You will be embedded in the Nanoscale Microscopy Group, nested within the Cardiovascular Research group in the University of Leeds (working with Dr Izzy Jayasinghe and Prof Ed White). This is one of the UK’s leading clusters of researchers examining the cellular mechanisms of cardiovascular diseases. You will form bridges between this group, Prof Nikita Gamper of the Leeds Neuroscience group, Badrilla Ltd which is one of the leading specialists of analytical reagents for the cardiovascular sciences, and our international collaborators in Australia, New Zealand and USA.
Benefits of being in the DiMeN DTP: This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research. We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Tom Sheard in our group is currently funded by the DiMeN DTP and has enjoyed supplementary funding from the DTP for a 1-month placement in Auckland, New Zealand, to study human hearts.
1. Jayasinghe, I. et al. True molecular scale visualisation of variable clustering properties of ryanodine receptors. Cell Reports. 2018; 22 (2), 557-567
2. Ouyang, K. et al. Ca2+ sparks and secretion in dorsal root ganglion neurons. PNAS. 2005; 102 (34) 12259-12264
3. Jayasinghe, I. et al. Shining new light on the structural determinants of cardiac couplon function. Frontiers in Physiology. doi: 10.3389/fphys.2018.01472
Our new review article to mark 10 years since the first super-resolution imaging experiments on cardiac muscle: Abstract: Remodelling of the membranes and protein clustering patterns during the pathogenesis of cardiomyopathies has renewed the interest in spatial visualisation of these structures in cardiomyocytes. Coincidental emergence of single molecule (super-resolution) imaging and tomographic electron microscopy tools in the last decade have led to a number of new observations on the structural features of the couplons, the primary sites of excitation-contraction coupling in the heart. In particular, super-resolution and tomographic electron micrographs have revised and refined the classical views of the nanoscale geometries of couplons, t-tubules and the organisation of the principal calcium handling proteins in both healthy and failing hearts. These methods have also allowed the visualisation of some features which were too small to be detected with conventional microscopy tools. With new analytical capabilities such as single-protein mapping, in situ protein quantification, correlative and live cell imaging we are now observing an unprecedented interest in adapting these research tools across the cardiac biophysical research discipline. In this article, we review the depth of the new insights that have been enabled by these tools toward understanding the structure and function of the cardiac couplon. We outline the major challenges that remain in these experiments and emerging avenues of research which will be enabled by these technologies. Read the full text usingthis link
Nanodomains are naturally assembled signaling stations, which facilitate fast and highly regulated signaling within and between cells. Calcium (Ca2+) nanodomains known as junctional membrane complexes (JMCs) transduce fast and highly synchronized intracellular signals, which are required by a variety of cell types. Common to most such nanodomains are clustered assemblies of the principal intracellular Ca2+ release channels, ryanodine eceptors (RyRs). JMCs found in cardiac muscle cells have been studied extensively as self-assembled clusters of RyR. While known to form crystalline arrays in vitro, the organization of RyRs in situ within the JMCs has been less clear. The development of single-molecule localization microscopy (SMLM or super-resolution) optical methods have transformed our ability to visualize and accurately quantify the spatial geometries and sizes of RyR clusters. The recent application of the novel DNA-PAINT super-resolution technology has exploited an unprecedented optical resolution of 10–15 nm to visualize the natural arrays of RyRs within JMCs. In this chapter, we review the key insights into the in situ RyR assembly within cardiac nanodomains that have been gained over the last decade with the utility of super-resolution microscopy and the major considerations in interpreting and validating such image data.
To request a preprint version of the chapter, please contact the authors via Researchgate.
Full text of the chapter can be accessed via this direct link.
Isuru attended the second annual Edinburgh Super Resolution Imaging Consortium (ESRIC) symposium, held this year at the Institute of Genetics and Molecular Medicine (IGMM) of the University of Edinburgh (UoE). His talk on the Molecular-scale imaging of ryanodine receptors at both the cell surfaces and interiors with the adaptation of DNA-PAINT was well-received by a range of researchers based in Edinburgh and regionally in Europe.
Highlights from this meeting included a number of world class investigations led by research fellows and academics in UoE and Heriot Watt University. Of note, were Dr Colin Rickman’s talk on using naturally occurring enzymes as super-resolution imaging probes, Dr Lynn Paterson’s adaptation of optofluidic devices and optical tweezers for developing novel optical tools for cell biology. The plenary speaker was Prof Christophe Zimmer (from Institut Pasteur) who spoke about the adaptation of artificial neuronal networks (a tool called ANNA-PALM) to speed up super-resolution microscopy and demonstrate high throughput imaging of structures such as microtubules, nuclear pore complexes and mitochondria. We now eagerly anticipate his paper on ANNA-PALM out in press very soon.
The conference was organised by Dr Ann Wheeler and colleagues of the ESRIC and showcased their world class line up of microscopy platforms including a state-of-the-art Nikon STORM and SIM instrument and a Leica STED system.
On the back of our recent publication in Cell Reports, our research has enjoyed a wide ranging body of TV coverage. This included 20+ online newspapers and science & technology websites. Among this coverage, was a brief recording for the local television station Made in Leeds which was featured on the 6:30pm news on 11/1/2018.
Here is the link to a clip where Isuru is explaining the context and the value of the super-resolution microscopy technology in studying both healthy and disease physiology of the heart. Featured in the video, was Miriam during one of her imaging experiments.
Recent work of researchers from the Universities of Exeter, Leeds and Cambridge has been featured in a number of press reports.
In particular, the Yorkshire Evening Post refer to the recently published paper in Cell Reports in their statement that “A pioneering new technique could boost the understanding of the causes of heart disease, a study suggests.”
Professor Christian Soeller, based at the Living Systems Institute at the University of Exeter expands upon the pioneering nature of this research, stating: “Slightly more than a decade ago nobody thought that we would ever see individual molecules with light, the resolution just seemed insufficient to resolve such fine detail. Since then an astonishing array of new tricks has been devised. In our latest advance, the use of synthetic DNA has been critical – the deep understanding of the chemistry of DNA we have today makes it an enormously versatile tool.”
Dr Isuru Jayasinghe, at the University of Leeds, has provided context to this research in regard to heart failure, having said that: “This new super-resolution microscopy tool gives us the perfect window to visually examine the individual protein changes within heart cells’ molecular machinery which lead to heart failure. At present, none of the treatments or therapies provided to heart failure patients specifically target the signalling stations – nanodomains – within the cell, which the evidence overwhelmingly suggests are a major cause of heart failure. We believe that by visualising these signalling structures at this level of detail using super-resolution microscopy we can help guide investigations into how we can target or repair these molecular machines and thus, in the long term, help patients to overcome heart disease.”
Here at the University of Leeds, Dr Isuru Jayasinghe’s group is utilising multiple super-resolution microscopy techniques to reveal further the fine structural properties of the heart within health and disease.