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Probing local ROS distributions in Calcium signaling microdomains after myocardial infarction

Periodic Reporting for period 1 - MICROS (Probing local ROS distributions in Calcium signaling microdomains after myocardial infarction)

Reporting period: 2017-07-18 to 2019-07-17

Context of the project: Heart and vessel diseases remain the major killer worldwide. Heart disease causes suffering and disability. It also has an important societal cost. The most common cause is disease of the blood supply to the heart causing a heart attack and a myocardial infarction (MI). After a MI, the injured heart adapts but is unable to maintain sufficient pump function to provide the necessary blood supply to the body. At the level of the heart muscle cells, this loss of pump function comes with deregulation of the calcium levels, which are the signal for contraction. The interplay of calcium and other signaling pathways, notably reactive oxygen species (ROS), is still unclear to the scientific and medical community. Insights into this signaling could contribute to the design of new strategies for treatment.
Objectives: We proposed to develop a novel method for the study of local calcium release and its regulation through redox signaling in heart muscle cells. This highly challenging project had as a major conjoint objective to build new skills and competences for the researcher.
For the project, we had to measure the local calcium signals within heart muscle cells and measure redox signals. This required extensive methodological development. The calcium signals occur in very small places within the cells (microdomains smaller than 2 micrometer) and are very brief (< 50 ms upstroke). We used different approaches and followed up on opportunities.

(1) Calcium signaling. To enable tracking of this fast and local calcium signaling we developed and optimized a dual-camera based confocal microscopy device. By simultaneously tracking the cell motion, we can map cell-signaling parameters during the full contraction cycle. A collaboration within the host institution led to development of original methods for unbiased analysis. The combination of fast confocal imaging and the novel advanced analysis algorithms allowed us to study localized calcium events in cardiac myocytes, in relation to the cell structure and the organization of phosphorylation. The fellow is preparing a manuscript to report on the latter findings. Importantly, she could apply her novel imaging algorithms for cell identification in the study of tissues from heart disease. Her approach and work with the team contributed to major advances in unbiased image analysis in several studies (Sci Rep, 2019; J Am Coll Cardiol, 2019)

(2) ROS. We used rare-earth doped nanoparticles, which the fellow had developed in previous works, as quantitative ROS sensors. We could show a proof of concept of their membrane localization on the cardiac cells. These ROS sensors have a very narrow absorption band (at 396±5 nm) and require a specific laser light illumination, which we implemented. The optical design of the available confocal system was however not fully compatible with this new light path, preventing the desired combined imaging of the calcium and ROS signals. As a contingency measure, the fellow eventually tested a 2-photon illumination strategy, which is unprecedented for these probes. The preliminary results confirmed that the multiphoton approach is a promising alternative for the continuation of this project.

(3) Skills and competences. The project has challenged the methodological limits of the imaging system and the ER has invested considerable time in developing novel approaches. Throughout this process, she had the possibility to improve on her experimental design, quality of collected data and analysis methods, working across disciplines in biophysics and imaging. She has built experience in communication and through presenting her work, participation in conferences and workshop, built her network. She has submitted an application for a next postdoctoral position where she can use her acquired skills and experience.
The project established an innovative method in the field of cellular cardiac physiology: simultaneous imaging of fast calcium signaling (125 frame per second) and tracking of cell membrane motion. The researcher developed original computational methods to process the images and analyze sub-cellular signaling in cardiac myocytes. In a future publication, the researcher will report on the method and make the algorithms freely available to the scientific community.
These novel methods have provided insight into the interplay between cell structure and organization of phosphorylation for calcium handling and myocyte physiology. In a long-term perspective the work can open new avenues for more local and targeted drug design.
Mapping calcium handling in relation to cardiac myocyte cell structure
Cell structure and calcium signaling microdomain in human cardiac myocytes