QUMOCA · Quantitative Modeling of Calcium Signaling in Vascular Smooth Muscle Nanojunctions

Quantitative Modeling of Calcium Signaling in Vascular Smooth Muscle Nanojunctions

Summary of results and conclusions Research activity carried out by the researcher over the two-year tenure of this Marie Curie International Incoming Fellowship, permitted the elucidation of a basic and important mechanism by which ionic calcium (Ca2+) is communicated from the extra-cellular environment to the endoplasmic reticulum (ER), the principal intra-cellular Ca2+ storing organelle in all mammalian cells. Due to its pivotal role in cell metabolism and communication, the ER with its multitude of functions has recently attracted a high level of attention in biomedical research. One major challenge is to gain insight into mechanisms of inter-organelle communication that is based on specialized membrane contact sites with as yet ill defined nanoscale architecture. The results from this work comprise a complete set of functional measurements to characterize local changes in Ca2+ concentrations ([Ca2+]i) within specialized nanospaces of vascular endothelial cells. The collected data around the above-mentioned mechanism were obtained with refined imaging techniques, were to-date unavailable, and led to the identification of the main channel and transporter proteins involved in the maintenance of cellular Ca2+ homeostasis and function of the ER. By means of careful experiments involving a series of pharmacological tools and genetic manipulation, we were able to characterize features of the temporal changes in the [Ca2+]i, which allowed us clearly to identify the key molecules for highly privileged refilling of ER Ca2+ from the extracellular space. In parallel, an ultrastructural characterization of the intra-cellular peripheral nanoscale environment for the relevant Ca2+ transport was performed, and imaging approaches for quantitative analysis of the involved nanoscale membrane architecture were developed. In the interest of achieving the goals set out in the proposal of generating a clear picture of the ER Ca2+ refilling mechanism and machinery, we opted initially to carry out the bulk of our study in cultured endothelial cells (in particular, we employed the EA.hy926 cell line derived from the human umbilical vein endothelium) and to transfer the acquired knowledge to continue the study in intact tissue at a later stage. During the initial characterization of this vascular endothelial cell line, it was uncovered that the membrane potential of these cells not only was a major determinant of endothelial Ca2+ handling, but was noticeably influenced by common cell culture conditions. Specifically, the use of antibiotics such as amphotericin B was identified to interfere profoundly with cell metabolism and signalling mechanism. Since the membrane potential was identified as a factor that enables fine-tuning of the ER Ca2+ refilling machinery, and since the literature appears devoid of systematic studies on this issue, we decided to delve deeper into the impact of common culture techniques. A local collaboration with Dr. Brigitte Pelzmann’s group, who provided the electrophysiology expertise for accurate measurements of the membrane potential, led to a publication, in which the researcher appears as senior and corresponding author (see Publications section below). Collectively, our findings lead us to infer that plasma membrane (PM)-ER nanojunctions are critical elements of cellular Ca2+ homeostasis and consequently important for metabolic processes causally linked to or associated with ER Ca2+ handling. Having deduced from our functional measurements that these junctions must play a fundamental role, we then went on to observe them in the intra-cellular peripheral architecture, by means of transmission electron microscopy imaging and subsequent quantitative image analysis of the obtained micrographs. The conclusion from these findings is that Ca2+ communication from the outside to the interior of vascular endothelial cells can and does take place via nano-junctional spaces between the cell membrane and the ER. Moreover, this transport appears to be chiefly due to two sets of proteins, the Na+/Ca2+ exchangers and the Orai channels. Highlights of the achieved progress in understanding vascular cell Ca2+ handling are (also see attached graphical abstract): 1. Na+/Ca2+ exchangers and highly Ca2+ selective ion channels of the Orai family collaborate within nanojunctions to maintain ER Ca2+ levels at even minute alterations in global cytosolic Ca2+; 2. The cell membrane potential tunes the individual contribution of nanojunctional Ca2+ transport systems and enables the cell to switch between distinct ER refilling mechanisms; 3. The nanojunctional contact sites between the ER and the plasma membrane are heterogenous and highly dynamic in nature. The collected data, which encompass [Ca2+]i features of the ER Ca2+ refilling process, ultrastructural characterization of PM-ER junctions, along with information from specific scientific literature, has generated a solid basis for the development of quantitative models leading to visualization of the junctional ionic transients, as yet inaccessible by microscopy. These models will enable invaluable insights into key mechanisms of human pathophysiology, specifically into processes leading to ER stress and tissue dysfunction. During Q4 of the 2nd year, we started investigation of this Ca2+ refilling mechanism in intact endothelial tissue by investigating the spatio-temporal features of [Ca2+ ]i in the inferior vena cava of mice. Preliminary data on this tissue have been collected and analyzed and, while still underway, these experiments have on the one hand confirmed findings obtained in vascular cell culture and, on the other, yielded novel insights on Ca2+ signaling potentially peculiar to the vascular endothelium. The findings from this most recent part of the study will be the basis of a follow-up study and funding application (to the Austrian National Research fund FWF, and to the local consortium BioTechMed) with focus on vascular PM-ER Ca2+ communication at the level of multicellular systems with the researcher as the PI. The project activities have prepared the ground for formation of a local research network and fund raising activities to continue research in the field of nanoscale inter-membrane communication. Please, also see attached documents for: Graphical abstract (referred to in the Summary text) Project logo (referred to in the Summary text)

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