Contribution of TRPV4 Channels in the Intrinsic Calcium Signaling Profiles of the Mouse Lung Microvascular Endothelium
Authors: Sen Pallavi , Choi Chung-Sik , Taylor Mark 
Published: May 2023

Endothelial calcium signaling in lung microvasculature is known to play an important role in maintaining lung endothelial barrier integrity. We previously characterized an intrinsic dynamic endothelial Ca2+ signaling pattern of the mouse lung microcirculation. Recently, the transient receptor potential vanilloid 4 (TRPV4), a non-selective calcium permeant cation channel has been implicated in various lung pathologies involving edema and inflammation. While acute activation of these stretch and temperature sensitive plasma membrane channels have caused barrier disruption, their underlying contribution to intrinsic microvascular endothelial Ca2+ signaling profiles under different physical forces or temperatures is unknown. In this study, we assess the role of TRPV4 channels in inherent Ca2+ signaling dynamics of the pulmonary microcirculation under different conditions of stretch and temperature. Lung slices isolated from C57BL/6 mice were loaded with GFP-based Ca2+ indicator Cal-520AM and imaged using confocal microscopy. The slices were left unstretched or radially stretched to 2X their resting area and assessed for signaling events at 25°C, 37°C and 40°C. After baseline recording, these slices were treated with TRPV4 channel antagonist GSK2193874 (300nM) to examine the reduction of the signaling events contributed by the channel. All of these slices were then fixed and immunostained to track TRPV4 channel expression and distribution. The recorded image sequences were analyzed using ImageJ and our custom signal tracking algorithm S8, for quantification of Ca2+ event parameters of frequency, amplitude, duration and spread. Immunofluorescence staining of TRPV4 channels showed positive expression consistently with the endothelium. The stretched lung slices themselves showed lesser duration but slightly higher spread and frequency of events as opposed to unstretched lung slices under basal conditions. The stretched slices subjected to 40°C showed higher values of event amplitude, duration and spread, compared to their counterparts’ basal dynamics recorded at 25°C and 37°C. Addition of TRPV4 antagonist diminished event amplitude, duration and spread of events for stretched lung slices at 40°C to a greater extent than the ones subjected to room temperature and 37°C. Unstretched slices did not seem to show any relative change in their basal dynamics at elevated temperatures as well as when subjected to the channel inhibitor.In summary, we found more contribution of TRPV4 channel mediated Ca2+ signaling events when lung slices were subjected to stretch and raised temperature, as observed with channel blocker. We conclude that these channels contribute minimally to basal dynamics under normal conditions and are activated to a relatively higher extent in a distinct way with the stimulations. NIH P01 HL066299, R01 HL155288, NIH S10 0D020149 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.