Quantification of Trpm7 channel protein expressions in the carotid body from lean and obese mice

RATIONALE: The carotid body (CB), a small organ located in the neck, which plays a significant role in sensing hypoxia and regulating the sympathetic nervous system, Overactivation of CB can contribute to increased sympathetic activity often observed in obesity, leading to potential complications like hypertension and metabolic dysfunction. We have shown high levels of expression of Trpm7 mRNA in the CB of diet induced obese (DIO) mice, which can potentially lead to augmented CB chemoreflex. We have also shown that Trpm7 expression in CB is regulated by leptin and that TRPM7 blockade treats hypertension in DIO. In the present study we have examined and quantified the Trpm7 channel expression in the CBs at the protein level in lean and DIO mice, using the immunofluorescence intensity signal in IMARIS. Methods: Carotid artery bifurcations with the CBs were isolated from lean and DIO mice and standard procedures were followed to prepare the CB sections (15 micron) for immunofluorescence. In this study, following primary antibodies were used; (a) to label CB type I cells - chicken anti-tyrosine hydroxylase (TH), (b) to label TRPM7 channels/receptors - rabbit anti-TRPM7, and (c) to label CB type II cells - rat anti-glial fibrillary acidic protein (GFAP). Fluorescent images were captured with a Zeiss Spinning disk confocal microscope and all images were taken using the same scanning parameters. All images were taken at 40x oil-immersion objective and 4.56 μm interval with 20-slice z-stack. Images were saved in the “czi” format, and processed in ZEN blue software and TRPM7 intensity was analyzed in Imaris 10.2 software. We quantified the fluorescence intensity of TRPM7 expression using a surface object which was created in the 3D view to define the region of interest (ROI) and normalized the TRPM7 intensity with total type I cell TH and type II cell GFAP intensities in the CB. CBs from C57BL/6J lean (n=2) and DIO (n=3) male mice were used and minimum of three images were analyzed per mouse. Result: Immunofluorescence data confirmed the presence of TRPM7 channel protein expressions in the CBs of both lean and DIO mice. Quantitative analysis of fluorescence intensity indicated noticeable and significant increase of TRPM7 channel expression in the CBs of DIO compared to lean mice. Conclusion: Overall, the study supported our previous observation of the Trpm7 channel mRNA expression. TRPM7 signaling in the CB could be a target for the treatment of obesity induced hypertension. Grant support: NIH R01 HL133100 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.