Antagonism of TRPV4 channels decreases mechanically activated current in rat small dorsal root ganglion neurons
Authors: Fukazawa Ayumi , Hori Amane , Hotta Norio , Katanosaka Kimiaki , Estrada Juan , Ishizawa Rie , Kim Han-Kyul , Iwamoto Gary , Smith Scott , Vongpatanasin Wanpen , Mizuno Masaki 
Published: May 2023

A mechanical stimulus to skeletal muscle during exercise activates the sympathetic nervous system via the skeletal muscle mechanoreflex. However, to date, the receptor mechanisms responsible for mechanotransduction in skeletal muscle afferents have not been fully identified. Transient receptor potential vanilloid 4 (TRPV4), a multimodally activated, nonselective cation channel, is sensitive to mechanical stimuli such as shear stress, osmotic pressure and stretch in various organs. Therefore, it is hypothesized that TRPV4 contributes to mechanotransduction in thin-fiber primary afferents innervating skeletal muscle. Purpose: The aim of this investigation was to determine 1) the extent to which TRPV4 and the afferent marker peripherin are co-localized in dorsal root ganglion (DRG) neurons subserving thin-fiber skeletal muscle afferents using a retrograde tracing technique, and 2) the impact of a selective TRPV4 antagonist, HC067047 on mechanotransduction in small DRG neurons. Methods: To label muscle afferent DRG neurons, dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate, (Dil, 60 mg/mL, 3 μL total) was injected into the white portion of the gastrocnemius muscle. Five days after the injection, L4-L6 DRGs were harvested from rats (Sprague-Dawley (SD), n = 3, body weight: 318.0 ± 8.7 g). The number of positively stained small-diameter DRG neurons (i.e. less than 30 μm in diameter) were counted and the percentage of peripherin- or DiI-positive neurons in TRPV4-positive neurons was calculated. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons (SD, n = 10, body weight: 352.1 ± 52.4 g), mechanically-activated (MA) current amplitude and mechanical threshold were measured before and 5 min after the application of either HEPES-buffered solution as a control or 1 μM HC067047 solution. Results: Fluorescence immunostaining demonstrated that 20.1 ± 10.1% of TRPV4 positive neurons were expressed with Dil-labeled small DRG neurons. Of these, 9.5 ± 6.1% and 10.6 ± 7.2% were peripherin-positive and -negative neurons, respectively. In in vitro whole-cell patch clamp recordings, MA current amplitude was significantly decreased 5 min after the application of HC067047 ( P < 0.01), while mechanical threshold did not change. Conclusion: The data demonstrates that 1) TRPV4 is expressed in DRGs innervating group IV muscle afferent C-fibers, and 2) HC067047 suppresses the response to mechanical stimulation in DRG neurons. These findings suggest that TRPV4 channels play an important role as mechanoreceptors contributing to mechanotransduction in thin muscle afferents. Supported by NIH HL-151632, JSPS 21J20070 & 22KK0154, and UMF 202131013 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.