Reevaluation of Piezo1 as a gut RNA sensor

Piezo1 is a stretch-gated ion channel required for mechanosensation in many organ systems. Recent findings point to a new role for Piezo1 in the gut, suggesting that it is a sensor of microbial single-stranded RNA (ssRNA) rather than mechanical force. If true, this would redefine the scope of Piezo biology. Here, we sought to replicate the central finding that fecal ssRNA is a natural agonist of Piezo1. While we observe that fecal extracts and ssRNA can stimulate calcium influx in certain cell lines, this response is independent of Piezo1. Additionally, sterilized dietary extracts devoid of gut biome RNA show similar cell line-specific stimulatory activity to fecal extracts. Together, our data highlight potential confounds inherent to gut-derived extracts, exclude Piezo1 as a receptor for ssRNA in the gut, and support a dedicated role for Piezo channels in mechanosensing.

For measuring Piezo1 activity during exposure to ssRNA40, we performed in 94 vitro fluorescent calcium imaging on N2a cell cultures with the Fluo-4 AM ester dye. 95 Using ssRNA40 from the same supplier and dosage as prior studies (10 µg/mL) 96 (Sugisawa et al., 2020), we found that application of ssRNA40 did not elicit a detectable 97 increase in fluorescence, even when the imaging time frame was extended up to 3 98 minutes ( Figure 1A and Video S1). As a positive control for Piezo1 activation, we used 99 Yoda1, which is known to induced Piezo1-dependent calcium transients (Syeda et al., mechanosensitive flow responses, and we tested ssRNA41 to examine sequence-105 specific effects of ssRNA ( Figure 1A). ssRNA41 is the same length and sequence as 106 ssRNA40, except all uridine residues are substituted with adenosine; unlike ssRNA40, 107 this molecule is not a TLR7/8 agonist (Heil et al., 2004). After autofluorescence 108 subtraction, there was no significant difference in N2a cell calcium activity between 109 these negative controls and ssRNA40 ( Figure 1C). 110 In addition to directly activating Piezo1, ssRNA40 was reported to delay the 111 inactivation of Piezo1 mechanically evoked currents (Sugisawa et al., 2020), similar to 112 Yoda1 (Syeda et al., 2015). We tested this claim by whole-cell voltage-clamp recordings 113 of N2a cells during simultaneous mechanical stimulation of the plasma membrane. 114 Mechanical stimuli were administered with a nanomotor probe to indent the cell surface 115 in 1 µm increments (Video S2). The stimulation elicited a rapidly inactivating inward 116 current, which is characteristic to the Piezo family of ion channels ( Figure 1D). As  Figures 1D and 1E). By contrast, 10 µg/mL ssRNA40 in the bath solution 120 showed no measurable change from the vehicle control on the amplitude, activation 121 threshold, or inactivation rate of mechanically evoked currents ( Figures 1D and 1E). represented on a fire color scale and is superimposed on a grayscale baseline fluorescence image. Cells 130 were exposed to buffer only (vehicle) or 10 µg/mL ssRNA40 or ssRNA41 for up to 3 minutes, followed by 131 30 µM Yoda1 and 10 µM ionomycin. Scale bar is 200 µm.

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(B) Example calcium imaging traces of ssRNA40 and ssRNA41, each followed by Yoda1 and ionomycin 133 control treatments. Only cells that responded to Yoda1 (functionally expressing Piezo1) were analyzed.

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(B) Example calcium imaging traces of Piezo1-transfected HEK293 cells during different treatments.

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Yoda1 was applied 90 seconds after any given RNA sample, and only cells that responded to Yoda1

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(D) Dose-response curve of ssRNA40 treatment on Piezo1-transfected GCaMP6s-expressing HEK293 184 cells. After 1 minute of baseline measurement, ssRNA40 was administered for 3 minutes followed by 185 ionomycin for 1 minute. A random selection of cells was analyzed from each recording. The responses 186 are normalized, with the ionomycin response being ΔF/F 0 = 1. n = 25 cells per dose plotted as mean ± 187 95% CI. One-way ANOVA with Bonferroni correction: n.s. p ≥ 0.05.

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See also Figure S1.     However, we found that the autofluorescence was mostly eliminated if the feces were 234 diluted to at least 5 mg/mL -we refer to this diluted filtered sample as "fecal extract".

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Applying fecal extract to Piezo1-transfected HEK293 cells triggered a substantial 236 calcium response ( Figure 3A and Video S3). More dilute preparations of extract yielded 237 little or no calcium influx. Interestingly, across several imaging trials, we noticed that the 238 response to 5 mg/mL fecal extract was variable; not all cells in the field of view would 239 necessarily respond, and the response onset was often 30 -60 seconds after the fecal 240 extract was first added (Figures 3A and 3B). Notably, however, we also found that

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Treating the samples with RNase A eliminated the low molecular weight nucleic acid (< 500 bp). The 289 crude fecal extract additionally had a high molecular weight smear (500 -5,000 bp) that was unaffected 290 by RNase A treatment, which is likely DNA.     Calcium response to fecal and dietary extracts is cell line-specific 343 We were concerned about the potential confounding effect of fecal/dietary   (Figures 6B and 6C). Notably, gadolinium did not 396 27 diminish the response to fecal RNA, but it seemed to have a nonspecific effect on the 397 decay rate of the calcium signal ( Figures 6E and 6F). Gadolinium completely abolished 398 the Yoda1 response, confirming successful Piezo1 inhibition (Figures 6H and 6I). 399 Together, these experiments demonstrate that while RIN14B cells do respond to a 400 variety of ssRNAs, their response is not dependent on Piezo1.   (Table S1). We anticipate these data will be 421 useful for more deeply assessing the fidelity of the RIN14B line as a model of gut    In this study, we set out to confirm the discovery that Piezo1 is a sensor of fecal 459 microbiome ssRNA. We were not able to detect ssRNA-evoked changes in Piezo1        control Ringer's solution that was tested had an osmolality of ~336 mmol/kg and 7.5 pH.

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In comparison, fecal extract was ~332 mmol/kg and 7.5 pH, and dietary extract was 756 ~334 mmol/kg and 7.5 pH. These measurements indicate that the extracts did not 757 substantially affect the osmolality or pH of the solutions. which was the same concentration used in the Sugisawa study ( Figure 3D).

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Additionally, fecal RNA was purified using the same methodology and