TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

Reviewer #1 (Public review):

Summary:

This comprehensive study employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure regulation in a mouse model of TGFβ2 -induced ocular hypertension. TGFβ2 upregulated expression of TRPV4 and PIEZO1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 activation induced TM contractility whereas pharmacological inhibition suppressed TGFβ2-induced hypercontractility and abrogated ocular hypertension in eyes overexpressing TGFβ2. Trpv4-/- mice resisted TGFβ2-driven increases in IOP. These data establish a fundamental role of TGFβ as a modulator of mechanosensing and identifies TRPV4 channel as a common mechanism for TM contractility and pathological ocular hypertension.

Strengths:

The manuscript is very well written and details the important function of TRPV4 in TM cell function. These data provide novel therapeutic targets and potential for disease-altering therapeutics.

Weaknesses:

The experimental rigor and design of the noctural IOP experiments was weak with low n values and differing methods of IOP measurement (conscious versus anesthetized). The same method of IOP measurement needs to be used for all measurements to make any conclusions on the circadian patterns of IOP in each condition.

Reviewer #2 (Public review):

The manuscript by Christopher N. Rudzitis et al. describes the role of TGFβ2 in the transcription and functional expression of mechanosensitive channel isoforms, alongside studies on TM contractility in biomimetic hydrogels and intraocular pressure. Overall, it is a very interesting study, nicely designed, and will contribute to the available literature on TRPV4 sensitivity to mechanical forces.

I have the following comment for the authors to address.

Figure 1A-C.
Often there is a difference between the massage and transcript data. I recommend the authors to confirm with qPCR data with another mode of protein measurements.
Does direct TRPV4 activation also induce the expression of these markers? Does inhibition of TRPV4, after TGF-β treatment, prevent the expression of these markers? Is TRPV4 acting downstream of this response?

Figure 1D. Beta tubulin is not a membrane marker. Having staining of b tubulin in membrane fraction shows contamination from the cytoplasm.
Does the overall expression also increase?

Figure 4A: it is not very clear. I recommend including a zoom image or better resolution image.

Figure 5B and 6B.
Why there is a difference between groups in pre-injection panel. As Figure 5A, in pre-injection, there is no difference between LV-TGFβ and LV-control while in 5B there is a significant difference between these groups.
Discussion section.

Line 279, . "TRPV4 channels in cells treated with TGFβ2 are likely to be constitutively active" ... needs to be discussed further.

Line 280: "The residual contractility in HC-06-treated cells may reflect TGFβ2-mediated contributions from Piezo1."
Piezo1 has a low threshold for mechanosensitivity. How do the authors discuss the observation that, in the presence of Piezo1, TRPV4 has a more prominent mechanosensory function? Is this tied to TGFβ signalling?

Author response:

We thank the editors and reviewers for the constructive assessment. We plan to address the comments as follows:

Reviewer #1 (Public review):

We are generating a new cohort of Lv-TGFB2 overexpressing mice in which IOP will be compared under the anesthesia conditions that are identical for diurnal and nocturnal states. Parenthetically, we used the awake (diurnal) and isoflurane (nocturnal) anesthesia to mirror the conditions in the Patel et al (2021) PNAS study.

Reviewer #2 (Public review):

We are not sure what the Reviewer means by the “difference between the message and transcript data” and are not sure whether providing evidence about the TRPV4-dependence of the expression of fibrotic genes and canonical TGFb2 pathway genes fits within the scope of our study (which focuses on the TGFB2-dependence of TRPV4 expression and IOP regulation). We propose to address this by including new data about the TGFb2- and TRPV4 dependence of TRPV4 and Piezo1 expression. We could include information about the effect of TGFB2 on fibrosis-related genes from a (submitted study) in which we used RNASeq to investigate TGFB2 and TGFB2 + HC067047-dependence of gene expression in TM cells on a confidential basis but not include it in the revised manuscript.

- Re: b-tubulin comment [b-tubulin associates with the plasma membrane by binding to integral membrane proteins in the plasma and organellar membranes, through palmitoylation and attachment to linker proteins and as an integral component of exocytotic vesicles (Wolff, BBA 2009; Hogerheide et al., PNAS 2017). Together with b-actin and Gapdh it is often used as a loading control to assess cellular TRPV4 protein expression (e.g., https://www.cellsignal.com/products/primary-antibodies/trpv4-antibody/65893; Grove et al., Science Signaling 2019 and Moore et al., PNAS 2013). Our qPCR and RNASeq studies show that TGFB2 does not affect b-tubulin expression]

- We will provide a higher resolution image for Fig. 4A

- Will address the Fig 5A and 6A comment [We thank the Reviewer for noticing the ambiguity and revised Figure Legends to clarify that “pre-injection” in Figures 5B and 6B refers to IOP measurements before the intracameral injection of HC-06 not pre-injection of lentiviral constructs].

- We will address the issue of constitutive TRPV4 activity and Piezo1 involvement in the revised Discussion.

We hope this is sufficient information at this point but would be more than happy to provide more information if needed.

Thank you, we are very impressed by the eLife review protocols.