Poster L20 - Pulling at the Heartstrings: The Role of Mechanically-Activated Piezo1/2 Channels in Vascular Smooth Muscle Cell Mechanics

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Eleanor L. McArthur

Amgen Scholar Summer Intern
Washington University in St. Louis
St. Louis, Missouri, United States

Co-Author(s)

Stephen Wheat

Intern
Washington University in St. Loius, United States

Last Author(s)

GE

Garrett Easson

Post Doctoral Researcher
Washington University in St. Louis, United States

Primary Investigator(s)

Jessica Wagenseil, DSc (she/her/hers)

Principal Investigator
Washington University in St Louis
St. Louis, Missouri, United States

Introduction: Supravalvular aortic stenosis (SVAS) affects 1 in every 120,000 live births, and currently, surgical intervention is the only treatment for this vascular disease [1]. SVAS is the narrowing of the ascending aorta above the aortic valve that limits blood flow to the body. It is characterized by decreased elastin levels and increased vascular smooth muscle cell (VSMC) proliferation [2] that causes the thickening and increased rigidity of the aortic wall. This phenotype change in the aorta causes increased strain in VSMCs.
Previous single-cell RNA-seq findings [5] in an SVAS mouse model identified the mechanically-activated ion channel Piezo2 as upregulated in VSMCs. Piezo2 and Piezo1 potentially have a role in VSMC mechanotransduction [3]. Once Piezo1/2 have been activated due to strain, calcium enters the cell and causes calcium signaling pathways to occur. The proposed downstream targets from Piezo1/2 calcium signaling include connective tissue growth factor (CTGF) and matrix metalloproteinase-3 (MMP3). Ctgf overexpression is indicated in VSMC proliferation and migration, as well as increased collagen expression [4], while MMP3 production suggests extracellular matrix (ECM) remodeling.
To investigate the effects of Piezo1/2 activation and downstream signaling, we induced strain in VSMCs through hypo-osmotic conditions that caused the cells to mildly swell. We hypothesized that increased strain on VSMCs will drive the Piezo1/2 pathway and inhibition of both channels will diminish the biological response to strain.

Materials and

Methods: Live calcium staining on VSMCs occurred using Fluo4-AM and Fura-Red AM calcium indicators to image cell response to different conditions. Cells with low intracellular calcium appear red, while cells with high intracellular calcium appear green. Cells were imaged with a confocal microscope. Baseline cells were imaged for 1 minute and treated cells for 10 minutes.
Cells were compared under iso-osmotic and then hypo-osmotic conditions. In a separate experiment, cells with imaged in iso- and hypo-osmotic conditions with and without Piezo2 inhibitor, D-GsMTx4. Iso-osmotic conditions were 300 mOsm and hypo-osmotic conditions were 100 mOsm.

Downstream signaling pathways were interrogated by long-term osmotic stimulation of the cells. MOVAS cells were grown to confluency in a 12-well plate and then treated with different osmotic/inhibitor conditions over 3 days for 1 hour per day. Four different trials were performed: Iso-osmotic/hypo-osmotic, Piezo1 inhibitor (GsMTx4), Piezo2 inhibitor (D-GsMTx4), and both Piezo1/2 inhibitors. Inhibitors were added at concentrations of 500nM. Twenty-four hours after the third day of conditions, cells were collected. Ctgf and Mmp3 expression were measured using qPCR. Expression was normalized to the housekeeping gene, Gapdh, and fold change was calculated by the delta delta Ct method.

Results, Conclusions, and Discussions: Confocal calcium imaging demonstrated that hypo-osmotic conditions initiate a calcium response in VSMCs. There was a diminished calcium response in the cells treated with Piezo2 inhibitor compared to the control. These results implicate that Piezo2 is involved in mediating calcium response in VSMCs (Figure 1).

qPCR results were not statistically different between groups for Ctgf expression (Figure 2). Although not statistically significant, Piezo2-inhibited cells appear to have decreased Ctgf expression compared to the cells treated with vehicle as well as the cells treated with Piezo1 inhibitor. Future studies should investigate the connection between Piezo2 inhibition and decreased Ctgf expression by increasing the sample size of this study.
qPCR results indicate increased expression of Mmp3 with Piezo1 inhibition (Figure 3). It appears that Mmp3 expression is largely Piezo1-mediated.
Additionally, osmolarity did not have a significant impact on the expression of Mmp3, except when both Piezo1/2 were inhibited. When Piezo1/2 were inhibited, Mmp3 expression increased in iso-osmotic conditions and decreased in hypo-osmotic conditions. Although significant differences were found, two samples from the Mmp3 expression experiment were excluded as outliers due to abnormally high values when calculated using the delta delta Ct method. Future work could increase the sample size and ensure repeatability of the results.

The implications of this study are essential in understanding the Piezo1/2 pathway and its role in SVAS. The diminished calcium response observed with Piezo2 inhibition demonstrates the importance of further investigation. While the qPCR results for Ctgf expression were not statistically significant, further research is needed to study the impact of Piezo2 inhibition on Ctgf expression. Likewise, a larger sample size is needed to understand Mmp3 expression in iso-osmotic and hypo-osmotic conditions. These results are important in determining the downstream pathways of VSMCs’ response to strain, which will be essential to discovering non-surgical treatments for SVAS.

Acknowledgements (Optional): Thank you to all the members of the Wagenseil Vascular Mechanics Laboratory for all their guidance and assistance with this project.
Funding provided by the National Institutes of Health (NIH Grant RO1HL166448).

References:
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2. Urbán Z, Riazi S, Seidl TL, Katahira J, Smoot LB, Chitayat D, Boyd CD, Hinek A. Connection between elastin haploinsufficiency and increased cell proliferation in patients with supravalvular aortic stenosis and Williams-Beuren syndrome. Am J Hum Genet. 2002 Jul;71(1):30-44. doi: 10.1086/341035. Epub 2002 May 6. PMID: 12016585; PMCID: PMC384991.
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