Dantrolene inhibits lysophosphatidylcholine-induced valve interstitial cell calcific nodule formation via blockade of the ryanodine receptor

Figures

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  Figure 1. Experimental overview. Valve interstitial cells were sterilely dissected from porcine (Sus scrofa) aortic valves. Experiments were divided into either short or long time courses. For short time course experiments, cells were treated with dantrolene or DMSO vehicle overnight and then exposed to LPC or ethanol vehicle for less than 24 h the next day. For long time course experiments cells were treated with dantrolene or DMSO vehicle and LPC or ethanol vehicle at the same time. Media were changed every 3 days. Created with BioRender.com.

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  Figure 2. Dantrolene allows for nontoxic inhibition of calcium signaling in porcine aortic valve interstitial cells. (A) An alamarBlue serial dilution fitted to four parameter logistic regression calculates the cellular median lethal dose of dantrolene at 297.8 μM. (n = 8 per point; data are representative of two independent experiments) (B) Dantrolene reduces increased cellular metabolism due to LPC. (Dead group was treated with 10% EtOH; n = 12 per group) (C–E) Live/Dead staining confirms the results of the alamarBlue assay in (A). (F) Dantrolene inhibits LPC-induced calcium flux. (n = 6–18 per group) (G) Representative curves from (F). (*indicates P < 0.05 via Tukey's test between the indicated groups; **indicates P < 0.05 between all the indicated comparisons by Tukey's test; scale bar is 100 μM; Dan, dantrolene; LPC, α-lysophosphatidylcholine; RFU, relative fluorescent units; DMSO, dimethyl sulfoxide; EtOH, ethanol).

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  Figure 3. Dantrolene reduces in vitro calcific nodule formation by paVICs due to LPC and OM. Dantrolene reduces total calcified area (A), nodule number per well (B), and average nodule surface area (C). (D) Vehicle-only control (0.1% ethanol and 0.6% DMSO). (E–L) Red-yellow stain is alizarin red. (E) LPC in DMSO-treated paVICs. 10 μM LPC with (F) 60 μM, (G) 30 μM, and (H) 10 μM dantrolene. (I) OM in DMSO-treated paVICs. (J) 60 μM and (K) 30 μM dantrolene with OM. (L) Breakthrough nodules can be seen in 10 μM dantrolene with OM. (n = 4 per group for all experiments in this figure; (C) shows each nodule as a point; * indicated P < 0.05 from DMSO-treated control via Dunnett's test; arrows indicate areas of calcification; scale bar is 100 μM; paVICs, porcine aortic valve interstitial cells; LPC, α-lysophosphatidylcholine; OM, osteogenic medium).

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  Figure 4. Dantrolene induces a number of phenotypical changes compared to cells treated with LPC. (A) Dantrolene inhibits LPC-mediated apoptosis. (n = 8–12 per group) (B) Dantrolene has no effect on LPC-induced ROS (n = 6 per group; **indicates significances from untreated control in dantrolene-treated [blue] group; # indicates significance reached DMSO-treated [red] group) but (C) inhibits ROS due to tert-butyl hydroperoxide. (n = 3 per group) (D) representative fluorescent intensity histograms from (B). (E,H) Dantrolene inhibits ACTA2 expression at 1 and 3 days compared to LPC. (F,I) Dantrolene upregulates RUNX2 expression in paVICs. (G,J) Dantrolene upregulates SOX9 expression in paVICs. (n = 6 for day 1 timepoints and n = 3 for day 3 timepoints; *indicates P < 0.05 via Tukey's test between the indicated groups; paVIC, porcine aortic valve interstitial cell; LPC, α-lysophosphatidylcholine; RLU, relative luminescence units; ROS, reactive oxygen species; Dan, dantrolene; DMSO, dimethyl sulfoxide; EtOH, ethanol; TBP, tert-butyl hydroperoxide).

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  Figure 5. Dantrolene induces cytoprotective changes in paVICs compared to LPC. (A) Principal component analysis shows grouping of the vehicle, LPC controls, and dantrolene-treated replicates. (B) Sample distance heat map. (C) Dantrolene and LPC have commonly and uniquely upregulated genes. (D) Dantrolene and LPC alone do not share any downregulated genes. (E–G) Volcano plots of differentially regulated genes between LPC, dantrolene, and combination treatments. (H) Heat map of the top 20 differentially regulated genes. (n = 3 per condition; paVIC, porcine aortic valve interstitial cell; LPC, α-lysophosphatidylcholine).

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  Figure 6. KEGG enrichment analysis of (A) upregulated and (B) downregulated genes. (x-axis shows number of genes differentially regulated; LPC, α-lysophosphatidylcholine, Dan, dantrolene).

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  Figure 7. Dantrolene prevents paVIC calcific nodule formation due to LPC. Dantrolene inhibits RyR3-mediated calcium flux due to LPC and reduces LPC-associated apoptosis, SMA transcription, and progressions of paVICs to calcific nodules in vitro. Of note, it does not prevent LPC-associated ROS. Further, dantrolene causes global gene transcription changes relating to inflammation, cell motility, and cell metabolism. Created with BioRender.com. (paVIC, porcine aortic valve interstitial cell; LPC, α-lysophosphatidylcholine; RyR, ryanodine receptor; SMA, smooth muscle actin).