Vitamin D Receptor Deficiency Upregulates Pulmonary Artery Kv7 Channel Activity

Figures

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  Figure 1. Right ventricular hypertrophy and pulmonary vascular function in Vdr−/− mice. (A) Representative genotyping of WT, Vdr−/−, and Vdr+/− mice. Vdr−/− animals have an insert in the Vdr gene that increases the base pairs, resulting in amplification of the gene. (B) Fulton index (RV/(LV+S)). (C) Maximum PA contraction to KCl. (D–G) Relaxant responses in WT, Vdr+/−, and Vdr−/− PAs to (D) acetylcholine, (E) sodium nitroprusside, and (F,G) retigabine. Panel F shows an original representative trace of the retigabine dose–response curve. Results are means ± standard error of the mean (n = 9 WT, 9 Vdr−/− and 4 Vdr+/−). * p < 0.05, ** p < 0.01 vs. WT using a two-way ANOVA test followed by a Bonferroni post hoc test.

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  Figure 2. Kv7 current in Vdr−/− pulmonary artery smooth muscle cells (PASMCs). (A,B) K+ current–voltage relationships measured before and after the addition of XE991 (10 µmol/L). Insets show representative current traces measured at +20 mV in the absence and presence of XE991 in (A) WT and (B) Vdr−/− PASMCs. (C) XE991-sensitive current obtained by subtracting the K+ current in the presence of XE991 from the current in the absence of the drug. Insets show representative XE991-sensitive current traces measured at +20 mV. Results are means ± standard error of the mean (n = 4 animals and 8 cells per group). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT using a two-way ANOVA test followed by a Bonferroni post hoc test.

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  Figure 3. Lung histology. (A) Representative lung section images stained with hematoxylin and eosin and examined by light microscopy; the insets show elastin visualized by its green autofluorescence. (B) Percentage of muscular (black), partially muscular (grey), and non-muscular (white) vessels (n = 4 mice per group, 1–5 vessels analyzed per mouse). (C,D) PA medial wall thickness of 75–250 µm and <75 µm, respectively.

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  Figure 4. KCNE4 vitamin D response element (VDRE) and Kv7 channel expression in Vdr knockout mice. (A) Predicted VDRE in the promoter region of the KCNE4 gene conserved between humans and mice. (B) Vitamin D receptor (VDR) and retinoid X receptor (RXR) protein interface signatures and the estimated binding specificities in the VDRE of the KCNE4 gene as sequence logo provided by 3D-footprint. Larger letters indicate greater affinity, and smaller letters indicate the allowable variability. (C–E) Averaged densitometric protein expression of (C) Kv7.1, (D) Kv7.4, and (E) KCNE4. Results are means ± standard error of the mean (n = 6–7 animals). * p < 0.05 vs. WT using a t-test. Original Western Blotting data can be found in Figure S1.