Aging Affects KV7 Channels and Perivascular Adipose Tissue-Mediated Vascular Tone

Figures

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  Figure 1. Effects of aging on regulation of arterial tone by a1-agonists Methoxamine (ME) and perivascular adipose tissue (PVAT). (A) Original traces showing a1-agonist-induced contractions in (−) PVAT and (+) PVAT mesenteric artery rings isolated from young mice. (B) Concentration-response relationships for a1-agonist-induced contractions in (−) PVAT (n=10, N=5) or (+) PVAT (n=10, N=4) mesenteric arteries from young animals. (C) Original traces showing aging effects on a1-agonist-induced contractions in (−) PVAT and (+) PVAT mesenteric artery rings isolated from 12-month-old mice. (D) Concentration-response relationships for a1-agonist-induced contractions of (+) PVAT (n=6, N=2) and (−) PVAT (n=9, N=2) artery rings isolated from 12-month-old mice. (E) Original traces showing aging effects on a1-agonist-induced contractions in (−) PVAT and (+) PVAT mesenteric artery rings isolated from 16-month-old mice. (F) Cumulative concentration-response relationships to a1-agonist in (−) PVAT (n=7, N=3) and (+) PVAT (n=9, N=3) mesenteric arteries in 16-month-old mice. *p<0.05. Two-way ANOVA followed by Bonferroni post hoc test. Data are mean and SEM.

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  Figure 2. Relaxation of (−) PVAT mesenteric artery rings by KCNQ channels activators flupirtine and retigabine. (A) Representative traces showing the relaxation induced by 0.01–30μm flupirtine in rings isolated from young, 12-months, and 24-month-old mice. Mesenteric arteries were precontracted by 1μm phenylephrine (PE). (B) Concentration-response relationships for flupirtine-induced relaxation in mesenteric arteries in young (n=12, N=3), 12-month-old (n=10, N=4), and 24-month-old (n=9, N=3) mice. (C) Representative traces showing the relaxation induced by 0.01–30μm retigabine in rings isolated from young, 12-, and 24-month-old mice. (D) Concentration-response relationships for retigabine-induced relaxation in mesenteric arteries in young (n=12, N=3), and 24-month-old (n=7, N=2) mice. *p<0.05. Two-way ANOVA followed by Tukey post hoc test. Data are mean and SEM.

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  Figure 3. Relaxation of (−) PVAT mesenteric artery rings by novel KCNQ channel openers QO58 and QO58-lysine. (A) Original traces showing the effects of 3μM XE991 on QO58-induced relaxation in (−) PVAT mesenteric artery rings compared with control rings without XE991. (B) Concentration-response relationships for QO58-induced relaxation in (−) PVAT mesenteric arteries from young wild-type animals after pre-incubation with 3μm XE991 (n=10, N=4) or in the absence of XE991 (n=6, N=3). (C) Original traces showing the effects of 3μm XE991 on QO58-lysine-induced relaxation in (−) PVAT mesenteric artery rings compared with control rings without XE991. (D) Concentration-response relationships for QO58-lysine-induced relaxation in (−) PVAT mesenteric arteries from young animals after pre-incubation with 3μm XE991 (n=10, N=4) or in the absence of XE991 (n=9, N=3). (E) Concentration-response relationships for QO58-induced relaxation in mesenteric arteries in young (n=11, N=2) and 16-month-old (n=13, N=2) mice. *p<0.05. Unpaired t test. Data are mean and SEM.

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  Figure 4. KCNQ channel openers effects on KCl-induced contraction in (−) PVAT mesenteric artery rings. (A) Original recordings showing the effects of 0.01–30μm retigabine, 0.01–30μm flupirtine on arterial tone of isolated mesenteric artery rings without (−) PVAT. Vessels were precontracted with 60mM KCl. (B) Concentration-response relationships for flupirtine- (n=6, N=2) and retigabine-induced relaxation (n=7, N=2) in (−) PVAT mesenteric arteries from young wild-type animals. (C) Original recordings showing the effects of 0.01–30μm QO58 and 0.01–30μm QO58-lysine on arterial tone of isolated mesenteric artery rings without (−) PVAT. (D) Concentration-response relationships for QO58 (n=9, N=3) and QO58-lysine-induced relaxation (n=8, N=4) in (−) PVAT mesenteric arteries from young wild-type animals. (E) Original recordings showing the effects of vehicle (DMSO or PSS) on arterial tone of isolated mesenteric artery rings without (−) PVAT. (F) Concentration-response relationships for DMSO (n=5, N=2) and PSS (n=5, N=2) in (−) PVAT mesenteric arteries from young wild-type animals. *p<0.05. paired sample t test. Data are mean and SEM.

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  Figure 5. Differential gene expression in mesenteric arteries and PVAT from young and old mice. (A) Principal component analysis in samples isolated from young vs. old animals. (B) Heatmap showing the expression of candidate genes involved in pathways regulating KCNQ channels (N=8 for 12-month-old; N=6 for 16-month-old; N=3 for 24-month-old; N=4 for young mice). Data are fragments per kilobase per million mapped fragments. (C) Relative mRNA levels for Cacna2d3 (N=4 for young; N=8 for 12-month-old; N=6 for 16-month-old; N=3 for 24-month-old mice). (D) Relative mRNA levels for Kcnk2 (N=4 for young; N=8 for 12-month-old; N=6 for 16-month-old; N=3 for 24-month-old mice). (E) Relative mRNA levels for P2rx5 (N=4 for young; N=8 for 12-month-old; N=6 for 16-month-old; N=3 for 24-month-old mice). (F) Number of differentially expressed genes in PVAT isolated from mesenteric arteries in young and old mice. (G) Volcano plot displaying statistical significance (adjusted value of p) versus magnitude of change (fold change) for differently expressed genes in PVAT isolated from 12-month-old (N=4) vs. young mice (N=4). Top 5 differentially expressed genes are marked. *p<0.05. Wald test. Data are mean and…

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  Figure 6. Representative schematic of downregulated genes and pathways in PVAT isolated from mesenteric arteries of 12-month-old mice. By decreased expression of genes belonging to Ppar and Rcr signaling pathway, the ubiquitination, fatty acid (FA) transport, adipocyte differentiation, and FAs oxidation are decreased. In addition, the downregulated glycolysis, TCA cycle, and fatty acid biosynthesis can decrease production of perivascular fat tissue-derived relaxation factors (N=4 for young; N=4 for 12-month-old). MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; PA, phosphatidic acid; TAG, triacylglycerol; DAG, diacylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine. Abbreviations can be found in Supplementary Table S4.