Dual thick and thin filament linked regulation of stretch- and L-NAME-induced tone in young and senescent murine basilar artery

Abstract

Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC₂₀)-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone. In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 μmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC₂₀-phosphorylation. In contrast, senescent BAs (s-BAs; >24 months) developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC₂₀-phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1^+/- mice generated stretch-induced tone already at an age of 20-21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1^+/- o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-▵Ex2^-/-) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC₂₀ phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC₂₀, predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon.

Keywords: basilar artery; caldesmon; non-muscle myosin; senescence; stretch-induced tone.

FIGURE 1

Stretch-, L-NAME-, and agonist-induced force in young murine basilar arteries obtained from WT and MYPT1-T696A/+ -mice. (A–C) Original force tracings and statistical summary from measurement of stretch-induced tone stretch-induced tone in y-BAs from WT-animals treated with 100 μmol/L of pan-NOS-inhibitor L-NAME in the presence of 3 μmol/L Y27632 (gray) or vehicle (H₂O; black). Depicted p-values represent results from 5 independent experiments (***p < 0.001; n.s.—not significant, p > 0.05; one way ANOVA n = 5). (D–F) Original force tracings and statistical evaluation of stretch-induced tone, L-NAME- and agonist-induced tone of y-BAs from WT (black) and MYPT1-T696A/+ -mice (red) under control conditions (controls) and after application of 100 μmol/l L-NAME (n = 6–7; *p < 0.05; unpaired t-test; n.s.—not significant). Bars represent mean ± SEM.

FIGURE 2

Basal and L-NAME-induced phosphorylation of pMLC₂₀-S19, pMYPT1-T696, pMYPT1-T853, pMYPT1-S695 in basilar arteries from young WT and MYPT1-T696A/+ -mice. Original chemiluminograms (A) and statistical evaluation (B–E) of the immunoreactive signals of lysates of y-BAs from WT and MYPT1-T696A/+ mice transferred on nitrocellulose membranes and probed with antibodies against pMLC₂₀-S19, pMYPT1-T696, pMYPT1-T853, and pMYPT1-S695. Statistical figures represent the ratio of the immunoreactive signal of pMLC₂₀-S19 normalized to β-Actin or pMYPT1-T696, pMYPT1-T853, and pMYPT1-S695 normalized to MYPT1-total (n = 4). Arteries were treated using same experimental protocol as in Figure 1. Statistic comparison on (B) represents evaluation using two-way ANOVA (***p < 0.0001; n = 4). ***p < 0.0001, *p < 0.05 represents evaluation by unpaired t-test (n = 4). n.s.—not significant.

FIGURE 3

Ca²⁺-activated contraction of basilar arteries from young WT- and MYPT1-T696A/+-mice. Original force tracing (A) and statistical summary (B) depicting Ca²⁺-activated contraction of young BA from WT- and MYPT1-T696A/+-mice (n = 4–5). Tone normalized to pCa 4.3 accepted as 100%.

FIGURE 4

Stretch-, L-NAME-, and U46619-induced tone in arterial rings from senescent basilar arteries from WT and MYPT1-T696A/+ -mice under control conditions and under inhibition of ROK. (A,B) Original force tracings of the experiments performed with s-BAs from WT and MYPT1-T696A/+ -mice pretreated with vehicle [1% H₂O; (A)] or 3 μmol/L Y27632 (B) and followed by inhibition of endogenous NO by treatment with 100 μmol/l L-NAME and cumulative application of U46619 (conc. 0.001–3 μmol/L). (C) Statistical evaluation of stretch-induced, L-NAME-induced and maximal tone (n = 8). (D) Statistical evaluation of tone induced by cumulative application of U46619 (n = 8). Data are represented as absolute force ±SEM; n.s. p > 0.05; two-way ANOVA. §p < 0.0001, stretch-induced tone WT controls (no treatment) vs. stretch-induced tone WT after 10 min treatment with 3 μmol/L Y27632, or stretch-induced tone L-NAME WT no treatment vs. stretch-induced tone L-NAME WT after 10 min treatment with 3 μmol/L Y27632, or stretch-induced tone L-NAME MYPT1-T696A/+ no treatment vs. stretch-induced tone L-NAME L-NAME MYPT1-T696A/+ after 10 min treatment with 3 μmol/L Y27632. *p < 0.05 F_max WT vs. F_max WT after treatment with Y27632; and **p < 0.001 stretch-induced tone MYPT1-T696A/+ controls (no treatment) vs. stretch-induced tone MYPT1-T696A/+ after 10 min treatment with 3 μmol/L Y27632 or F_max MYPT1-T696A/+ vs. F_max MYPT1-T696A/+ after treatment with Y27632. All values were calculated by unpaired t-test. n.s.—not significant; pEC₅₀; ***p < 0.0001; two-way ANOVA.

FIGURE 5

Effect of ROK inhibition on MLC₂₀-S19, MYPT1-T696, and MYPT1-T853 in senescent basilar arteries from WT and MYPT1-T696A/+ mice. (A) Original western blots and statistical evaluation of the phosphorylation of (B) MLC₂₀-S19 (pMLC₂₀), and MYPT1 at (C) T696 (pMYPT1-T696) and (D) T853 (pMYPT1-T853) in s-BAs from WT and MYPT1-T696A/+ mice under control conditions (no stimulations), and after preincubation with 3 μmol/L Y27632, 100 μmol/l L-NAME or Y27632 plus L-NAME. Data are represented as ratio of pMYPT1-T696/853 toward MYPT1-total or MLC₂₀-S19 toward GAPDH (n = 6). **p < 0.01, *p < 0.05, n.s.—not significant; two-way ANOVA. n.s.—not significant; ***p < 0.0001 and p = 0.06 have been calculated by using unpaired t-test.

FIGURE 6

Stretch-, L-NAME-, and U46619-induced tone in senescent basilar arteries from WT and MYPT1-T696A/+ -mice under inhibition of cross-bridge cycling of non-muscle myosin. (A,B) Original force tracings representing the effect of the inhibitor of cross-bridge cycling of non-muscle myosin, blebbistatin on tone. Vessels were pretreated with the active (−) and inactive (+) enantiomers of blebbistatin and further incubated with 100 μmol/l L-NAME and cumulative application of U46619. (C) Statistic evaluation of stretch-induced, L-NAME-induced and maximal tone in presence of (+) or (−) blebbistatin (n = 5). (D) Statistic evaluation of tone induced by cumulative application of U46619 (n = 5). Data represented as absolute force ±SEM; n.s. p > 0.05; two-way ANOVA. *p < 0.05 and **p < 0.001 calculated by unpaired t-test.

FIGURE 7

Phosphorylation of pMYPT1-T696, pMYPT1-T853, and pMLC₂₀-S19 in senescent basilar arteries from WT and MYPT1-T696A/+ mice under inhibition of cross-bridge cycling of non-muscle myosin. (A–D) Original Western blots (A) and statistic evaluation of the phosphorylation of pMLC₂₀-S19 (B), pMYPT1-T696 (C) and pMYPT1-T853 (D) in s-BAs from WT and MYPT1-T696A/+ mice under control conditions and after inhibition of cross-bridge cycling of non-muscle myosin by blebbistatin (−). Preparations mounted as previously and treated by vehicle (0.3% DMSO; controls), or 10 μmol/L blebbistatin (−), or 100 μmol/l L-NAME, or blebbistatin (−) plus L-NAME. Data represented as ratio of pMYPT1-T696/853 toward MYPT1-total or MLC₂₀-S19 toward GAPDH (n = 5). n.s.—not significant; unpaired t-test.

FIGURE 8

Phosphorylation of NM-II-S1943, MYPT1-T853, and PAK-T423 in basilar arteries from young and senescent mice under control conditions and after inhibition of ROK. (A–C) Original western blots and statistic evaluation of the phosphorylation of MYPT1 at T853 (A,B), non-muscle-myosin II (NM-Myosin) at S1943 (C,D) and PAK-T423 (E,F) in y- and s-BAs from WT animals (n = 10–6). Preparations were treated either by vehicle (0.5% H₂O; controls) or by 3 μmol/L Y27632, shock-frozen and subjected to Western blot as described in “Methods.” **p < 0.01 and *p < 0.05; control y-BAs vs. control s-BAs; two-way ANOVA. **p < 0.001 and *p < 0.05; unpaired t-test.

FIGURE 9

Effect of Caldesmon targeting on tone maintenance in basilar arteries from old mice. (A) Representative Western blot of expression of h-CaD in WT and heterozygous s-BAs from 2 litters (B) statistical evaluation from arteries of four independent measurements (n = 3 litters with WT and Het littermates, n = 1 WT and Het from different litters). M, marker; W, wilde type; H, heterozygotes. Caldesmon expression the Ponceau Red stained actin band. (B) Statistic evaluation. (C,D) Original force tracings representing the effect of caldesmon mutation on tone. (E) Statistic evaluation of stretch-induced, L-NAME-induced and maximal tone in BAs from WT and Cald1^+/− BAs (n = 6–4). (F) Statistic evaluation of tone induced by cumulative application of U46619 (n = 6–4). Data represented as absolute force ±SEM; *p < 0.05; n.s. p > 0.05; two-way ANOVA.

FIGURE 10

Effect of ablation of exon2 (Ex2) of Caldesmon on tone maintenance basilar arteries (A) Original luminogram and statistic evaluation showing caldesmon expression in s-BAs obtained from wild type animals, homozygous animals lacking the strong myosin-binding domain of caldesmon encoded by Exon 2 (−/−) and heterozygous animals (+/−) (n = 3-3-2). (B) Analysis of two independent experiments confirming the lack of myosin binding of the truncated caldesmon isolated from Cald1 −/− mice (left panel), whereas actin-binding was not affected (right panel). (C,D) Original force tracings representing the effect of ablation of Exon2 in caldesmon protein on tone. (E) Left: Statistic evaluation of stretch-induced (n = 7), L-NAME-induced (n = 3–7), and maximal tone (n = 3–7) in BAs from WT (CaD_ΔEx2 +/+) and CaD ΔEx2^−/− BAs under control conditions. Right: Statistic evaluation of stretch-induced (n = 4–3), L-NAME-induced (n = 4–3), and maximal tone (n = 4–3) in BAs from same groups in the presence of 3 μmol/L Y27632. (F) Statistic evaluation of tone induced by cumulative application of U46619 (n = 6–4). Data represented as absolute force ±SEM.

FIGURE 11

G/F actin ratio in basilar arteries from senescent WT and MYPT1-T696A/+ animals under basal conditions. Localization of F-actin in basilar arteries from senescent WT mice after Calyculin stimulation (A) Original Western blot representing the global actin-immunoreactivity in supernatant (S) used as read out for globular (G) -actin fraction and pellet (P) used as read out for fibrillar (F) -actin in s-BAs from WT and MYPT1-T696A/+ animals. Supernatant and pellet fractions were obtained by ultracentrifugation as described in methods. (B) Statistic evaluation of n = 6 s-BAs, each group. n.s.—not significant; p > 0.05; two-way ANOVA. Confocal images y-BAs (C) and s-BAs (D) arteries from WT animals stained with phalloidin for F-actin and Hoechst for Nuclei. Transmission light images are denoted as “T-light.” Vessels were isolated, mounted and normalized as described in methods and treated for 30 min with 0.1 μmol/L Calyculin. After stimulation, preparations were fixed and F-actin was stained with Fluor™ 555 conjugated phalloidin, nuclei were stained with Hoechst 33342 (see methods). Representative images from four independent vessels/animals.