RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na+/H+ exchanger

Abstract

Smooth muscle contraction is triggered when Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC₂₀). However, blood vessels from Mlck-deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC₂₀ to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from Rsk2-deficient mice were dilated and showed reduced myogenic tone and RLC₂₀ phosphorylation. RSK2 phosphorylated Ser¹⁹ in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na⁺/H⁺ exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca²⁺ that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in Rsk2-deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca²⁺ transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, Rsk2-deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.

Fig. 1.. Myogenic- and phenylephrine-induced vasoconstriction with associated RSK2 and RLC₂₀ phosphorylation in WT and Rsk2KO mesenteric arteries measured by pressurization and Western blotting.

(A) Scheme showing the phosphorylation cascade for activation of RSK2. (B) Cartoon showing the apparatus for controlled luminal pressurization of mouse mesenteric artery cascades to obtain sufficient material for biochemical analysis of phosphorylation events. (C) Typical traces showing myogenic constriction of WT and Rsk2^KO mesenteric arteries in response to increases in intraluminal pressure in Ca²⁺-containing and Ca²⁺-free Krebs solution. (D) Summary of myogenic responses of Rsk2^KO and WT arteries at 40, 60, 80, and 100 mmHg. P < 0.001, P < 0.01, P < 0.01, and P < 0.01, respectively, determined by two-way analysis of variance (ANOVA). Rsk2^KO: n = 3 mice and 6 arteries; WT: n = 3 mice and 6 arteries. (E) Phenylephrine (PE) concentration responses of Rsk2^KO and WT third- and fourth-order mesenteric arteries pressurized to 80 mmHg. ***P = 0.005 determined by two-way ANOVA. Rsk2^KO: n = 3 mice and 7 arteries; WT: n = 3 mice and 6 arteries. EC₅₀ values for phenylephrine-induced contractions (bar graph) were not significantly different. P values were determined by two-tailed homoscedastic Student’s t test. (F) Time course of RSK2 phosphorylation (normalized to total RSK2) at the MEK/ERK Thr⁵⁷⁷ site and at the PDK Ser²²⁷ site following a pressure step from 20 to 80 mmHg in WT arteries. Data are means ± SEM; n = 6 mice and 6 arteries for each time point for Ser²²⁷ and 5 mice and 5 arteries for each time point for Thr⁵⁷⁷. *P < 0.05 for each time point compared to the corresponding control phosphorylation for Ser²²⁷; ^#P < 0.05 for each time point compared to the corresponding control phosphorylation for Thr⁵⁷⁷, two-tailed homoscedastic Student’s t test. (G) Time course of RLC₂₀ Ser¹⁹ phosphorylation following a pressure step from 20 to 80 mmHg in WT and Rsk2^KO artery arcades. Data are means ± SEM; n = 11 WT mice and 11 arteries for each time point and n = 4 Rsk2^KO mice and 4 arteries for each time point. **P < 0.01 for each time point after an increase in pressure, compared to Ser¹⁹ phosphorylation in 0-mmHg pressure WT arteries; ^#P < 0.05 for each time point compared to Ser¹⁹ phosphorylation in 0-mmHg pressure Rsk2^KO arteries, two-tailed homoscedastic Student’s t test.

Fig. 2.. The RSK inhibitor LJH685 induces relaxation of myogenic tone in mesenteric arteries and suppresses RSK2 phosphorylation in WT smooth muscle cells.

(A and B) Tension trace and summary showing relaxation of myogenic tone in response to increasing concentrations of LJH685 in a mesenteric artery in Krebs bicarbonate buffer and pressurized to 60 mmHg. Subsequent treatment with Ca²⁺-free solution induced maximal vessel dilation. Dimethyl sulfoxide (DMSO) diluent concentrations were matched to LJH685 concentrations. n = 3 to 7 arteries per group. P = 0.04 for DMSO compared to LJH685 treatment, two-way ANOVA. (C) RSK2 Ser²²⁷ phosphorylation (normalized to total RSK2) in response to serum application in serum-starved mouse aortic smooth muscle cells or preincubation with LJH685, the PDK1 inhibitor GSK2334470, and the ERK1/2 inhibitor U0126. Rsk2^KO smooth muscle cells served as a control. ***P < 0.001 control compared to serum stimulation, two-tailed homoscedastic Student’s t test. Data are means ± SEM; n = 3 biological replicates per group.

Fig. 3.. Western blot analysis for agonist activation of RSK2, RLC₂₀, and MYPT1 phosphorylation in mouse mesenteric artery arcades.

Phosphorylation of Ser²²⁷ and Thr⁵⁷⁷ in RSK2 of Ser¹⁹ in RLC₂₀ and Thr⁸⁵³ in MYPT1 (the ROCK site) under resting conditions compared to responses over time to ET-1, AngII, the thromboxane analog U46619, and phenylephrine. The doublets seen for RSK2 and MYPT1 in the Western blots (WB) likely reflect RSK2 isoforms (National Center for Biotechnology Information accession numbers NM 001346675.1 and NM 148925.2) and the two MYPT1 isoforms. Data are means ± SEM; n = 3 mice per agonist group and 3 artery samples per time point. *P < 0.05 for each time point compared to the corresponding control (0 s) for each agonist. P values were determined by two-tailed homoscedastic Student’s t test.

Fig. 4.. RSK2 phosphorylation of RLC₂₀, effect on MLCP activity and immunoprecipitation, and fractionation assays to assess RSK2 association with actin.

(A) Phosphorylation of purified RLC₂₀ protein at Ser¹⁹ by recombinant active RSK2. n = 3 independent experiments each carried out in triplicate, homoscedastic Student’s t test. MLCK served as a positive control. (B) Effect of RSK2- or ROCK2-mediated phosphorylation of MYPT1 on in vitro MLCP activity. Bars show values of relative MLCP activity compared to the sample without adenosine 5′-(3-thio)triphosphate (ATPγS) thiophosphorylation. Two independent experiments each carried out in triplicate. (C) Phosphorylation of MYPT1 Thr⁸⁵³ (normalized to total MYPT1) over time in response to an increase in pressure from 0 to 80 mmHg in arteries from WT and Rsk2^KO mice. WT: n = 8 mice; Rsk2^KO: n = 5 mice. WT: **P < 0.01; Rsk2^KO: ^#P < 0.05, ^##P < 0.01, two-tailed homoscedastic Student’s t test. There was no significant difference between WT compared to Rsk2^KO at any time point. (D) Representative WB of RSK2 immunoprecipitation (IP) of actin from aortic smooth muscle cell lysates from WT and Rsk2^KO mice (n = 3 biological replicates). (E) WB of fractions from lysates of mesenteric arteries at 0- or 80-mmHg pressure, showing RSK2 and MLCK in the 200,000g pellet. Myosin (MYH11) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mark the cytoskeletal (200,000g pellet) and cytosolic fractions (supernatant), respectively; histone3 and caveolin2 mark the nuclear and membrane fractions and unbroken cells in the crude pellet (n = 4 mice and 4 artery arcades). (F) WB analysis of the effect of actin depolymerization by latrunculin B (LatB) plus cytochalasin D (CycD) or of actin polymerization by jasplakinolide (Jasplak) on the distribution of RSK2 and actin in the supernatant and high-speed spin pellets from mouse aortic smooth muscle cells. *P < 0.05 compared to control, two-tailed homoscedastic Student’s t test. n = 4 biological replicates. The concentrations of RSK2 in the supernatant were not significantly changed by treatment.

Fig. 5.. RSK2 regulation of Na⁺/H⁺ exchanger activity as assessed by phosphorylation and immunoprecipitation measurements.

(A) WB showing phosphorylation of NHE-1 Ser⁷⁰³ (normalized to total NHE-1) in WT and Rsk2^KO mesenteric arteries. Data are means ± SEM; n = 3 mice per group; **P < 0.01. (B) WB showing phosphorylation of NHE-1 Ser⁷⁰³ (normalized to total NHE-1) in untreated WT mesenteric arteries or those treated with the RSK inhibitor LJH685 or LJI308. Data are means ± SEM; n = 3 mice per group. *P < 0.05 for LJH685 and LJI308 compared to untreated arteries. The doublet for phospho–NHE-1 likely reflects NHE-1 isoforms that are frequently detected by the more reactive phospho–NHE-1 antibody than the less reactive total NHE-1 antibody and under the conditions used for electrophoresis. (C) Time course of NHE-1 Ser⁷⁰³ phosphorylation normalized to total NHE-1 in response to increased arterial intraluminal pressure. Data are means ± SEM; n = 6 mice and 6 artery samples per time point. *P < 0.05, **P < 0.01, time points compared to 0 s, two-tailed homoscedastic Student’s t test. (D) Representative WB assay showing NHE-1 immunoprecipitation from mesenteric artery homogenates by RSK2 antibody (n = 4 mesenteric arcades from four mice analyzed separately). The lack of an NHE-1 band in the total lysate is due to the low intensity used to scan the membrane to not saturate the NHE-1–immunoprecipitated band.

Fig. 6.. RSK2 activation of the Na⁺/H⁺ exchanger increases intracellular pH and Ca²⁺.

(A) RSK2-regulated NHE-1 activity was monitored with the ratiometric fluorescent pH indicator BCECF-AM loaded into WT and Rsk2^KO mesenteric arteries. Bar graphs show conversion of intensity ratios to pH with typical fluorescent images of arteries. n = 3 mice per genotype and 6 arteries. *P < 0.05, for 20-mmHg pressure compared to 60-mmHg pressure and for Na⁺-free compared to re-addition of Na⁺ for WT arteries; ^#P < 0.02, for 20 mmHg compared to 60 mmHg in Rsk2^KO arteries, two-tailed homoscedastic Student’s t test. The last three pH values for WT and Rsk2^KO arteries upon re-addition of Na⁺ are averaged in the bar graph. (B) Cytosolic Ca²⁺ measured in WT and Rsk2^KO aortic smooth muscle cells treated with sodium acetate (NaAc) to stimulate NHE-1, with or without the NHE-1 inhibitor cariporide. n = 3 biological replicates per genotype. P < 0.0002 for [Ca²⁺] in WT cells before and after treatment with NaAc, two-tailed homoscedastic Student’s t test. (C) Typical example of Ca²⁺ transient analysis in WT mesenteric arteries in the presence of 10 μM ryanodine to block Ca²⁺ release from the ryanodine-sensitive Ca²⁺ stores. Gray-scale images showing smooth muscle cells in an artery. Typical Ca²⁺ fluorescent intensity images of all the event sites in the field summed over 1000 ms. F/F₀ traces of cytoplasmic Ca²⁺ transients. Each color represents a trace from a region in a different cell. (D) Summary of Ca²⁺ events at 20- and 60-mmHg intraluminal pressure in the presence and absence of cariporide and/or ryanodine. Data are means ± SEM. *P < 0.05, ***P < 0.001, n = 4 mice and 6 arteries without ryanodine. *P < 0.05, **P < 0.01, n = 4 mice and 6 arteries with ryanodine treatment.

Fig. 7.. Blood pressure and cardiac function measurements in Rsk2^KO mice.

(A) Tail cuff measurements in male and female WT and Rsk2^KO mice. n = 9 Rsk2^KO mice, n = 15 WT mice. Systolic blood pressure mean ± SEM. P < 0.003 for female Rsk2^KO mice compared to WT female mice; P < 0.001 for male Rsk2^KO mice compared to WT male mice, two-tailed homoscedastic Student’s t test. Radiotelemetric measurements of blood pressure in WT and Rsk2^KO blood pressure over 24 hours. n = 7 WT males, n = 3 Rsk2^KO males. Data are means ± SEM for systolic blood pressures. (B) Heart/body weights; n = 8 WT and 9 Rsk2^KO mice. Male and female mice were averaged together because there was no statistically significant difference in the ratios. MRI analysis, n = 3 mice per group. (C) Typical MR images used to calculate cardiac wall thickness and ejection fraction. Left column, diastole; middle column, systole; right column, longitudinal view, diastole. (D) Histological cardiac sections of WT and Rsk2^KO mice. n = 3 mice per group. (E) Scheme showing RSK2 signaling in smooth muscle and its contribution to vasoconstriction, the myogenic response, and blood pressure regulation. GPCRs and pressure-sensitive mechanosensors activate RSK2 through activation of ERK1/2 or another, as yet unidentified kinase to activate PDK. Active RSK2 phosphorylates and activates two targets, RLC₂₀ and NHE-1, leading to alkalinization of the cell and increased [Ca²⁺]_i, which, in turn, augments MLCK activity and promotes vasoconstriction.