Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Abstract

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.

Fig. 1.

Targeted deletion of myosin light chain kinase (Mlck) gene in mesenteric arteries. A: Western blot of MLCK in mesenteric arteries from smooth muscle-specific MLCK knockout [MLCK^SMKO; knockout (KO)] and control (CTR) mice collected at day 16 after tamoxifen injection. β-Actin was used as a loading control. B: immunofluorescence analysis of MLCK expression in mesenteric arteries from MLCK^SMKO and CTR mice. At day 16 after tamoxifen injection, arteries were embedded in optimum cutting temperature and sectioned. Frozen sections were fixed with acetone and then stained with K36 antibody and α-smooth muscle actin antibody. Scale bar=50 μm. C: representative Western blots of contractile and regulatory proteins [integrin-linked kinase (ILK), Rho-associated coiled-coil-forming protein kinase II (ROCK II), myosin phosphatase targeting subunit of the RLC phosphatase (MYPT1), smooth muscle myosin heavy chain (SM-MHC)] in mesenteric arteries from MLCK^SMKO and CTR mice collected at day 16 after tamoxifen injection. β-Actin was used as a loading control. D: expression of MLCK (N=10) and other proteins (N=3) in KO arteries is expressed as the ratio relative to that of CTR.

Fig. 2.

Gross morphology of mesenteric vessels of MLCK^SMKO mice and CTR mice. The mesenteric vasculature from a CTR mouse (A, C) and a MLCK^SMKO mouse (B, D) was photographed at 16 days after tamoxifen treatment. Images were obtained at comparable anatomical sites (A, B, scale bar=5 mm; C, D, scale bar=2 mm). Arteries (A) and veins (V) are indicated. E: diameters of secondary branches of the mesenteric vein were measured from photographs as in C and D. Values are means ± SE of CTR (N=4) and MLCK^SMKO (N=4); **P < 0.01. F: internal circumferences of second-order branches of mesenteric arteries of CTR and MLCK^SMKO mice were calculated by the Myograph normalization procedure. IC₁₀₀ is the internal circumference corresponding to a transmural pressure of 100 mmHg. Values are means ± SE for CTR (N=4) and MLCK^SMKO (N=12).

Fig. 3.

Histological sections of control and MLCK-deficient arteries. Fresh tissues of aorta, femoral artery, heart, and mesenteric artery from CTR and MLCK^SMKO mice were fixed with 4% formalin and dehydrated in a graded series of ethanol solutions followed by standard paraffin section and hematoxylin and eosin staining. Scale bars=50 μm.

Fig. 4.

Reduced contraction and regulatory light chain (RLC) phosphorylation in mesenteric arteries from MLCK^SMKO mice. Dose-response curves of KCl (A), norepinephrine (NE; D), phenylephrine (PE), ANG II, and endothelin-1 (ET-1; G–I) in fresh mesenteric artery segments from MLCK^SMKO (KO) and CTR mice analyzed by wire myography are shown. Bars are means ± SE (N=4); *P < 0.05, **P < 0.01. After stimulation with 124 mM KCl (B, C) or 1 μM NE (E, F), mesenteric arteries were quickly frozen at indicated times for sample preparation. RLC phosphorylation (RLC-p) was measured by Western blots of glycerol/urea PAGE gels (B, E). RLC-p level was expressed as percentage of total RLC (C, F; N=4). All values represent means ± SE; *P < 0.05, **P < 0.01 compared with MLCK^SMKO values at the indicated time.

Fig. 5.

Reduced physiological blood pressures of MLCK^SMKO mice. A: systolic blood pressures in CTR and MLCK^SMKO mice were measured by the tail-cuff method. Daily measurements were performed for 15 days following the initial tamoxifen injection. Each arrow indicates an injection of tamoxifen; N=3 (female mice). B: effect of intravenous injection of NE (25 μg/kg), PE (10 μg/kg), and ET-1 (1 nmol/kg) on blood pressures of CTR and MLCK^SMKO mice. Responses are plotted as the maximum change in pressure (ΔmmHg) after injection. Values are means ± SE; N=3. *P < 0.05, **P < 0.01 compared with CTR values.

Fig. 6.

Attenuation of Mlck abolished salt-induced hypertension. CTR and MLCK^SMKO mice received sequential treatments of uninephrectomy and implantation of a DOCA pellet for induction of hypertension. MLCK attenuation was induced by 5 consecutive daily injections of tamoxifen (arrows). A: daily systolic blood pressure was measured with the tail-cuff method for 23 days (d). B: statistical analysis of systolic blood pressure in CTR and MLCK^SMKO mice (KO) before (white bars) and 7 days after (black bars) DOCA-salt treatment and 2 wk after induction of DOCA-salt-treated mice with tamoxifen (gray bars). NS, no statistical significance. Values of blood pressure are means ± SE; N=3 (female mice). **P < 0.01.