Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries

Abstract

New findings: What is the central question of this study? DAPK3 contributes to the Ca²⁺ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system.

Abstract: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca²⁺ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca²⁺ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

Keywords: cerebrovascular; myogenic reactivity; vascular smooth muscle.

FIGURE 1

HS38 treatment relaxes rat posterior cerebral arteries constricted with serotonin. Posterior cerebral arteries were mounted at 10 mmHg and constricted with continuous exposure to serotonin hydrochloride (5‐HT, 1 μM). Increasing concentrations of HS38 (0.1–20 μM) were applied to the bath chamber. (a) Representative recordings of the change in outer diameter for 5‐HT constricted vessels subjected to sequential application of HS38. (b) Cumulative data showing vessel constriction in the presence or absence of HS38 relative to the maximum passive diameter observed in 0 Ca²⁺ solution. Vehicle and HS38 treatment groups were found to be significantly different (two‐way ANOVA, P = 0.0086). Data are displayed as mean with SD; vessels were obtained from n = 4 different male animals.

FIGURE 2

HS38 administration attenuates the myogenic response of rat posterior cerebral arteries and elicits a decrease in passive vessel diameter. The myogenic reactivity of posterior cerebral arteries was monitored in the absence (a, DMSO vehicle) or presence of DAPK3 inhibitor (b, HS38, 10 μM). (i) Representative recordings of the change in outer diameter for vessels subjected to sequential pressure steps (10–120 mmHg) in the presence of normal Krebs buffer (NB), vehicle or DAPK3 inhibitor, and then calcium‐free buffer (0 Ca²⁺). (ii) Cumulative data showing the vessel constriction relative to the maximum passive diameter observed in 0 Ca²⁺ solution. (iii) Magnitude of active myogenic constriction. No significant impact on percentage dilatation (a, ii) or myogenic constriction (a, iii) was identified for vehicle/DMSO treatment; P = 0.999 and P = 0.744, respectively, by two‐way ANOVA with Šidák's multiple comparisons test. HS38 treatment did have a significant impact on percentage dilatation above 60 mmHg (b, ii) and myogenic constriction above 80 mmHg (b, iii); P = 0.0006 and P = 0.030, respectively by two‐way ANOVA with Šidák's multiple comparisons tests (a, P = 0.023; b, P = 0.009; c, P = 0.021; d, P = 0.041; e, P = 0.023; f, P = 0.0025; g, P = 0.0006). Data are displayed as means with SD; vessels were obtained from n = 3 different male animals. (c, i) Representative recordings illustrating the change in outer diameter for vessels in calcium‐free Krebs buffer (0 Ca²⁺). Arteries were incubated at 10 mmHg for 30 min in the presence of vehicle (DMSO) or HS38 (10 μM), and then pressure steps (20–120 mmHg) were developed over an additional 60 min. (c, ii) Cumulative data show the vessel constriction relative to the maximum passive diameter observed in 0 Ca²⁺ solution. Vehicle and HS38 treatment groups were found to be significantly different (two‐way ANOVA, P = 0.0001). Data are presented as means with SD; vessels were obtained from n = 4 different male animals.

FIGURE 3

Myogenic responses of human cerebral arteries are attenuated with HS38 treatment. (a) Pial arteries (∼300–500 μm in diameter) were collected from four unique human brain tissue samples and immunoblotted for DAPK3 and smooth muscle actin (αSMA) as a loading control. (b) Representative recordings demonstrate the response of human pial arteries, outer vessel diameters, subjected to sequential 10–120 mmHg pressure steps in normal Krebs buffer (Control), with DAPK3 inhibitor (HS38, 10 μM), and in calcium‐free Krebs buffer (0 Ca²⁺). (c, d) Cumulative data provided to show the vessel constriction relative to the maximum passive diameter observed in 0 Ca²⁺ solution (c) and the magnitude of active myogenic constriction (d). HS38 treatment had a significant impact on percentage dilatation and active myogenic constriction at pressures above 100 mmHg; P = 0.013 and P = 0.024, respectively, by two‐way ANOVA with Šidák's multiple comparisons test (a, P = 0.007; b, P = 0.006; c, P = 0.013; d, P = 0.010. Data are presented as means with SD for vessels obtained from three different patients.

FIGURE 4

HS38 treatment of posterior cerebral arteries displaying active myogenic tone provides transient vasodilatation. (a) Representative vessel responses (outer diameter) to prolonged exposure with HS38. Vessels displayed active myogenic tone (MT) when pressurized to 80 mmHg. HS38 administration resulted in vessel dilatation (MD) and then spontaneous recovery of myogenic reactivity (MR). (b) Cumulative data show vessel dilatation in response to HS38; the percentage dilatation at MD and MR were calculated relative to the myogenic response observed at MT. Vessel dilatation at MD was significantly different from MT (P = 0.029) and MR (P = 0.014) while vessel dilatation at MR was not different from MT (P = 0.377); one‐way ANOVA and Tukey's multiple comparisons test. (c) Pressurized PCA vessels were flash‐frozen at the points denoted by the MT, MD and MR labels, and LC20 phosphorylation was determined by Phos‐tag SDS‐PAGE. A sample of rat tail artery (RTA) treated with microcystin was used to identify the phosphorylated LC20 (0P, unphosphorylated; 1P, monophosphorylated; 2P, dephosphorylated). (d) Cumulative data for LC20 phosphorylation stoichiometry are provided. Results were analysed by one‐way ANOVA and Tukey's multiple comparison test. LC20 phosphorylation at MD was not significantly different from MT (P = 0.189) or MR (P = 0.202); however, LC20 phosphorylation at MR was significantly different from MT (P = 0.017). Data are presented as means with SD; PCA vessels were obtained from n = 3–4 different male animals.

FIGURE 5

HS94 treatment of posterior cerebral arteries elicits transient vasodilatation. (a) Representative vessel responses (outer diameter) to prolonged exposure with HS94. Vessels displayed active myogenic tone (MT) when pressurized to 80 mmHg. HS94 administration resulted in vessel dilatation (MD) and then spontaneous recovery of myogenic reactivity (MR). (b) Cumulative data show vessel dilatation in response to HS94. Vessel dilatation at MD was significantly different from MT (P = 0.010) and MR (P = 0.034) while vessel dilatation after recovery at MR was not different from baseline tone at MT (P = 0.982); one‐way ANOVA and Tukey's multiple comparisons test. (c, d) Cumulative data for LC20 phosphorylation stoichiometry are provided for pressurized PCA vessels treated with HS94 or vehicle (DMSO), respectively. Vessels were flash‐frozen at the points denoted in (a), and LC20 phosphorylation stoichiometry was determined by Phos‐tag SDS‐PAGE as described in Figure 4. Results were analysed by one‐way ANOVA and Tukey's multiple comparison test. No significant differences in LC20 phosphorylation were identified: HS94 treatment: MD vs. MT (P = 0.594), MD vs. MR (P = 0.555), MT vs. MR (P = 0.998); DMSO treatment: MD vs. MT (P = 0.971), MD vs. MR (P = 0.245), MT vs. MR (P = 0.069). Data are presented as means with SD; PCA vessels were obtained from n = 3–4 different male animals.

FIGURE 6

Effect of DAPK3 silencing on cytoskeletal architecture and FAK phosphorylation of vascular smooth muscle cells. (a) CASMCs were fixed and stained with AlexaFluor488‐phalloidin to examine F‐actin cytoskeletal organization following 16 h administration of small molecule inhibitor (HS38, 50 μM or DMSO vehicle control) or siRNA (siDAPK3 or scrambled (Scr)‐siRNA). Representative images from three independent experiments are shown with fluorescence signals for phalloidin‐F‐actin (green) and DAPI nuclear stain (blue). (b, c) Western blotting and densitometric quantification of total cell lysates was also used to examine FAK phosphorylation: DAPK3 and pY397‐FAK levels following treatment with siDAPK3 (b) and pY397‐FAK levels after HS38 administration (c). Data are means with SD from n = 3 different experiments using separate passages of cells. Significantly different from corresponding control, Student's t‐test, ***P < 0.0001.