Smooth muscle Ca²⁺ sensitization causes hypercontractility of middle cerebral arteries in mice bearing the familial hemiplegic migraine type 2 associated mutation

Abstract

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2^+/−G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca²⁺ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2^+/−G301R and WT showed similar contractile responses. The Atp1a2^+/−G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca²⁺ change was smaller than in WT. The enhanced constriction of Atp1a2^+/−G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca²⁺]_i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2^+/−G301R mice had reduced resting blood flow through MCA in comparison with WT mice. FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca²⁺, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Introduction

Familial hemiplegic migraine type 2 (FHM2) is an autosomal dominant form of migraine caused by mutations of the Na,K-ATPase α2 isoform.^1,2 FHM2 is a ‘classic’ migraine with aura, which is linked to a propagating wave of neuronal depolarization similar to cortical spreading depression³ followed with long-lasting depression.⁴ This spreading depolarization is often reported to cause long-lasting oligemia.⁵ Although this response is often biphasic and highly variable,³ recent studies of FHM2 patients with prolonged aura suggested initial multifocal hypoperfusion in the affected hemisphere.^6,7 This initial hypoperfusion is followed by hyperperfusion and is in line with the reports on cerebral blood flow changes during migraine attack over the last ∼30 years.⁸ The mechanism by which spreading depression leads to changes in cerebral blood flow is not fully understood.

A broad spectrum of functional abnormalities of the Na,K-ATPase α2 isoform have been identified for more than 80 FHM2-associated mutations.² These mutations lead to changes in the enzymes properties, and may cause haploinsufficiency.^1,2 In contrast to the ubiquitously expressed α1 isoform that serves a housekeeping role for Na⁺/K⁺ homeostasis, the α2 isoform is characterized by a tissue-specific expression and is suggested to be involved in a number of signalling pathways.⁹ In the brain, the α2 isoform is mostly expressed in glia and the vasculature. Since glia is an important modulator of neuronal activity, attention was previously given to glia in studies of the mechanisms important in FHM2 with limited interest in vascular (dys)function.^1,2,10

Importantly, the vascular Na,K-ATPase α2 isoform has been suggested to control [Ca²⁺]_i signalling by forming a signalosome with other Ca²⁺ transport proteins such as the Na,Ca-exchanger (NCX).¹¹ In accordance with this model, potentiation of [Ca²⁺]_i signalling upon α2 isoform inhibition is suggested to be responsible for the pro-contractile action of ouabain in the vasculature.¹² However, another Na,K-ATPase inhibitor, digoxin does not potentiate vascular contraction,^13,14 suggesting that the Na,K-ATPase-dependent modulation of arterial contractility cannot be limited to a direct effect on ion homeostasis.^9,12 The Na,K-ATPase has previously been shown to initiate several signalling pathways including cSrc kinase signaling.¹⁵ This is supported by Y418 phosphorylation-dependent activation of cSrc kinase upon ouabain application,^16–22 although the mechanism for this cSrc activation is debated.^23–25 Of note, acute application of ouabain can activate a cSrc-dependent pathway in rat mesenteric arteries, and modulate intercellular communication in the vascular wall.²⁶ Chronic ouabain treatment also activates cSrc in rat mesenteric arteries.¹⁴ Src kinase family signalling plays an important role in cerebrovascular myogenic contraction^27–29 and vasospasm,³⁰ although a detailed mechanistic understanding is lacking.

Mice bearing one of the mutations found in FHM2 patients, i.e. G301R,³¹ were recently generated and their behavioural and neuronal properties characterized.³² Homozygous knock-in mutant mice die after birth, while heterozygous mice (Atp1a2^+/−G301R) demonstrate FHM2-relevant disease traits, including mood depression, stress-induced anhedonia, obsessive-compulsive disorder and increased acoustic startle response.³² Moreover, electrocorticographic recordings in vivo showed that regeneration after induction of cortical spreading depression was significantly reduced in brains from Atp1a2^+/−G301R mice, suggesting that the spontaneous activity is more likely affected in these mice than the evoked activity.³²

In the current study, it is hypothesized that these Atp1a2^+/−G301R mice have abnormal cerebrovascular function, affecting cerebral blood flow. The Atp1a2^+/−G301R mice were found to have increased vascular contractility specific for cerebral compared to systemic mesenteric arteries. Accordingly, resting cerebral blood flow was reduced in these mice. This hypercontractility is suggested to be a factor behind the mechanism of aura-associated regional hypoperfusion in the brain, linked to the significance of cSrc-dependent sensitization of vascular smooth muscle cells to Ca²⁺.

Methods

A detailed ‘Methods’ is available in the Supplementary Information file.

All experiments conformed to guidelines from the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes and were approved by and conducted with permission from the Animal Experiments Inspectorate of the Danish Ministry of Environment and Food. Animal experiments were reported in accordance with the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines (www.nc3rs.org.uk/arrive-guidelines).

Atp1a2^+/−G301R mice

The Atp1a2^+/−G301R mice were generated by introduction of the G301R mutation in Atp1a2 gene, as described previously.³² Homozygote pups died immediately after birth, resembling the lethal phenotype previously reported for other genetic disturbances in the Atp1a2 gene.^1,2,33 Heterozygous Atp1a2^+/−G301R (n = 82) and wild type (WT) (n = 86) mice ∼12–16 weeks old were used in the current study. A previous study found some sex-coupled differences in behavioural tests.³² However, in the current study, an equal number of males and females were used, as no sex-coupled difference was seen, and data from male and females were pooled in the final analyses. Notably, the lack of sex difference suggests that female sex hormone/s may have a modulatory action on migraine prevalence via neuronal/astrocytic signalling³² rather than via a vascular wall effect. Middle cerebral arteries and mesenteric small arteries from littermate Atp1a2^+/−G301R and WT mice were used.

Quantitative polymerase chain reaction

The quantitative polymerase chain reaction (qPCR) was carried out using Taqman probe (FAM) technology. Gene expression (the total Atp1a2 expression or expression of WT Atp1a2 allele only) was normalized to reference gene (Gapdh) and presented as a ΔCt value. Comparison of gene expression was derived from subtraction of averaged WT ΔCt in the experiment from ΔCt value in the analysed probe (either WT or Atp1a2^+/−G301R), producing ΔΔCt. Relative gene expression was calculated as 2^–ΔΔCt.

Semi-quantification of cSrc and MYPT phosphorylation

Arterial segments were fixed, lysated and used for Western blotting as previously described.²⁶ Membranes for identification of proteins were cut at approximately 100 kDa, 50 kDa and 35 kDa, and the upper segment used for MYPT detection (expected band ∼ 130 kDa), middle segment for cSrc detection (expected band ∼ 65 kDa), and the lower segment for the reference protein thioredoxin 2 detection (expected band ∼ 12 kDa). The membranes were incubated with primary antibodies overnight at +4℃ and then with horseradish-peroxidase-conjugated secondary antibody (1:2000; Dako, Denmark) for 1.5 h. Bound antibodies were detected by an enhanced chemiluminescence kit (ECL, Amersham, UK).

Proteins (MYPT or cSrc) were semi-quantified by measuring band intensities using ImageJ software (NIH, USA). Protein phosphorylation was semi-quantified as a ratio between phospho-protein and total protein. The average measurements from WT group under resting conditions (i.e. in the absence of drugs) were set to 100% to compare the effects of drug interventions detected from the same gel. The total cSrc and MYPT protein expression were individually normalized to the corresponding band intensity of thioredoxin 2 from the same load, and semi-quantified by setting normalized intensity measured for WT group to 100%.

Whole-mount staining of arterial segments

The brain and mesentery were removed from perfusion-fixed mice³⁴ and arteries dissected out. Tissues were incubated at room temperature for 2 h in blocking buffer (1% BSA and 0.2% Triton in PBS), further washed and incubated overnight with primary antibody in blocking buffer at 4℃, washed again and incubated in secondary antibody in 0.1% Triton in PBS for 2 h at room temperature. Tissue was subsequently mounted in anti-fade mounting media; with the media of select preparations also containing 0.002% propidium iodide to clarify cell layer patency. Tissue was imaged with uniform confocal settings. Controls involved peptide block of primary antibody in a 1:10 (v/v) excess of the immunizing peptide; and secondary only was used as a ‘zero’ setting.

Isometric force measurement of isolated arteries

Mice were sacrificed by cervical dislocation, and the brain and mesentery dissected into ice-cold physiological salt solution. Arteries were cleaned of connective tissue and mounted in a wire myograph (Danish Myo Technology A/S, Denmark) for isometric force measurements as previously described.²⁶ The study was performed on endothelium intact arteries, unless otherwise stated.

Simultaneous measurements of isometric force and [Ca²⁺]_i

Ratiometric [Ca²⁺]_i measurements using fura 2-acetoxymethyl ester were obtained simultaneously with force measurements in a myograph as described elswhere.³⁵ It has been previously shown that this loading protocol enables measurements of smooth muscle [Ca²⁺]_i with negligible contribution from endothelial cells.³⁶ [Ca²⁺]_i was expressed as the ratio of fluorescence during excitation at 340 nm and 380 nm.

Simultaneous measurements of isometric force and membrane potential

Smooth muscle membrane potential in the intact vascular wall was measured in arteries mounted in an isometric myograph (Danish Myo Technology A/S, Denmark) as previously described.³⁷ Membrane potential was averaged over a 1 min recording, at least 1 min after U46619 application.

Morphometric measurements

Arteries were mounted in a wire myograph and morphometric measurements performed using a light microscope (40× water immersion lens) as previously described.^38,39 Measurements were obtained at three different points on either side of the vessel.

Isobaric tone of isolated cerebral arteries

A segment of middle cerebral artery was dissected and cannulated using glass micro cannulas and mounted in a pressure myograph (111P, DMT), as previously described.³⁵ The pressure-step protocol was repeated for arteries under control conditions in PSS and after 15 min incubation in Ca²⁺-free PSS in the presence of 30 µM papaverine and 10 µM Y27632. The outer diameters obtained under these conditions were considered active (AD) and passive diameters (PD), respectively. The degree of tone at each pressure level was quantified as [1 – (AD/PD)].

Laser speckle analysis

Laser speckle (LS) imaging experiments were performed in order to assess resting blood flow in cerebral arteries in vivo (Figure 1(a) and Supplemental Figure 1(a)). A segmentation algorithm^40,41 was applied to extract diameter and corresponding speckle contrast values for selected arteries (Supplemental Figure 1(b)). These values were then averaged over the vessel segment and used to estimate volumetric blood flow dynamics (blood flow index).

Figure 1.

Atp1a2^+/−G301R mice have reduced resting blood flow through branches of middle cerebral artery, in comparison with WT. Whole-brain averaged Laser Speckle intensity image showing cerebral blood vessels (a). White arrows identify branches of middle cerebral artery (Supplemental Figure 1(a)) that were used for Laser Speckle analyses. Analyses did not found any significant changes in arterial diameter (b; n = 6–7), while blood flow index (an estimate volumetric blood flow dynamics) was significantly reduced in Atp1a2^+/−G301R mice in comparison with WT (C; *P < 0.05).

pNaKtide peptide

The pNaKtide peptide²² (HD Biosciences, China) is composed of 20 amino acids from the N–domain of the Na,K-ATPase and a leader peptide sequence of 13 amino acids that allows cell membrane penetration. Prior studies have shown that it is effective in blocking the Na,K-ATPase-dependent cSrc activation.^{20,22,26,42–44}

Data analyses

Microsoft Excel and GraphPad Prism software (v.5.02) were used for graphing and statistical analysis. Data are summarized as the mean value ± SEM of the sample group. Concentration-response curves were fitted to experimental data using four-parameter, non-linear regression curve fitting. From these curves, pD₂ (-log to the concentration required to produce a half-maximal response), the Hill slope and maximal response were derived and compared using an extra sum-of-squares F test to assess the overall significance for a regression model.^45,46 Area under the concentration-response curve (AUC) was calculated based on trapezoidal rule. Significant differences between means were determined by either one-way or two-way ANOVA, where appropriate, followed by Bonferroni correction for multiple comparison or by Student t-test. A probability (P) level of <0.05 was considered significant.

Results

Mice bearing G301R mutation have reduced brain perfusion

Estimation of diameter and blood flow in branches of middle cerebral artery in vivo (Supplemental Figure 1(a)) in anaesthetized mice demonstrated significantly reduced resting blood flow in Atp1a2^+/−G301R mice in comparison with WT, although no significant difference in diameter was seen (Figure 1).

G301R mutation modifies expression of the Na,K-ATPase in the vascular wall

The total amount of the α₂ isoform Na,K-ATPase mRNA in middle cerebral artery isolated from the Atp1a2^+/−G301R mice was similar to that from WT (Figure 2(a)). However, when primers amplifying only the WT allele were used, cerebral artery from the Atp1a2^+/−G301R mice had ∼50% of mRNA transcripted from the WT allele (Figure 2(b)).

Figure 2.

The α₂ isoform Na,K-ATPase haploinsufficiency of middle cerebral arteries from Atp1a2^+/−G301R mice. Quantitative PCR results showed an unchanged expression of total Atp1a2 mRNA (n = 6) in comparison with WT (n = 8) (a). In arteries from the Atp1a2^+/−G301R mice, approximately a half of the α₂ isoform mRNA is from the WT allele and the rest is from the mutated G301R allele, as it is shown with primers amplifying only the WT allele (b; n = 6–8). Whole-mount staining indicated the presence of α₂ isoform protein in both endothelial cells and smooth muscles (c; representative images). Semi-quantitative fluorescence demonstrated a reduction of the α₂ isoform in middle cerebral arteries from the Atp1a2^+/−G301R mice in comparison with WT (d; n = 5). The α₁ isoform Na,K-ATPase is reduced in endothelial cells from the Atp1a2^+/−G301R mice in comparison with WT (e, representative images; and f, averaged results; n = 5). In inserts, ‘+peptide’ indicates a control experiment where primary antibody was blocked with an excess of the immunizing peptide. ‘2° only’ shows staining with secondary antibody only that was used as a ‘zero’ setting. * and ***, P < 0.05 and 0.001 vs. WT (one-way ANOVA).

Consistent with previous reports on FHM2-associated α₂-isoform haploinsufficiency,² we found an ∼50% reduction in α₂ isoform protein in cerebral arteries from the Atp1a2^+/−G301R mice in comparison with WT (Figure 2(c) and (d)). This reduction was seen in both endothelial and smooth muscle cells. The α₁ isoform Na,K-ATPase (Figure 2(e) and (f)) and the NCX (Supplemental Figure 2) were also significantly reduced in cerebral arteries from the Atp1a2^+/−G301R mice.

Middle cerebral arteries from Atp1a2^+/−G301R mice demonstrated stronger agonist-induced constriction and depolarization in comparison with WT

Middle cerebral arteries from Atp1a2^+/−G301R mice mounted in isometric myograph had larger lumen diameter (P < 0.026) than arteries from WT; 175 ± 5 µm (n = 33) in comparison 161 ± 4 µm (n = 41), respectively. Media thickness and media-to-lumen ratio were also increased in arteries from Atp1a2^+/−G301R mice in comparison to WT (Supplemental Figure 3). Pressurized arteries from Atp1a2^+/−G301R mice had increased diameter in Ca²⁺-free solution (Supplemental Figure 4(a)). In the presence of Ca²⁺, pressurized cerebral arteries developed myogenic tone, which was similar in arteries from WT and Atp1a2^+/−G301R mice (Supplemental Figure 4(a) and (b)).

The thromboxane A₂ agonist, U46619, constricted and depolarized mouse middle cerebral arteries (Figure 3). This constriction was significantly potentiated in arteries from Atp1a2^+/−G301R mice compared with WT (Figure 3(a)). Since endothelium-dependent relaxation of the two groups were similar (Supplemental Figure 4(c)), and contractile responses of cerebral arteries from the Atp1a2^+/−G301R mice remained stronger than WT after removal of the endothelium (Suppl. Fig. 4D), the differences in the contractile responses are unlikely to be due to changes in endothelial function. The rest of the study was performed with endothelium intact arteries.

Figure 3.

Middle cerebral but not small mesenteric arteries from Atp1a2^+/−G301R mice had increased agonist-induced constriction and depolarization in comparison with WT. Concentration-response curves to U46619 of middle cerebral arteries (a) from Atp1a2^+/−G301R (n = 7) and WT mice (n = 9). F test indicated a significant difference between experimental groups (***, P < 0.05). No difference in U46619-induced constriction was seen between mesenteric small arteries from Atp1a2^+/−G301R and WT mice (b; n = 6). A representative simultaneous recording of wall tension and membrane potential in WT middle cerebral artery stimulated with U46619 (c). Wall tension (d) and membrane potentials (e) under resting conditions and after 0.3 µM U46619 stimulation of middle cerebral arteries from Atp1a2^+/−G301R (n = 6) and WT mice (n = 8). The experimental protocol is similar to data shown in c. *, P < 0.05, comparison between Atp1a2^+/-G301R and WT groups; +++, P < 0.001, the effect of U46619 (two-way ANOVA).

When wall tension and membrane potential were measured simultaneously (Figure 3(c)), no difference between the groups was observed under resting conditions (Figure 3(d) and (e)). However, in the presence of 0.3 µM U46619, a stronger constriction of cerebral arteries from Atp1a2^+/−G301R (Figure 3(d)) was associated with larger smooth muscle depolarization (Figure 3(e)).

The enhanced constriction of Atp1a2^+/−G301R cerebral arteries is associated with increased sensitization to [Ca²⁺]_i

To test whether the increased depolarization of the arteries from Atp1a2^+/−G301R are associated with elevated Ca²⁺ influx, [Ca²⁺]_i was measured simultaneously with wall tension (Figure 4). No significant difference in the resting [Ca²⁺]_i between the groups was seen. Surprisingly, the increased vasoconstriction of the arteries from Atp1a2^+/−G301R (Figure 4(a)) was associated with a smaller increase in [Ca²⁺]_i in comparison with arteries from WT (Figure 4(b)). This relation between wall tension and [Ca²⁺]_i suggests an increased sensitization of contractile machinery to Ca²⁺. Indeed, when wall tension is plotted as a function of [Ca²⁺]_i (Figure 4(c)), arteries from Atp1a2^+/−G301R demonstrated a steeper slope, i.e. an increased sensitization to [Ca²⁺]_i.

Figure 4.

Potentiated constriction to U46619 of middle cerebral arteries from Atp1a2^+/−G301R mice is associated with smaller changes in [Ca²⁺]_i suggesting higher sensitivity to Ca²⁺. U46619 concentration-dependent vasoconstriction (a) of middle cerebral arteries from Atp1a2^+/−G301R mice (n = 6) was significantly (***, P < 0.01; F test) potentiated in comparison with WT (n = 5). However, changes of [Ca²⁺]_i (b) were less pronounced (***, P < 0.01; F test) in arteries from Atp1a2^+/−G301R (n = 6) compared to WT (n = 5). Arteries from Atp1a2^+/−G301R mice had a steeper relation between [Ca²⁺]_i and wall tension (**, P < 0.01 for slopes based on linear regression analysis) in comparison with WT (c; re-plotted data from a and b). The relation between [Ca²⁺]_i and membrane potential (d; re-plotted data from Figures 2(d) and 3(b)) suggested a lesser [Ca²⁺]_i rise in arteries from Atp1a2^+/−G301R despite the larger depolarization.

Interestingly, in the presence of 0.3 µM U46619 arteries from Atp1a2^+/−G301R mice experienced a reduced [Ca²⁺]_i increase despite a larger membrane depolarization (Figure 4(d)). This suggests that arteries from Atp1a2^+/−G301R mice have reduced voltage-dependent Ca²⁺ influx in compared to WT.

An increased Ca²⁺ sensitization of middle cerebral arteries from Atp1a2^+/−G301R mice is not agonist-specific

To test whether the observed Ca²⁺ sensitization was specific for U46619 induced constriction, we stimulated middle cerebral arteries with endothelin (Figure 5(a) to (c)). Arteries from Atp1a2^+/−G301R mice constricted to endothelin stronger than those from WT (Figure 5(a)). However, endothelin-induced [Ca²⁺]_i increase was not significantly different between arteries from WT and Atp1a2^+/−G301R mice (Figure 5(b)). The slope of wall tension as a function of [Ca²⁺]_i was not significantly different between the groups, although there was a tendency for increased Ca²⁺-sensitivity in cerebral arteries from Atp1a2^+/−G301R mice (Figure 5(c); P = 0.13).

Figure 5.

Increased Ca²⁺ sensitization of middle cerebral arteries from Atp1a2^+/−G301R mice is independent of vasoconstrictor type. Constriction of WT (n = 5) and Atp1a2^+/−G301R (n = 6) arteries to endothelin (a) and simultaneous recordings of [Ca²⁺]_i expressed as a ratio of Fura-2 fluorescence at 340 and 380 nm (b). The relation between [Ca²⁺]_i and wall tension at different concentrations of endothelin (c; data re-plotted from a and b). Constriction (d) and simultaneous changes in [Ca²⁺]_i (e) in response to K⁺-induced depolarization. Changes in relation between [Ca²⁺]_i and wall tension in response to K⁺-induced depolarization (f). ** and *** indicates P < 0.01 and 0.001; n = 5–6.

Arteries from Atp1a2^+/−G301R mice constricted also to a greater extent to K⁺-induced depolarization (in the presence of 1 µM phentolamine) than WT (Figure 5(d)), but this was associated with an attenuated [Ca²⁺]_i increase (Figure 5(e)). This led to a steeper slope of [Ca²⁺]_i – wall tension relation for cerebral arteries from Atp1a2^+/−G301R mice in comparison with WT, suggesting increased Ca²⁺-sensitization (Figure 5(f)).

Contractile responses of cerebral arteries from Atp1a2^+/−G301R and WT mice became similar in the presence of ouabain, but not digoxin

Constriction of middle cerebral arteries from the two groups of mice was compared before and after pre-treatment with Na,K-ATPase inhibitors, digoxin and ouabain (Supplemental Figure 5). Digoxin (10 µM) potentiated slightly (P = 0.059) contractile response of arteries from WT, but this was without any effect (P = 0.483) in arteries from Atp1a^2+/−G301R mice (Supplemental Figure 5(a)). The difference in contractile responses between the groups (i.e. a stronger constriction in arteries from Atp1a^2+/−G301R compared to WT) was not affected by digoxin (Supplemental Figure 5(a)). Ouabain (10 µM) potentiated cerebral artery constriction from both groups (Supplemental Figure 5(b)), while 0.1 µM (n = 6) and 1 µM (n = 10) ouabain was without significant effect (data not shown). In the presence of 10 µM ouabain, no difference in the constriction of cerebral arteries from Atp1a^2+/−G301R and WT mice was seen (Supplemental Figure 5(b)).

Inhibition of cSrc kinase abolished the difference in contractile responses of cerebral arteries from the Atp1a2^+/−G301R and wild type mice

Pre-incubation of cerebral arteries with 3 µM PP2 abolished the difference in contractile responses between the groups (Figure 6(a)). PP2 suppressed constriction of cerebral arteries and there was a tendency for a larger effect of PP2 inhibition on the arteries from Atp1a2^+/−G301R mice in comparison with WT (Figure 6(a)). This was associated with reduced [Ca²⁺]_i changes, which were similar between the groups in the presence of PP2 (Figure 6(b)). Consistent with this, the relationship between wall tension and [Ca²⁺]_i was not different between cerebral arteries from Atp1a2^+/−G301R and WT mice in the presence of PP2 (Figure 6(c)).

Figure 6.

Inhibition of cSrc kinase abolished the increased force development, [Ca²⁺]_i, and Ca²⁺ sensitivity in cerebral arteries from Atp1a2^+/−G301R mice. PP2 (3 µM) significantly suppressed constriction of cerebral arteries from Atp1a2^+/−G301R (***, P < 0.001; n = 5) and WT (+++, P < 0.001; n = 5), and abolished the difference between the groups seen under control conditions (##, P < 0.01) (a). This was associated with suppression of [Ca²⁺]_i changes in response to U46619 stimulation (b). Under control conditions [Ca²⁺]_i changes were larger (###, P < 0.001; n = 5) in WT than in Atp1a2^+/−G301R, but PP2 significantly suppressed responses in Atp1a2^+/−G301R (***, P < 0.001; n = 5) and WT (+++, P < 0.001; n = 5) groups and the difference between the groups was abolished. The relation between [Ca²⁺]_i and wall tension (c) was re-plotted from the data obtained in the presence of PP2 and shown in A and B. No difference in the slopes was found. pNaKtide (2 µM) also suppressed contraction of cerebral arteries from Atp1a2^{+/− G301R} (***, P < 0.001; n = 6–7) and WT (+++, P < 0.001; n = 6–7) mice and abolished the difference between the groups seen under control conditions (###, P < 0.001) (d). [Ca²⁺]_i changes in response to U46619 were less in cerebral arteries from Atp1a2^+/-G301R mice in comparison with WT (###, P < 0.001; n = 6–7) (e). [Ca²⁺]_i changes were significantly suppressed by pNaKtide in Atp1a2^+/-G301R (***, P < 0.001; n = 6–7) and WT (+++, P < 0.001; n = 6–7) groups. The relation between [Ca²⁺]_i and wall tension (f) in the presence of pNaKtide was re-plotted from the data shown in D and E; no difference between the slopes was found. Inserts in A, B, D, E shows differences in area under concentration-response curves (AUC) before and after treatment with PP2 (a, b) and pNaKtide (d, e). & indicates P < 0.05.

pNaKtide (2 µM) also suppressed contraction and abolished the difference in contractile responses between the groups (Figure 6(d)). The effect of pNaKtide on wall tension was significantly larger for cerebral arteries from Atp1a2^+/−G301R mice in comparison with arteries from WT, but this was associated with similar suppression of [Ca²⁺]_i changes (Figure 6(e)). Under control conditions [Ca²⁺]_i, changes were larger in cerebral arteries from WT than in Atp1a2^+/−G301R (Figure 4(b)), but this difference was abolished in the presence of pNaKtide (Figure 6(e)). The relationship between [Ca²⁺]_i and wall tension in the presence of pNaKtide was similar for cerebral arteries from Atp1a2^+/−G301R and WT mice, suggesting a similar level of sensitization for Ca²⁺ under these conditions (Figure 7(f)).

Figure 7.

Semi-quantitative analyses of expression and phosphorylation of cSrc kinase and MYPT1 proteins in middle cerebral arteries from Atp1a2^+/−G301R and WT mice. Representative WT Western blot where MYPT 1 and cSrc expression and phosphorylation were studied (a). Thioredoxin 2 (Trx2) was used as loading control. Total cSrc kinase expression (normalized to Trx2 band density) was reduced in cerebral arteries from Atp1a2^+/−G301R compared to WT mice (b; n = 6–9). Averaged results suggest an increased cSrc phosphorylation (calculated as a ratio between phosphorylated and total cSrc, and normalised to averaged values for WT under resting conditions) in arteries from Atp1a2^+/−G301R mice compared to WT after U46619 (10⁻⁵ M) stimulation (C; n = 5–11). Incubation with pNaKtide (2 µM) abolished this difference. Expression of total MYPT1 (normalized to Trx2 band density) was not different between the groups (n = 4–12; d, representative Western blot; e, averaged data). U46619 significantly increased MYPT1 phosphorylation at Thr850 (n = 4–12; f) (calculated as a ratio of phosphorylated and total MYPT1, and normalised to averaged values for WT under resting conditions). This potentiation is stronger in cerebral arteries from Atp1a2^+/−G301R than WT. In the presence of pNaKtide this potentiation was abolished, and there was no difference between arteries from Atp1a2^+/−G301R and WT. *, ** and ***, P < 0.05, < 0.01 and < 0.001 (two-way ANOVA).

Enhanced phosphorylation of cSrc and MYPT in middle cerebral arteries from Atp1a2^+/−G301R compared to WT mice

Despite a slightly reduced total expression of cSrc kinase in middle cerebral arteries from Atp1a2^+/−G301R mice (Figure 7(b)), no significant difference in phosphorylated cSrc was found between cerebral arteries from Atp1a2^+/−G301R and WT mice under resting conditions (Figure 7(c)). When cerebral arteries were stimulated with U46619, the phosphorylation of cSrc was significantly increased and this increase was stronger in arteries from Atp1a2^+/−G301R than in WT mice. Moreover, pNaKtide significantly suppressed cSrc phosphorylation induced by U46619 (Figure 7(c)).

The expression of myosin phosphatase targeting protein 1 (MYPT) was similar between cerebral arteries from Atp1a2^+/−G301R and WT mice (Figure 7(d)), as well as MYPT1 phosphorylation under resting conditions (Figure 7(d)). U46619 induced MYPT1 phosphorylation, which was significantly larger in arteries from Atp1a2^+/−G301R compared to WT mice (Figure 7(d)). This difference in U46619-induced MYPT1 phosphorylation was abolished by pNaKtide.

Contractile responses of mesenteric small arteries did not differ between Atp1a2^+/−G301R and WT mice

In contrast to middle cerebral arteries, mesenteric arteries from the Atp1a2^+/−G301R and WT mice showed similar agonist-induced constriction (Figure 3(b)). Ouabain significantly potentiated constriction of both groups (Supplemental Figure 6(a) and (b)). In the presence of ouabain, there was no significant difference in constriction of mesenteric arteries from the Atp1a2^+/−G301R and WT mice.

Mesenteric arteries from the Atp1a2^+/−G301R and WT mice constricted in a similar manner to different contractile agonists, e.g. noradrenaline (NA; Supplemental Figure 7(a)) and U46619 (Supplemental Figure 7(b)). Incubation with pNaKtide was without any significant effect on contractile response of arteries from Atp1a2^+/−G301R; however, it suppressed the constriction of arteries from WT mice (Supplemental Figure 7). This difference in the effects of pNaKtide between the groups was not due to endothelial function, since it was similarly affected by pNaKtide in both groups, i.e. pNaKtide was without any effect on relaxation to 3 µM ACh (Supplemental Figure 7(c)) and suppressed relaxation to 0.1 mM ACh (Supplemental Figure 7(d)).

In contrast to cerebral arteries (Figure 2), whole-mount immunostaining of mesenteric arteries demonstrated an elevation of both α₁ and α₂ isoforms of the Na,K-ATPase in the Atp1a2^+/−G301R mice in comparison with WT (Supplemental Figure 8(a) to (d)). However, the expression of NCX in mesenteric arteries from the Atp1a2^+/−G301R mice was reduced (Supplemental Figure 8(e) to (f)), similar to cerebral arteries (Supplemental Figure 2).

No difference in total cSrc and its phosphorylation levels was found between the Atp1a2^+/−G301R and WT mice (Supplemental Figure 9(a) to (c)). Stimulation with NA increased cSrc phosphorylation in both groups without any difference between them (Supplemental Figure 9(c)). Incubation with pNaKtide suppressed cSrc phosphorylation.

Total MYPT1 expression and baseline MYPT1 phosphorylation were also the same in mesenteric arteries from the Atp1a2^+/−G301R and WT mice (Supplemental Figure 9(a), (d) and (e)). NA elevated MYPT1 phosphorylation, and this increase was the same for both Atp1a2^+/−G301R and WT mice. Incubation with pNaKtide reduced NA-induced MYPT1 phosphorylation in mesenteric arteries from both groups (Supplemental Figure 9(d) and (e)).

Discussion

Mutation of the Na,K-ATPase α2 isoform causes FHM2 in humans. In this study, increased middle cerebral artery contractility in mice with a mutation in the Na,K-ATPase α2 isoform was found. This was found to be associated with complex changes in excitation-contraction coupling. These changes can be summarized (Supplemental Figure 10) as cerebral artery smooth muscle cells from the Atp1a2^+/−G301R mice being depolarized more to agonist stimulation, with this being paradoxically associated with a lesser increase in [Ca²⁺]_i compared to WT. Furthermore, the reduced [Ca²⁺]_i increase was unexpectedly associated with an increased force development. These findings suggest that an activation of cSrc kinase may be the mechanistic link, which can explain the complex changes in the excitation-contraction coupling (Supplemental Figure 10). A second important finding of this study is that the increased contractility may be confined to the cerebral, over systemic mesenteric arteries.

FHM2 is characterized with perfusion abnormalities in the brain

Biphasic changes in cerebral blood flow during a migraine attack in FHM2 patients are established.^6,47 Although it is highly variable between reports,^47,48 an initial hypoperfusion (i.e. vasoconstriction) in the affected hemisphere is linked to spreading depolarization of neural tissue and followed by hyperperfusion.^3,49 Vasoconstriction has been hypothesized to be a consequence of abnormal neuronal excitation,^8,50 although it might also be a result of increased concentration of contractile factors in the circulation.^51–53 Thus, plasma thromboxane A₂ is known to be elevated in migraine patients.⁵¹ Moreover, interical plasma concentration of endothelin-1, and especially its ictal concentration at the beginning of attack, is also known to be elevated in migraine with aura.^52,53

How spreading depression leads to cerebral artery constriction is unknown. It has been hypothesized that an abnormally high accumulation of [K⁺]_o and/or other metabolites, neuromediators, or hyperactivation of perivascular innervation could contribute to regional vasoconstriction in the brain.³ Data from the present study suggest that cerebral artery smooth muscle cells with the FHM2-associated mutation have increased sensitivity to [Ca²⁺]_i. Thus, stimuli, which are usually subthreshold for a normal subject, might lead to regional cerebral vasoconstriction and hypoperfusion in FHM2 patients, and in this mouse model. This also implies that changes in perfusion might at least be partly independent of altered neuronal activity, to originate within the vascular wall. Consistent with this, we found that the Atp1a2^+/−G301R mice have reduced perfusion through middle cerebral arteries under resting condition. We did not find any significant difference in arterial diameter in vivo. This suggests a possible elevation of downstream resistance of microvessels exposed to circulatory and perivascular vasoactive factors in vivo.

Atp1a2^+/−G301R mouse model for vascular haploinsufficiency of the Na,K-ATPase α2 isoform

Previously, attention was given to abnormal function of the mutated Na,K-ATPase α2 isoform in glial cells in FHM2.^2,54 However, it is also suggested that the FHM2 disease phenotype cannot be explained by impaired astrocytic function alone, as the α2 isoform constitutes a relatively minor component (∼20%) of the total astrocytic Na,K-ATPase,⁵⁵ and even less (∼10%) in terms of activity.⁵⁶ An ∼50% reduction in the Na,K-ATPase α2 isoform in all brain regions of the Atp1a2^+/−G301R mice has been reported.³² This is consistent with other reports on haploinsufficiency of the FHM2-associated mutations.^54,57 The present study also found a reduction of α2 Na,K-ATPase protein expression in middle cerebral arteries from Atp1a2^+/−G301R mice of ∼50%. The present and other findings^32,54,57 are not in agreement with another report where no significant reduction of the Na,K-ATPase α2 isoform was found in Atp1a2^+/−G301R mouse hippocampus.⁵⁸ The reason for this discrepancy is unknown, but may represent variability within the brain, to reflect the current data comparison of cerebral and systemic (mesenteric) vascular beds.

Surprisingly, an increased α2 Na,K-ATPase expression in mesenteric arteries from the Atp1a2^+/−G301R mice was found. This suggests that α2 isoform maturation, transport, insertion and / or degradation are controlled differently in cerebral and mesenteric arteries. Since the present study is unable to discriminate between mutated and WT proteins, we do not know how much of the mutated protein is expressed, and what the functional consequence of this is in these arteries. However, based on similar contractile responses, we suggest that the functional contribution of the α2 isoform may be unchanged in mesenteric arteries from Atp1a2^+/−G301R mice. It is unknown which factor(s) are responsible for the differences in the expression profile of these two vascular beds, but notably, the G301R mutant was previously shown to be expressed differently in different cell types, e.g. the G301R mutant was normally expressed in oocytes,⁵⁹ but not in neurons³² and oocytes⁵⁸ in other studies, alluding to differences in endothelial and smooth muscle phenotype and function between different arteries and states.⁶⁰

The G301R mutation is associated with changes in the expression of membrane transporters

The present study found that expression of the α1 isoform was also suppressed in cerebral arteries from Atp1a2^+/−G301R mice. This is in contrast to previous reports where the expression of other isoforms of the Na,K-ATPase was not affected by changes in the expression of the α2 isoform.^32,54 The reason for this is unclear, but an increase of cSrc activation, discussed below, could potentially contribute to changes in the smooth muscle phenotype and the expression profile.⁶¹ The present study also found a reduction of NCX expression, which is consistent with suppression of the NCX after siRNA-induced downregulation of the α2 isoform in rat mesenteric arteries.³⁵ Accordingly, chronic ouabain treatment upregulated Na,K-ATPase α2 isoform expression in rat mesenteric artery smooth muscle cells, and this was associated with increased NCX expression.⁶² Collectively, this suggests that expression of the Na,K-ATPase α2 isoform and the NCX is closely linked and might be associated with smooth muscle cell phenotype and vascular remodelling of cerebral arteries, per the present data.

It has been suggested that the Na,K-ATPase modulates expression of other proteins via the cSrc kinase pathway.¹⁴ In the present study, changes in cSrc kinase expression and activation in cerebral arteries of Atp1a2^+/−G301R mice were found. The mechanism for cSrc-dependent transcriptional regulation is unclear. Notably, tyrosine phosphorylation of STATs (signal transducers and activators of transcription) has been shown to trigger their activation and translocation to the nucleus where it can subsequently upregulate nuclear factor of activated T-cells (NFAT) expression.⁶³ The calcineurin/NFAT pathway can modulate expression of Ca²⁺ transport proteins including the NCX.⁶⁴ It is however unclear whether changes in NCX expression in the present study are dependent on cSrc kinase activation or mediated via altered Ca²⁺ homeostasis. Further work with chronic inhibition of NFAT and cSrc kinase is necessary to test these possibilities.

Expression changes in cerebral arteries of Atp1a2^+/−G301R mice are associated with an increased arterial contractility due to sensitization to Ca²⁺

The increased contractility of cerebral arteries cannot simply be explained by reduced pumping activity of the Na,K-ATPase α2 isoform, or by the secondarily reduced NCX. Expression changes and pharmacological manipulation of the Na,K-ATPase α2 isoform can affect vascular tone via modulation of [Ca²⁺]_i through an interaction of the Na,K-ATPase with the NCX.^9,12 In this scenario, suppression of Na,K-ATPase ion pumping activity was suggested to suppress the Na⁺-dependent net Ca²⁺ extrusion leading to increased [Ca²⁺]_i and potentiation of vasoconstriction.^35,65 This is not consistent with the present results, where increased contraction of cerebral arteries of Atp1a2^+/−G301R mice is associated with reduced [Ca²⁺]_i. Previous studies reported that reduced expression of the NCX leads to reduced net Ca²⁺ influx and suppressed arterial contraction.^66,67 Consistently, the present work detected a reduction in the NCX to be associated with a smaller increase of [Ca²⁺]_i. It has been suggested that the reduced [Ca²⁺]_i is a result of functional suppression of the voltage-gated Ca²⁺ influx, consequent to the reduced NCX activity.⁶⁷ The present observation of reduced net Ca²⁺ influx in response to K⁺-induced depolarization and reduced net Ca²⁺ influx in spite of stronger depolarization supports this proposal.^66,67

Reduced [Ca²⁺]_i changes can also be achieved by hyperactivation of the plasma membrane Ca²⁺ ATPase, which has been shown to be activated by the Src kinase family in neurons.⁶⁸ The effect of cSrc kinase can also be mediated via membrane potential due to phosphorylation and inhibition of the voltage-gated and Ca²⁺-activated K⁺ channels.⁶⁹ Accordingly, the present data show a stronger agonist-induced depolarization of cerebral artery smooth muscles in Atp1a2^+/−G301R than in WT.

The difference in contractility between Atp1a2^+/−G301R and WT mice cannot be explained by changes in Ca²⁺ transport proteins, since in mesenteric arteries, the present data found a reduced NCX expression, which was not associated with any significant change in agonist-induced contraction. This suggests limited significance of the NCX for agonist-induced contraction of smooth muscles, at least in mesenteric arteries, although some importance of cerebral artery [Ca²⁺]_i homeostasis cannot be excluded. Reversal potential for the NCX in vascular smooth muscle cells is approximately −30 mV;⁷⁰ i.e. near the maximal depolarization level in response to maximal agonist stimulation. This suggests that the NCX cannot be the major contributor for Ca²⁺ influx during agonist stimulation and therefore the reduction in its expression did not affect contraction of mesenteric small arteries and cannot explain lower [Ca²⁺]_i changes in cerebral arteries from the Atp1a2^+/−G301R mice.

Stronger contractile responses of cerebral arteries from Atp1a2^+/−G301R mice may result from the increased media-to-lumen ratio found described herein. However, per below, the ‘normalizing’ effect of acute maximal activation of cSrc as well as inhibition of cSrc does not support this suggestion. The mechanism/s underlying cerebral artery remodelling in the present study is unclear, but may be due to changes in hemodynamic parameters, and/or to a cSrc-dependent phenotypic switch in smooth muscle cell signalling.

Elevated cSrc-dependent signalling is responsible for increased Ca²⁺ sensitivity

The Na,K-ATPase has been suggested to form a membrane microdomain with several proteins involved in intracellular signalling, including Src kinase.¹⁵ Ouabain binding to the Na,K-ATPase has been shown to activate cSrc kinase via autophosphorylation at Y418.^{22–24,26,71} There are two views on how the activity of cSrc kinase is modulated by the Na,K-ATPase. One suggestion is that cSrc kinase interacts physically with the Na,K-ATPase.^16–21,72 This view contrasts with the suggestion that changes in ATP consumption by the Na,K-ATPase play a key role for cSrc autophosphorylation.^23–25 The current study is consistent with an interaction between cSrc kinase and the Na,K-ATPase, but cannot distinguish between the two possibilities. Previous reports suggested the importance of either α1 or α2 Na,K-ATPase for cSrc-dependent signaling.^26,43 In the current study, it is not possible to distinguish between these options since the expression of both isoforms was suppressed in the cerebral arteries of Atp1a2^+/−G301R mice.

The present data suggest that potentiation of cerebral vasoconstriction is mediated by elevated cSrc kinase activation via the sensitization of the contractile machinery to [Ca²⁺]_i. The contribution of cSrc kinase to Ca²⁺-sensitization was suggested to be mediated via Rho kinase translocation and subsequent MYPT1 phosphorylation in studies of rat pulmonary arteries,⁷³ aorta,⁷⁴ and mouse mesenteric arteries.⁷⁵ Pharmacological analyses suggest that agonist-stimulated cSrc kinase activation and contribution to the arterial contraction is upstream to Rho kinase activation.⁷⁴ The mechanism important for this interaction is unknown, but leukaemia-associated RhoGEF, which is expressed in smooth muscle⁷⁶ and which can enhance RhoA, is known to be activated by tyrosine phosphorylation.⁷⁷ Moreover, ouabain was suggested to constitutively activate Rho kinase via a caspase-dependent cleavage pathway.⁷⁸ Accordingly, the present results suggest that elevated cSrc kinase activation in the Atp1a2^+/−G301R mice increases the Ca²⁺-sensitivity of cerebral arteries via MYPT1 phosphorylation. The role of Na,K-ATPase-cSrc-MYPT1 signalling was also supported by the effects of tyrosine kinase inhibition. The present work used two non-related inhibitors, a conventional cSrc kinase inhibitor, PP2 and a peptide that inhibits the Na,K-ATPase-dependent cSrc activation,^20,22,43 and both suppressed the contraction of cerebral arteries and partially antagonized MYPT1 phosphorylation. These findings argue against remodelling as a major contributor to elevated cerebrovascular contractility in Atp1a2^+/−G301R mice.

The present work found that although the expression of total cSrc protein was slightly reduced in cerebral arteries from the Atp1a2^+/−G301R mice, phosphorylated cSrc in the arteries from Atp1a2^+/−G301R mice was not significantly different under resting conditions and significantly increased upon agonist stimulation in comparison with WT. This supports the hypothesis that the reduction in Na,K-ATPase permits autophosphorylation of cSrc kinase.¹⁷ The observed reduction in total cSrc expression in cerebral arteries of Atp1a2^+/−G301R mice could be a result of compensatory downregulation when a significant part of the Na,K-ATPase was lost. Consistent with this, no difference in resting tone, [Ca²⁺]_i and membrane potential was seen in vitro. Agonist stimulation further activated cSrc kinase and this was significantly potentiated in cerebral arteries of the Atp1a2^+/−G301R mice; albeit probably due to reduced ‘buffering’ by the Na,K-ATPase. If the level of cSrc activation inversely correlates with the availability of the Na,K-ATPase, two predictions can be made. When the Na,K-ATPase is blocked with ouabain in arteries from the two groups of mice, no difference in contraction should be seen, and when cSrc kinase activity is blocked by PP2 or pNaKtide, no difference in contraction should be seen. Both predictions were confirmed in this study, supporting the hypothesis that elevated contractility of cerebral arteries in Atp1a2^+/−G301R mice is mediated by higher activation of cSrc kinase.

In the present study, digoxin did not activate cSrc signalling in smooth muscles¹⁴and nor did it abolish the difference in contraction of cerebral arteries. Notably, a tendency for digoxin to potentiate contraction of cerebral arteries from WT, but not Atp1a2^+/−G301R mice, suggests that inhibition of the Na,K-ATPase can affect arterial contraction in a mechanism other than that involving the cSrc activation pathway. This is possibly mediated via the NCX-dependent increase in [Ca²⁺]_i,^35,65 which cannot explain the difference between contractile responses cerebral arteries of Atp1a2^+/−G301R and WT mice. Importantly, the difference in contractile responses between the groups was observed at agonist concentrations where [Ca²⁺]_i reached sub-maximal levels. This suggest that the proposed cSrc-dependent Ca²⁺ sensitization has a potentiating action on [Ca²⁺]_i initiated constriction, and is of primary importance in association with vasoconstrictor stimulation, such as at the first stage of a migraine attack.

Limitations

Although FHM2 is a relatively rare form of migraine,¹ further insight into its pathology will contribute to a broader understanding of migraine in general. The increased Ca²⁺ sensitivity of cerebral arteries from mutated mice likely contributes to the aura-associated initial hypoperfusion. This cannot be generalized to all forms of migraine since FHM2 is the only migraine subtype known to be associated with mutations in the α2 isoform Na,K-ATPase. However, the principle can also be applied to other types of migraine where cerebrovascular hypercontractility associated with neuronal excitation may be elicited by other pathways.

This study has used an animal model, and it will be important to translate the findings to patients. It is also unclear whether aura-associated hypoperfusion can be elicited in mice, although we have seen some reduction in resting cerebral blood perfusion. Finally, the mechanisms responsible for regional specific translational modulation/s of the Na,K-ATPase α isoform, NCX and cSrc kinase remain to be elicited.

Conclusion

This study suggests a novel mechanism for hypoperfusion of the brain, which is an initial phase in the migraine attack of FHM2. The proposed mechanism suggests that the α2 isoform dependent regulation of cSrc kinase explains the Ca²⁺-sensitization of the contractile machinery in smooth muscle cells of cerebral arteries. The present work suggests that this signalling pathway has significant importance for cerebral artery contractility, but is of lesser significance for extra-cerebral systemic vessels.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Lundbeck Foundation [grant number R183-2014-3618]; the Novo Nordisk Foundation [grant numbers NNF14OC0012731 and NNF17OC0026198]; the Independent Research Fund Denmark – Medical Sciences [grant number 7025-00015B] the Brain Foundation, Australia.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

CS, LH, SK, RK and PBJ performed myograph experiments, CS and VVM made perfusion fixation, CS, VVM and EVB performed immunoblotting, NL and SLS made whole-mount staining, and data analysis. CS, LH, EVB, ZX, KLH, SLS, CA and VVM took part in the conception and designed the study. CS, ZX, SLS, CA and VVM completed the manuscript writing.

Supplementary material

Supplementary material for this paper can be found at the journal website: http://journals.sagepub.com/home/jcb