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) 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 Ca and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2 and WT showed similar contractile responses. The Atp1a2 MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca change was smaller than in WT. The enhanced constriction of Atp1a2 MCA was associated with increased cSrc activation, stronger sensitization to [Ca] and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2 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 Ca, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Involvement of the Na+,K+‐ATPase isoforms in control of cerebral perfusion

“…We therefore suggested that NVC was changed in FHM2 and tested this hypothesis in a knock‐in mouse model carrying a G301R mutation in the Na + ,K + ‐ATPase α2 isoform (Bøttger et al., ). We demonstrated that cerebral arteries from FHM2 mice have increased contractility that might compromise brain perfusion and suggested the underlying molecular mechanism (Staehr et al., ). These arteries might also exhibit abnormal relaxation in response to metabolic stimuli.…”

“…Accordingly, we demonstrated that activation of Src signalling by ouabain in mesenteric arteries sensitized smooth muscles to Ca 2+ and potentiated vasoconstriction (Bouzinova et al., ; Zhang et al., ). We also showed that knockdown of the α2 isoform in mouse cerebral arteries potentiated Ca 2+ sensitization via an increase in Src activation without changing total Src kinase and suggested that this is caused by reduced inhibition of Src by the Na + ,K + ‐ATPase (Staehr et al., ).…”

“…Some rodent studies support this signalling by showing an elevation of [Ca 2+ ] i and potentiation of arterial contraction upon inhibition of the Na + ,K + ‐ATPase α2 isoform with submicromolar and micromolar ouabain concentrations (Iwamoto et al., ; Zhang et al., ). However, several other studies failed to demonstrate direct modulation of [Ca 2+ ] i by the Na + ,K + ‐ATPase α2 isoform (Bouzinova et al., ; Matchkov et al., ; Staehr et al., ). A spatially restricted Ca 2+ signalling leading to an increased Ca 2+ loading of the SR was suggested to overcome this inconsistency (Blaustein et al., ), although further high‐resolution studies are needed.…”

“…Accordingly, reduced expression of the α2 isoform potentiated myogenic contraction of mesenteric arteries in a Ca 2+ ‐dependent manner (Zhang et al., ). In contrast, reduction of the α2 isoform in mouse middle cerebral arteries was associated with increased arterial contractility, although [Ca 2+ ] i responses were suppressed (Staehr et al., ). This suggests that the Na + ,K + ‐ATPase suppresses the mechanism that otherwise sensitizes vascular smooth muscle cells to [Ca 2+ ] i .…”

“…The Na + ,K + ‐ATPase‐dependent Src signalling in non‐vascular tissue depends on the α1 isoform, with insignificant, if any, contribution of the α2 isoform (Xie et al., ). It is therefore highly peculiar that Src signalling in the vasculature seems to be strongly dependent on the α2 isoform, in accordance with its high ouabain sensitivity and the dependence on the α2 isoform expression (Bouzinova et al., ; Hangaard et al., ; Staehr et al., ; Zhang et al., ; Zulian et al., ).…”