BKCa nitrosylation is associated with cerebral microvascular dysfunction in female 5x-FAD mice

Abstract

Vasculopathy and nitro-oxidative stress are present in patients with Alzheimer’s disease (AD) and may contribute to disease progression and severity. Large conductance calcium activated K+ channels (BKCa) plays an important role in vasodilatory responses and maintenance of myogenic tone in resistance arteries. Opening of BKCa channels occurs upstream from localized intracellular Ca2+ release events (Ca2+ sparks), and results in K+ efflux, vascular smooth muscle cell hyperpolarization and vasorelaxation. In a pro-nitro-oxidative scenario, BKCa can be modified, resulting in decreased activity and hypercontractility, which can compromise cerebral blood flow regulation, generating an environment that may accelerate neurodegeneration. We hypothesized that reductions in BKCa-dependent vasodilation in cerebral arteries, as consequence of nitro-oxidative stress, results in neurovascular dysfunction in the 5x-FAD model of AD. Methods: Posterior communicating arteries (PComA) from 5 months-old male and female 5x-FAD and wild-type (WT) littermates were isolated and studied ex vivo using pressure myography. Smooth muscle Ca2+ transients were evaluated by spinning-disk confocal microscopy. Oxidative stress was assessed by oxidized glutathione levels in the brain using a colorimetric enzymatic assay. BKCa expression was assessed by qPCR. Nitrosylated BKCa was evaluated using Western blot. Functional hyperemia were evaluated by laser speckle contrast imaging. Data are means±SEM, 5x-FAD vs WT, analyzed by two-tailed Student’s t-test or Mann-Whitney test. Results: In females, PComA from 5x-FAD showed higher spontaneous myogenic tone than WT (Myogenic tone: 24.48 ± 3.20 vs 16.09 ± 0.93%, p<0.05, N=7). Constriction to the BKCa blocker iberiotoxin (30 nM) was smaller in 5x-FAD than WT, suggesting lower basal BKCa activity (Vasoconstriction: -4.25 ± 0.43 vs -9.22 ± 2.56%, p<0.05; N=5), which was independent of alterations in intracellular Ca2+ transients or BKCa mRNA expression. These vascular changes were associated with higher levels of oxidized glutathione in female 5x-FAD (7.83 ± 0.62 vs 5.27 ± 0.74 μM, p<0.05, N=8) and of S-nitrosylation in the BKCa α-subunit (0.68 ± 0.04 vs 0.41 ± 0.03, p<0.05, N=5). Female 5x-FAD mice showed increased expression of iNOS mRNA ([2-ΔΔCT]: 10.64 ± 5.40 vs 0.74 ± 0.19, p<0.05, N=6) and impaired functional hyperemia responses after whisker stimulation (%increase: 3.82 ± 0.64 vs. 9.91 ± 1.41%, p<0.05, N=6). No significant differences were observed between male 5x-FAD and WT for all parameters above. Conclusion: Cerebrovascular impairments were more pronounced in female 5x-FAD mice, observed as an increase in spontaneous myogenic tone, likely due to reduction in smooth muscle cell BKCa activity associated to an increase in brain nitro-oxidative stress and a blunted neurovascular coupling response. Together, they identify post-translational modifications of BKCa as a putative target to improve cerebral microvascular function in AD. National Institutes of Health (R00 HL140106, R01 AG073230) and the Alzheimer's Association (AARGD-21-850835). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.