Genetic ablation of smooth muscle K IR 2.1 is inconsequential to the function of mouse cerebral arteries

Figures

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  Figure 1.. Genetic ablation of KIR2.1 does not eliminate inward K+ currents in cerebral arterial smooth muscle cells (SMCs). Whole-cell patch clamp electrophysiology was used to measure KIR current with voltage ramps from −100 to +20 mV in the absence and presence of Ba2+ in 60 mM K+. (a,b) Representative recordings of whole-cell and Ba2+-subtracted KIR currents in myocytes isolated from SMC KIR2.1−/− mice and non-induced Cre SMC controls. (c) Summary data compare peak inward current at −100 mV between groups (n = 9 SMCs from 6 mice in control group and n = 9 SMCs from 8 mice in knockout group; nested t-test).

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  Figure 2.. KIR2.1 is negligibly expressed in cerebral vascular smooth muscle cells (SMCs) while KIR2.2 is highly expressed at the cell membrane. Tamoxifen-induced KIR2.1 knockout significantly reduced subunit expression but levels remained detectable by immunofluorescence. (a) Fluorescent anti-KIR2.1 (green) exhibited a faint labeling pattern in cerebral arterial myocytes from SMC KIR2.1−/− and control mice with nuclei stained with DAPI (blue). (b) Summary of data compares fluorescence intensity (background subtracted) of KIR2.1 signal between groups (n = 10 cells from 5 animals in control group and n = 9 cells from 5 animals in knockout group; unpaired t-test). (c) Immunofluorescence labeling of SMCs for KIR2.2. (d) Summary of data compares background-subtracted fluorescence intensity of KIR2.2 signal between groups (n = 8 cells pooled from 4 animals/group; unpaired t-test). Two cells were analyzed per animal with background signal subtracted using 2° antibody control.

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  Figure 3.. KIR2.2 protein expression is unaltered in smooth muscle cell (SMC) KIR2.1−/− cerebral arteries. (a) Western blot of intact cerebral arteries confirmed that protein levels of KIR2.2 are not impacted by deletion of SMC KIR2.1. (b) Summary of data compares KIR2.2 protein levels between control and knockout mice (normalized to actin; n = 6 mice; unpaired t-test).

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  Figure 4.. KIR2.1 and KIR2.2 subunits are inversely expressed in cerebral endothelial and vascular smooth muscle cells. (a) KIR2.1 and (b) KIR2.2 subunit expression is shown as average cellular transcript counts per cell, as determined by single-cell RNA sequencing of the mouse brain vasculature. Data highlight differences in the dominant subunit between cell types. Abbreviations: PC – pericytes; SMC – smooth muscle cells; EC – endothelial cells; v – venous; c – capillary; a – arterial; aa – arteriolar. Figures provided by http://betsholtzlab.org/VascularSingleCells/database.html.33,34

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  Figure 5.. Myogenic responses and K+-induced dilation are intact in cerebral arteries of smooth muscle cell (SMC) KIR2.1−/− mice. Cerebral arteries from control and SMC KIR2.1−/− mice were cannulated and intravascular pressure was elevated stepwise while vasomotor responses were measured. (a) Representative diameter traces from endothelium-denuded vessels of control and SMC KIR2.1−/− mice show the effect of increasing pressure on myogenic tone. (b) Summary of data highlights limited impact of smooth muscle KIR2.1 knockout on myogenic tone development. Paired t-test was performed for 0 vs. 100 μM Ba2+ treatment (n = 10 vessels in the control group and n = 11 vessels in the SMC KIR2.1−/− group with 1 vessel/mouse; *P < 0.05). (c) K+-induced dilation, elicited by increasing extracellular K+ from 5 mM to 10 mM before and after treatment with 100-μM Ba2+, was intact in the knockout group (n = 6 vessels in the control group and SMC KIR2.1−/− group; one-way ANOVA with Sidak’s multiple comparisons test). (d) K+-induced dilation (5 mM K+ to 10 mM K+) was abrogated with exposure to low concentrations of Ba2+, implicating KIR2.2 as the mediator of this response based on its Ba2+ sensitivity profile (n…

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  Figure 6.. Region-specific brain perfusion is not altered in smooth muscle cell (SMC) KIR2.1−/− mice at rest and with increased systemic blood pressure. (a) Representative arterial spin-labeled MR brain perfusion maps. Scans were done in a posterior-to-anterior direction and the volume of brain scanned was divided into 5 coronal slices. Resting cerebral blood flow was measured in control and SMC KIR2.1−/− mice. Scans were repeated after blood pressure challenge with an intraperitoneal phenylephrine injection. Figure shows slices from 2 regions of the brain (red boxes) spanning cerebral nuclei, hippocampus, thalamus, and hypothalamus. (b) Baseline perfusion in several major brain structures was not significantly different between control and tamoxifen-induced mice. The blood pressure challenge caused a modest but significant rise in cerebral blood flow to a similar extent in control and SMC KIR2.1−/− animals. Unpaired t-test was performed for control (n = 7 mice) vs. SMC KIR2.1−/− (n = 11 mice) comparison; paired t-test was performed for baseline vs. phenylephrine-treatment. *P < 0.05 compared to baseline control.