α1 C S1928 Phosphorylation of Ca V 1.2 Channel Controls Vascular Reactivity and Blood Pressure

Figures

• 

  Figure 1. pS1928 controls CaV1.2 biophysical properties upon Ang II exposure. A, Representative nifedipine‐sensitive Ba2+ currents (I Ba) elicited by a single voltage step from −70 mV to +10 mV recorded from WT and SA arterial myocytes under control conditions and after exposure to 100 nmol/L Ang II. The right‐side panel shows summary scatter plots (WT: n=9 cells from 4 mice; SA ct: n=6 cells from 3 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0473). B, Exemplary single‐channel traces obtained in cell‐attached mode using a single voltage step from −80 mV to −30 mV recorded from WT and S1928A arterial myocytes under control conditions and after exposure to 100 nmol/L Ang II. Ca2+ was used as the charge carrier to visualize Ca2+‐dependent CaV1.2 cooperative events. Sections of the traces in the boxes are magnified below for a detailed appreciation of openings and cooperative events. The baseline level is highlighted by a (c) and unitary levels by (on). Summary data of (C) channel activity (nPo), (D) channel availability, (E) coupling frequency, and (F) coupling strength (κ) (WT ct: n=13 cells from 7 mice; WT Ang II: n=15 cells from 6 mice; SA…

• 

  Figure 2. Ang II increased α1C clustering requires pS1928. A, Exemplary superresolution total internal reflection fluorescence images of WT and SA arterial myocytes labeled for α1C with 2 magnified areas under control conditions and after exposure to 100 nmol/L Ang II (scale bars=5 μm→5 μm→200 nm). B, Plot of the calculated pair‐correlation function (g(r)) of α1C clusters in WT and SA arterial myocytes under control conditions and after 100 nmol/L Ang II exposure. Scatter plots of (C) α1C cluster radius (nm), (D) α1C cluster density (number of clusters/μm2) and (E) estimated α1C proteins per cluster (Ncluster) in WT and SA arterial myocytes under control conditions and after 100 nmol/Ls Ang II exposure (WT ct: n=27 cells from 7 mice; WT Ang II: n=34 cells from 7 mice; SA ct: n=38 cells from 5 mice; SA Ang II: n=50 cells from 7 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0268 for C; P=0.0676 for interaction and P=0.0108 for D; P=0.0342 for E). F, Cartoon of the modified PLA approach. Representative maximal projection images of PLA puncta for α1C‐α1C interactions in WT (G) and SA (H) male arterial myocytes under control conditions, after Ang II…

• 

  Figure 3. pS1928 mediates altered arterial myocyte excitation‐contraction coupling upon Ang II. A, Simulated Ang II‐mediated effects on arterial myocyte V M and [Ca2+] i . To simulate the gradual effects of Ang II over time, full modification of Ang II‐dependent model parameters is reached after 180 seconds (s) from the initial administration. The mathematical model was parameterized using a 100% increase in Ca2+ currents, 60% reduction in K+ currents and 75% increase in TRP currents upon Ang II exposure in WT cells, and 60% reduction in K+ currents and 75% increase in TRP currents with no change in Ca2+ currents upon Ang II exposure in SA cells. The changes in Ca2+, K+, and TRP currents are consistent with prior data and results here. 3 , 7 , 43 , 44 , 45 , 46 B, Representative traces of perforated whole‐cell recordings from WT and SA mesenteric arterial myocytes in current‐clamp mode with a gap‐free protocol and (C) summary data of V M under control conditions and after exposure to 100 nmol/L Ang II and 60 mmol/L KCl (WT n=9 cells from 3 mice; SA n=9 cells from 3 mice). HK+=60 mmol/L K+. Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.3882 in…

• 

  Figure 4. pS1928 sustains arterial constriction upon Ang II. A, Representative normalized diameter traces of WT and SA male mesenteric arteries pressurized to 60 mm Hg and treated with 100 nmol/L Ang II and 0 Ca2++ 1 μmol/L nifedipine. The peak and plateau tones are highlighted by arrows. Scatter plots of (B) basal tone, (C) Ang II‐induced peak diameter reduction, and (D) plateau tone (n=6 mice per group). Significance was assessed with unpaired t test for all plots. (E) Representative pseudocolored blood flow (ie, flux) images of WT and SA male mesenteric arteries through a laparotomy before and after exposure to Ang II and 0 Ca2+ plus a vasodilatory mix (described in the Methods section). (F) Exemplary normalized arterial diameter (upper panel) and normalized blood flux (lower panel) before and after exposure to increasing concentrations of Ang II (from log−10 mol/L to log−5 mol/L) and subsequent application of 0 Ca2+ plus a vasodilatory mix. G and H, Summary plots of basal tone and basal flux, respectively (n=6 mice per group). Significance was assessed with unpaired t test. I and J, Exemplary 3‐dimensional graphs highlighting the peak and 5‐minute normalized arterial diameter and blood…

• 

  Figure 5. Increased CaV1.2 activity and α1C clustering during hypertension. A, Scatter plots of (A) channel activity (nPo), (B) channel availability, (C) coupling frequency and (D) coupling strength (κ) in sham and hypertension WT and SA arterial myocytes (WT sham: n=15 cells from 5 mice; WT hypertension: n=16 cells from 7 mice; SA sham: n=11 cells from 4 mice; SA hypertension: n15 cells from 5 mice). Significance was assessed with nested 2‐way ANOVA with Bonferroni post hoc test (P=0.0054 for A; P=0.0068 for B; P=0.1721 for interaction and P=0.0099 for treatment for C; P=0.4589 for interaction and P=0.0001 for treatment for D). E, Representative super‐ TIRF images of sham and hypertension WT and SA arterial myocytes labeled for α1C with 2 magnified areas (scale bars = 5 μm → 200 nm). F, Plot of the calculated pair‐correlation function (g(r)) of α1C clusters in sham and hypertension WT and SA arterial myocytes. Scatter plots of (G) α1C cluster radius (nm), (H) α1C cluster density (number of cluster/μm2) and (I) estimated α1C proteins per cluster (Ncluster) in sham and hypertension WT and SA arterial myocytes (WT sham: n=16 cells from 5 mice; WT hypertension: n=37 cells from 6 mice; SA…

• 

  Figure 6. pS1928 regulates CaV1.2 function, vascular reactivity, and blood pressure during hypertension. Representative diameter recordings over a pressure range (from 10 to 100 mm Hg) and plot summary data of percentage myogenic tone obtained using mesenteric arteries from WT (A) and SA (B) sham and hypertension mice (WT sham: n=12 arteries from 5 mice; WT hypertension: n=9 arteries from 5 mice; SA sham: n=8 arteries from 5 mice; S1928A hypertension: n=7 arteries from 5 mice). Significance was assessed with nested t‐test. Plots of systemic (C) and diastolic (D) blood pressure at day 0 (blue circles) and day 7 (red circles) from sham and hypertensive WT and SA mice using radio telemetry (n=5 mice per group). Significance was assessed with 3‐way ANOVA with Bonferroni post hoc test (P=0.0017 for C; P=0.0299 for D). E, Scatter plot of ΔMAP from day 0 to day 7 in sham and hypertensive WT and SA mice (n=5 mice per group). Significance was assessed with 2‐way ANOVA with Bonferroni post hoc test (P=1.4×10−4). Data are mean±SEM. P values for relevant comparisons within each panel and supplemental material. ΔMAP, change in mean arterial pressure; HTN, hypertension; pS1928, serine 1928…

• 

  Figure 7. Model by which Ang II/PKC signaling promotes α1C pS1928 to modulate the α1C/CaV1.2 channel spatial and temporal properties leading to changes in global [Ca2+] i , myogenic tone, blood flow, and blood pressure during hypertension. Image created with Biorender.com. Ang II indicates angiotensin II; [Ca2+] i , intracellular Ca2+ concentration; PKC, protein kinase C; pS1928, serine 1928 phosphorylation; and S1928, serine 1928.