CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries

Abstract

The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle [Ca²⁺] is a key determinant of constriction, tied to L-type (Ca_V1.2) Ca²⁺ channels. While important, other Ca²⁺ channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca²⁺ channel (Ca_V3.1) using C57BL/6 wild type and Ca_V3.1^-/- mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca²⁺ imaging defined the Ca_V3.1^-/- phenotype relative to C57BL/6. Ca_V3.1^-/- mice had absent Ca_V3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20-60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca²⁺ wave generation, asynchronous events of Ca²⁺ release from the sarcoplasmic reticulum, insensitive to L-type Ca²⁺ channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP₃R1/Ca_V3.1 close physical association. IP₃R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a Ca_V3.1^-/- contractile phenotype. Findings indicate that Ca²⁺ influx through Ca_V3.1 contributes to myogenic tone at hyperpolarized voltages through Ca²⁺-induced Ca²⁺ release tied to the sarcoplasmic reticulum. This study helps establish Ca_V3.1 as a potential therapeutic target to control blood pressure.

Figure 1

Absence of Ca_V3.1 expression and current in SMCs isolated from mesenteric arteries of Ca_V3.1^−/− mice, and lower arterial blood pressure indices in Ca_V3.1^−/− mice compared to C57BL/6. (Aa) Polymerase chain reaction of Cacna1g gene (Ca_V3.1). DNA was extracted from ear notches (C57BL/6 and Ca_V3.1^−/− mice) and amplified; the different product sizes confirm the gene modification leading to functional knockout. Illustration created with BioRender.com. (Ab) Ca_V3.1 (green) in cerebral arterial myocytes from control mice with nuclei stained with DAPI (blue) detected with immunohistochemistry. This signal was absent in Ca_V3.1^−/− mice. Secondary antibody controls were negative for nonselective labelling (n = 4 cells pooled from 4 animals/group). (B) Averaged Ca_V currents were assessed by whole-cell patch clamp in C57BL/6 cells showing a residual current remaining (highlighted green) after blocking L-type and Ca_V3.2 currents by nifedipine and Ni²⁺, respectively. (C) Recordings of whole-cell Ca_V currents in Ca_V3.1^−/− cells showing no residual current after nifedipine and Ni²⁺ treatment. (D) Peak current (I) plots of whole-cell Ba²⁺ (10 mmol/L) current before and after the application of nifedipine to C57BL/6 and Ca_V3.1^−/− smooth muscle cells. n = 9 SMCs from 6 mice in control group and n = 9 SMCs from 8 mice in knockout group. (* P = 0.013, unpaired t test). (E) Systolic, diastolic, and mean arterial pressure (mmHg) of Ca_V3.1^−/− and C57BL/6 mice were measured using the CODA6 tail-cuff system. 25-min recordings daily for one week were performed on both groups (n = 5). (Systolic: *P = 0.028, Diastolic: **P = 0.005, MAP: **P = 0.008, unpaired t test).

Figure 2

Arteries from Ca_V3.1^−/− mice develop less myogenic tone at low intraluminal pressures. Isolated mesenteric arteries from Ca_V3.1^−/− and C57BL/6 mice underwent a pressure curve in two conditions: PSS containing Ca²⁺ and Ca²⁺ free PSS + 2 mM EGTA, a Ca²⁺-chelating agent. (A,B) Representative trace and summary data of changes in diameter in response to pressure curve (20–100 mmHg) in C57BL/6 and Ca_V3.1^−/−. (C) Summary data shows arteries from Ca_V3.1^−/− mice had lower myogenic tone in the pressure range from 20–60. (20 mmHg: **P = 0.002, 40 mmHg: *P = 0.014, 60 mmHg: **P = 0.008, 80 mmHg: P = 0.188, 100 mmHg: P = 0.108, unpaired t test). (D) Summary data of incremental distensibility shows no difference between groups. (n = 6 arteries from 6 animals for each experiment). (20 mmHg: P = 0.945, 40 mmHg: P = 0.651, 60 mmHg: P = 0.571, 80 mmHg: P = 0.793, 100 mmHg: P = 0.245, unpaired t test).

Figure 3

Ca_V3.1 deletion has no impact on phenylephrine-induced constriction. Increasing concentrations of phenylephrine were applied onto pressurized arteries isolated from C57BL/6 and Ca_V3.1^−/− mice in the presence and absence of nifedipine (L-type Ca²⁺ channel blocker). Experiments were conducted at an intraluminal pressure of 60 mmHg. (A,B) Representative traces (Left) and summary data (Right) of changes in diameter in response to phenylephrine showing a decrease in constriction in nifedipine-treated vessels from both strains. (C) %Maximal phenylephrine-induced constriction relative to KCl-induced constriction shows no significant difference in agonist-induced constriction between C57BL/6 and Ca_V3.1^−/− mice. (n = 6 arteries from 6 animals). P values for increasing PE concentrations in PSS: 0.529, 0.790, 0.763, 0.957, 0.554, 0.719 and in PSS + nifedipine: 0.565, 0.343, 0.074, 0.396, 0.925, 0.837 (Paired t test).

Figure 4

Functional roles of Ca_V3.1 and IP₃Rs in Ca²⁺ waves generation. Rapid Ca²⁺ imaging was performed on Fluo-8-loaded arteries from Ca_V3.1^−/− and C57BL/6 mice at an intraluminal pressure of 60 mmHg. (A) Representative traces from C57BL/6 and Ca_V3.1^−/− mesenteric arteries with and without nifedipine. (B) Summary data (n = 6 arteries from 6 mice). Number of cells firing (***P = 0.0002) and firing frequency (***P = 0.0001) were significantly reduced in Ca_V3.1^−/− when compared to C57BL/6. Nifedipine did not impact the number of cells firing (C57BL/6: P = 0.485, Ca_V3.1^−/− P = 0.980) or the firing frequency (C57BL/6: P = 0.093, Ca_V3.1^−/− P = 0.925) in either strain. P values were calculated using 2-way ANOVA. (C) Representative traces from C57BL/6 mesenteric arteries with and without 2-APB. (D) Summary data (n = 6 arteries from 6 mice). 2-APB (IP₃R inhibitor) decreased the number of cells firing and their firing frequency (****P < 0.0001 and ***P = 0.0002, respectively, paired t test) in mesenteric arteries from C57BL/6 mice. (E) Representative traces from C57BL/6 mesenteric arteries with and without xestospongin C. (F) Summary data (n = 5 arteries from 5 mice). xestospongin C (IP₃R inhibitor) decreased the number of cells firing and their firing frequency (*P < 0.011 and **P = 0.002, respectively, paired t test) in mesenteric arteries from C57BL/6 mice. F fluorescence intensity, F_o baseline fluorescence.

Figure 5

Ca_V3.1 channels colocalize with IP₃Rs. Proximity ligation assay was employed using isolated mesenteric arterial SMCs from C57BL/6 (n = 35 cells from 6 animals) and Ca_V3.1^−/− (n = 36 cells from 6 animals) mice to determine the close association (< 40 nm) of Ca_V3.1 and IP₃R1 proteins (red, denoted by white arrows). Nuclei were stained with DAPI (Blue). Control experiments used only one primary antibody or no primary antibody. Note, dots were averaged across cells within each animal, and represented as a data point in the bar graph to facilitate statistical comparison. **P = 0.0033.

Figure 6

IP₃R blockade has no impact on myogenic tone development in Cav3.1^−/−. Mesenteric arteries isolated from C57BL/6 and Ca_V3.1^−/− mice underwent stepwise pressure increases in control conditions (Ca²⁺ PSS), with nifedipine (Ca_V1.2 blocker) alone and with 2-APB or xestospongin C (IP₃R blockers). (A,D,G) Representative traces and (B,E,H) summary data of changes in mesenteric arteriolar diameter in response to pressure steps from C57BL/6 (A,G) and Ca_V3.1^−/− mice (D) are depicted. In C57BL/6 mice, pressure-induced constriction decreased after nifedipine, 2-APB, and xestospongin C treatment. In Ca_V3.1^−/− mice, the vasomotor response following nifedipine and 2-APB treatment was not different from nifedipine treatment only. (C,I) %Myogenic tone was reduced following 2-APB (20 mmHg: *P = 0.013, 40 mmHg: **P = 0.008, 60 mmHg: P = 0.124, 80 mmHg: P = 0.085, 100 mmHg: P = 0.102, paired t test) and xestospongin C (20 mmHg: *P = 0.015, 40 mmHg: **P = 0.001, 60 mmHg: *P = 0.02, 80 mmHg: *P = 0.013, 100 mmHg: P = 0.092, paired t test) treatment at 20–40 pressure range in C57BL/6 mice. (F) No changes in myogenic tone were observed following 2-APB treatment in Ca_V3.1^−/− mice. (20 mmHg: P = 0.182, 40 mmHg: P = 0.334, 60 mmHg: P = 0.535, 80 mmHg: P = 0.899, 100 mmHg: P = 0.245, paired t test) (n = 6 arteries from 6 animals for each experiment).

Figure 7

Ca_V3.x blockade diminishes myogenic tone development and Ca²⁺ wave generation in C57BL/6 mice. Mesenteric arteries isolated from C57BL/6 mice underwent stepwise pressure increases in control conditions (Ca²⁺ PSS), with nifedipine (Ca_V1.2 blocker) alone and with kurtoxin (150 nM, Ca_V3.x blocker). (A) Representative trace, and summary data of (B) arterial diameter and (C) % myogenic tone to pressure in C57BL/6 mesenteric vessels. Pressure-induced constriction decreased after nifedipine, and kurtoxin treatment. %Myogenic tone was reduced following kurtoxin (20 mmHg: *P = 0.04, 40 mmHg: **P = 0.008, 60 mmHg: P = 0.074, 80 mmHg: P = 0.105, 100 mmHg: P = 0.116, unpaired t test). (D) Representative trace of Ca²⁺ wave generation in C57BL/6 mesenteric arteries in the absence and presence of nifedipine + kurtoxin. (E) Unlike Fig. 4, where nifedipine had no effect on Ca²⁺ wave generation, summary data (n = 5 arteries from 5 mice) reveals that further addition of kurtoxin causes a marked attenuation in the number of cells firing and firing frequency (**P = 0.004 and **P = 0.002, respectively, paired t test). F fluorescence intensity, F_o baseline fluorescence.

Figure 8

High-voltage-activated Ca_V1.2 channels control [Ca²⁺]_i when intravascular pressure is elevated and membrane potential depolarized. In contrast, Ca_V3.1 channels display a hyperpolarized profile with more negative activation/inactivation properties compared to Cav1.2 channels. Ca_V3.1 channels foster Ca²⁺ wave generation likely through sarcoplasmic reticulum IP₃R activation as the two proteins lie in close proximity. Ca²⁺ waves are known to induce a Ca²⁺-calmodulin (CAM)-dependent activation of myosin light chain kinase (MLCK) which regulates myosin phosphorylation leading to myogenic tone control. Ca_V3.1 deletion is coupled to reduced blood pressure and hemodynamic control thus bearing clinical importance. Created with BioRender.com.