Ca_V3.1 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.

Introduction

Smooth muscle cells in the arterial wall actively contract to intravascular pressure, maintaining organ blood flow under dynamic conditions^1,2. This “myogenic response” was first described by Bayliss³ and is intimately tied to arterial depolarization, the activation of voltage-gated Ca²⁺ channels and the concomitant rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which complexes with calmodulin driving myosin light chain phosphorylation⁴. Three subclasses of voltage-gated Ca²⁺ channels (Ca_V1–3), are encoded in the mammalian genome and each displays unique voltage-dependent properties⁵. In arterial smooth muscle, Ca_V1.2 (L-type) Ca²⁺ channels are principally responsible for extracellular Ca²⁺ entry and their blockade is notable for attenuating a range of constrictor responses, including those induced by pressure⁶.

L-type Ca²⁺ channels are classified as high-voltage-activated and dominate the setting of smooth muscle [Ca²⁺]_i when intravascular pressure is elevated and arteries depolarized⁷. Their activity, however, markedly drops with hyperpolarization as pressure is reduced or when endothelial cell K⁺ channels are activated by selected agents⁸. As [Ca²⁺]_i remains a determinant of tone, even in the hyperpolarized state, it follows that other Ca²⁺ channels, ones with a leftward voltage profile, should be expressed in vascular smooth muscle⁶. T-type Ca²⁺ channels display activation/inactivation properties decidedly more negative to their L-type counterparts^9,10. Two subtypes (Ca_V3.1 and Ca_V3.2) are expressed in vascular smooth muscle, the latter linked to the activation of large-conductance Ca²⁺-activated K⁺ (BK) channels and a negative feedback response limiting arterial constriction¹⁰. This leaves Ca_V3.1 as to enabling myogenic tone at hyperpolarized voltages^9,11,12, presumptively through a mechanism where Ca²⁺ influx directly contributes to the cytosolic Ca²⁺ pool or indirectly triggers sarcoplasmic reticulum Ca²⁺ release in the form of asynchronous Ca²⁺ waves^13,14,15.

This study explored whether and by what mechanisms Ca_V3.1 channels enable myogenic tone development in mouse mesenteric arteries. This work entailed the use of C57BL/6 wild type and Ca_V3.1^−/− mice, and the integrated use of cellular (patch-clamp electrophysiology, immunofluorescence, and PLA), tissue (pressure myography and rapid Ca²⁺ imaging) and whole animal (metabolic caging and blood pressure) techniques. Initial assays confirmed the absence of Ca_V3.1 in mesenteric arterial smooth muscle of knockout animals. This absence aligned with a drop in systemic blood pressure and reduced myogenic tone at lower pressures compared to controls. Subsequent experiments revealed that Ca²⁺ wave generation was attenuated in Ca_V3.1^−/− arteries as this channel no longer resided near IP₃R1 and that IP₃R blockade in C57BL/6 arteries produced a Ca_V3.1^−/− phenotype. These findings highlight a role for Ca_V3.1 in myogenic tone development and hemodynamic control through the triggering of sarcoplasmic reticulum Ca²⁺ waves. They additionally reveal the potential therapeutic value of Ca_V3.1 in the control of hypertension.

Materials and methods

Animal and tissue preparation

Animal procedures were approved by the animal care committee at the University of Western Ontario ensuring compliance with federal and provincial standards and under consideration of the ARRIVE guidelines. Male C57BL/6 (wild type; Jackson labs) or Ca_V3.1^−/− (in-house colony) mice (16–20 weeks of age) were humanely euthanized via CO₂ asphyxiation. The mesentery was removed rapidly and placed in cold PBS solution (pH 7.4) containing (in mM): 138 NaCl, 3 KCl, 10 Na₂HPO₄, 2 NaH₂PO₄, 5 glucose, 0.1 CaCl₂, and 0.1 MgSO₄. Third and fourth-order mesenteric were isolated and cut into 2–3 mm segments and transferred to fresh cold PBS.

Polymerase chain reaction

Ear tissue was collected from C57BL/6 and Ca_V3.1^−/− mice (n = 3) and DNA was extracted using the QIAamp Fast DNA Tissue Kit (QIAGEN). 200 ng of DNA was amplified via polymerase chain reaction (PCR) using previously published Ca_V3.1 primers: C57BL/6 F, 5ʹ-ATACGTGGTTCGAGCGAGTC-3ʹ; WT R, 5ʹ-CGAAGGCCTGACGTAGAAAG-3ʹ; Ca_V3.1^−/− R, 5ʹ-CTGACTAGGGGAGGAGTAGAAG-3ʹ¹⁶. Gel electrophoresis was performed on PCR products at 95 V for 1 h on a 1.5% agarose gel. Gel was then imaged using the Bio-Rad ChemiDoc™ MP Imaging System and Image Lab 6.1 software (Bio-Rad).

Isolation of mesenteric arterial smooth muscle cells

Third and fourth-order mesenteric arteries were placed in an isolation medium (37 °C, 10 min) containing (in mM): 60 NaCl, 80 Na-glutamate, 5 KCl, 2 MgCl₂, 10 glucose and 10 HEPES with 1 mg/mL bovine serum albumin (BSA, pH 7.4). Vessels were then exposed to a two-step digestion process that began with 14-min incubation (37 °C) in media containing 0.5 mg/mL papain and 1.5 mg/mL dithioerythritol, followed by 10-min incubation in media containing 100 μM Ca²⁺, and collagenases type H (0.7 mg/mL) and type F (0.4 mg/mL). Following incubation, tissues were washed repeatedly with ice-cold isolation medium and triturated with a fire-polished pipette. Liberated cells were stored in ice-cold isolation medium for use the same day.

Immunohistochemistry

Ca_V3.1 expression was assessed in mesenteric arterial smooth muscle cells isolated from C57BL/6 and Ca_V3.1^−/− mice. Briefly, isolated cells were fixed onto a microscope cover glass in PBS (pH 7.4) containing 4% paraformaldehyde and 0.2% Tween 20. Fixed cells were blocked (1 h, 22 °C) with a quench solution (PBS supplemented with 3% donkey serum and 0.2% Tween 20) and subsequently incubated overnight (4 °C, humidified chamber) with rabbit anti-Ca_V3.1 primary antibody diluted in quench solution (1:100). In the following morning, cells were washed 3× in PBS-0.2% Tween 20 and then incubated (1 h, 22 °C) in a PBS-0.2% Tween 20 buffer containing Alexa Fluor 488 donkey anti-rabbit IgG-secondary antibody (1:200). After further washing, isolated cells and whole-mount preparations were mounted with Prolong Diamond Antifade Mountant with DAPI. Immunofluorescence was detected through a 63× oil immersion lens coupled to a Leica-TCS SP8 confocal microscope equipped with the appropriate filter sets. Smooth muscle cells isolated from C57BL/6 cerebral arteries were used as Ca_V3.1 positive controls. Secondary antibody controls were performed and were negative for nonselective labelling.

Electrophysiological recordings

Conventional patch-clamp electrophysiology was utilized to measure voltage-gated Ca²⁺ currents in smooth muscle cells isolated from mesenteric arteries. Cell averaged capacitance was 12–18 pF. Recording electrodes (pipette resistance, 5–8 MΩ) were fashioned from borosilicate glass using a micropipette puller (Narishige PP-830, Tokyo, Japan) and backfilled with pipette solution containing (in mM): 135 CsCl, 5Mg-ATP, 10 HEPES, and 10 EGTA (pH 7.2). To attain a whole-cell configuration, the pipette was lowered onto a cell while applying negative pressure to rupture the membrane and garner intracellular access. Cells were voltage clamped (holding potential: − 60 mV) and subjected to − 90 mV followed by 10 mV voltage steps (300 ms) starting from − 50 to 40 mV in a bath solution consisting of (mM): 110 NaCl, 1 CsCl, 1.2 MgCl₂, 10 glucose, and 10 HEPES plus 10 BaCl₂ (charge carrier). Delineation of vascular voltage-gated Ca²⁺ channels was performed by introducing 200 nM nifedipine to block L-type channels, followed by 50 µM Ni²⁺ to selectively block Ca_V3.2 channels without affecting Ca_V3.1. Currents were recorded using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) at room temperature (~ 22 °C). Data were filtered at 1 kHz, digitized at 5 kHz, and stored on a computer for offline analysis with Clampfit 10.3 software (Molecular Devices, Sunnyvale, CA). Current/voltage relationships were plotted as peak current density (pA/pF) at the different voltage steps.

Indirect calorimetry, activity, and inactivity

Comprehensive Lab Animal Monitoring System (CLAMS) interface using Oxymax software (Columbus Instruments, Columbus, OH) was utilized to measure the differences in O₂ consumption and CO₂ production, the cumulative amount of food and water consumed, respiratory exchange rate, activity (number of infrared beam breaks), and sleep epochs were measured in C57BL/6 and Ca_V3.1^−/− mice. Chambers were kept at 24 ± 1 °C with airflow of 0.5 L/min, and animals had ad libitum access to food and water. Metabolic parameters were recorded every 10 min for 48 h. Data from the same 12-h interval for each mouse was selected to standardize the data processing.

Blood pressure and heart rate assessment

Blood pressure measurements were performed on awake C57BL/6 and Ca_V3.1^−/− mice using the non-invasive CODA tail-cuff system (Kent Scientific, CT), as described, and following recommendations of the Subcommittee of Professional and Public Education of the American Heart Association Council on High Blood Pressure Research^17,18. To minimize anxiety, the animals were properly acclimatized in advance, and a heating platform was used to maintain body temperature. Mice (n = 5) were subjected to 25-min recordings daily for one week, and the weekly averages were recorded.

Pressure myography

Isolated mesenteric arteries were cannulated in an arteriograph and superfused with physiological salt solution (PSS; 5% CO₂, balance air) at 37 °C containing (in mM): 119 NaCl, 4.7 KCl, 1.7 KH₂PO₄, 1.2 MgSO₄, 1.6 CaCl₂, 10 glucose, and 20 NaHCO₃. To limit the influence of endothelial receptors, air bubbles were passed through the vessel lumen (1 min). Arterial diameters were monitored using an automated edge detection system (IonOptix, MA) and a 10× objective. Arteries were equilibrated at 15 mmHg, and contractile responsiveness was assessed by a brief (≈ 10 s) application of 60 mM KCl. After equilibration, intraluminal pressure was elevated from 20 to 100 mmHg in 20 mmHg increments for 10 min each, and arterial diameters were monitored in Ca²⁺ PSS (control), and in the presence of 0.3 µM nifedipine (L-type Ca²⁺ Channel blocker) alone or with 50 µM 2-APB or 3 µM xestospongin C (IP₃R blockers) or 150 nM kurtoxin (Ca_V3.x blocker). A final passive diameter assessment was conducted in Ca²⁺-free + 2 mM EGTA. Arteries that did not respond to superfused KCl (60 mM) or were insensitive or hypersensitive to pressure were excluded from experimentation. Percentage of maximum tone and incremental distensibility were calculated as follows:

$$\text{\% Maximum tone}=\frac{\text{Max diameter }-\text{ Diameter under control or treated conditions}}{\text{Max diameter}} \times 100,$$

$$\% \text{Incremental distensibility}=\frac{\mathrm{\Delta\, Diameter}}{\mathrm{\Delta\, Pressure}} \times 100.$$

Agonist-induced constriction

Endothelium-denuded mesenteric arteries were cannulated in a pressure myograph as explained above. Following the high potassium challenge, arteries were equilibrated at 60 mmHg, then subjected to the administration of phenylephrine (PE) into the bath solution. Increasing concentrations of PE (in M) 10^–7, 3 × 10^–7, 10^–6, 3 × 10^–6, 10^–5, and 3 × 10^–5 were superfused into the bath containing PSS in the absence (control) or presence of 0.3 µM nifedipine. Changes in diameter in response to each concentration were recorded and percentage of maximum constriction was calculated as follows:

$$\text{\% Maximum constriction}=\frac{D0-D}{D0-Dm} \times 100$$

where $D$ is the external diameter after each agonist concentration application, $D0$ is the external diameter in Ca²⁺ PSS, and $Dm$ is the external diameter after the highest concentration of agonist under control condition.

Calcium imaging

Freshly isolated arteries were incubated with the Ca²⁺ indicator Fluo-8 and placed on the stage of a Nikon swept-field confocal microscope with enclosed Agilent 3B laser attached to Andor camera (iXon Ultra). Fluo-8 working solution (19.1 µM) was freshly prepared by dissolving 5 μL of stock solution (1.91 mM) in 5 μL pluronic acid plus 490 μL HBSS buffer consisting of (in mM): 134 NaCl, 6 KCl, 1 MgCl₂, 2 CaCl₂ 10 HEPES, and 10 Glucose (pH 7.4). Incubation was done for 75 min at 37 °C in the dark. Vessels were then cannulated and equilibrated at an intraluminal pressure of 15 mmHg for 15 min in Ca²⁺ PSS solution. Intraluminal pressure was then raised to 60 mmHg, and Ca²⁺ waves were recorded in the presence and absence of 0.3 µM nifedipine (± 150 nM kurtoxin) or 50 µM 2-APB or 3 µM xestospongin C. Fluo-8-loaded arteries were excited at 488 nm and emission spectra at 510 nm viewed through a 60× water immersion objective (1.2 WI) and were monitored and analysed using Nikon NIS Elements software (AR 4.20.01). To limit laser-induced tissue injury, image acquisition was set to 45 s at 5 fps. A series of regions of interest (1 × 1 µm), created within the analysis software, was placed on 10 successive cells that were in sharp focus using the first visibly loaded smooth muscle cell as a starting point. A Ca²⁺ wave was defined as local fractional fluorescence ($\text{F}/{\text{F}}_{0}$) increase above the noise level of 1.1, which spans the whole cell and lasts for at least 1 s. Ca²⁺ waves were assessed by the number of firing cells in an array of the 10 adjacent cells and the frequency of Ca²⁺ waves propagation per cell per minute.

Proximity ligation assay

To test the spatial proximity of Ca_V3.1 and IP₃R, the Duolink in situ PLA detection kit was employed as previously described¹⁹. Briefly, freshly isolated mesenteric arterial smooth muscle cells underwent successive steps of fixation (10% paraformaldehyde in PBS, 15 min), permeabilization (0.2% Tween 20 in PBS, 15 min) and blocking (Duolink blocking solution, 1 h). Cells were then washed with PBS then incubated with primary antibodies (anti-Ca_V3.1, anti-IP₃R1) in Duolink antibody diluent solution at 4 °C overnight. Cells were then incubated with secondary Duolink PLA PLUS and MINUS probes for 1 h at 37 °C. If target proteins are within 40 nm of each other, synthetic oligonucleotides attached to the probes hybridize enabling their subsequent amplification and binding to complementary fluorescent oligonucleotide sequences, detected using Leica-TCS SP8 confocal microscope.

Statistical analysis

Data are expressed as means ± SD, and n indicates the number of cells, arteries, or animals. Power analysis was performed a priori to assess the sample size sufficient for obtaining statistical significance. No more than 1 experiment was performed on cells/tissues from any given animal. Where appropriate, paired/unpaired t-tests or two‐way analysis of variance (ANOVA) were performed to ascertain significant differences in mean values to a given condition/treatment. P values ≤ 0.05 were considered statistically significant.

Solutions and chemicals

Fluo-8 was acquired from Abcam. Primary antibodies against Ca_V3.1 and IP₃R1 were purchased from NovusBio and Alomone Laboratories, respectively. PCR kit was obtained from Qiagen. Secondary antibody, Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L), and 2-APB were obtained from ThermoFisher. Duolink PLA detection kits, nifedipine, PE hydrochloride, DAPI, donkey serum kurtoxin, and all other chemicals were obtained from Sigma-Aldrich unless stated otherwise. In cases where DMSO was used as a solvent, the maximal DMSO concentration after application did not exceed 0.5%. Please see the Major Resources Table in the Supplemental Materials.

Results

Characterization of Ca_V3.1^−/− genotype and phenotype

Genetic deletion of Ca_V3.1 channels was confirmed by PCR, immunohistochemical analysis and conventional whole-cell patch-clamp electrophysiology. In detail, PCR amplification of C57BL/6 and Cav3.1^−/− mouse DNA with Ca_V3.1 primers resulted in different sized PCR products. C57BL/6 mice had a PCR product of 288 bp corresponding to the wild type allele, while the PCR product for Ca_V3.1^−/− mice was seen at 385 bp (Fig. 1Aa). Figure 1Ab shows Ca_V3.1 protein expression is punctate in smooth muscle cells isolated from C57BL/6 but not Ca_V3.1^−/− mesenteric arteries (n = 4 mice per group). This analysis aligned with whole-cell electrophysiology, which noted dampened Ca_V3.1 activity in smooth muscle cells from Ca_V3.1^−/− mice relative to C57BL/6 controls (P = 0.013). Note, Ca_V3.1 activity was measured by first monitoring the total inward Ba²⁺ current, the collective sum of Ca_V1.2, Ca_V3.1, and Ca_V3.2 currents²⁰. Based on past studies, nifedipine and Ni²⁺ were then applied to abolish Ca_V1.2 (L-type) and Ca_V3.2 (T-type) activities, respectively, and the residual current was then assigned to Ca_V3.1^21,22. The current–voltage relationship of each Ca²⁺ channel is illustrated in Fig. 1B,C (Ca_V3.1 current in green), with peak current (at + 10 mV) summarized in Fig. 1D. Recordings were attained from mesenteric smooth muscle cells (9 cells per group) isolated from 6 C57BL/6 and 8 Ca_V3.1^−/− mice.

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).

Metabolic and blood pressure measurements in C57BL/6 and Cav3.1^−/− mice

Metabolic caging assessed O₂ consumption, CO₂ production, the respiratory exchange rate, along with cumulative food and water consumed during normal activity. No significant difference was observed among C57BL/6 and Ca_V3.1^−/− mice (Table 1) in these parameters. However, Ca_V3.1^−/− mice displayed a disrupted night-time sleeping pattern and were modestly but significantly heavier than the C57BL/6 controls. Subsequent tail-cuff measurements revealed that systolic, diastolic, and consequently mean arterial pressures were reduced in Ca_V3.1^−/− mice compared to C57BL/6 mice (Fig. 1E).

Table 1 There are no discernible metabolic differences among strains, except sleep time and weight.

Ca_V3.1 channels contribute to the myogenic response

Mesenteric arteries from C57BL/6 and Ca_V3.1^−/− mice were mounted in a myograph and exposed to increasing intraluminal pressures (20 to 100 mmHg) in physiological saline solutions, with Ca²⁺ and Ca²⁺-free + 2 mM EGTA. Traces and summative data are presented in Fig. 2A–C, and findings reveal that Ca_V3.1^−/− arteries displayed reduced myogenic tone compared to C57BL/6 controls, a trend that was statistically significant at lower intraluminal pressures (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). Arterial distensibility, a surrogate of vessel stiffness and defined as the percentage change in passive arteriolar diameter per change in intravascular pressure²³, was comparable among the two groups of arteries (Fig. 2D). Control experiments using PE as a vasoconstrictor noted a comparable vasomotor tone among C57BL/6 and Ca_V3.1^−/− arteries across a full concentration range (Fig. 3A,B). This statement applies equally to tone generated in the absence and presence of nifedipine, except at the higher agonist concentrations where the L-type Ca²⁺ channel blocker initially appeared to be less impactful in Ca_V3.1^−/− arteries (Fig. 3A,B). Note, however, when this data was normalized to the % maximal constriction, the nifedipine-sensitive and insensitive components of agonist-induced constriction were comparable among the two groups of arteries (Fig. 3C).

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).

Ca_V3.1 enable myogenic tone by facilitating Ca²⁺ wave generation

To assess whether Ca²⁺ flux through Ca_V3.1 triggers Ca²⁺ wave generation, mesenteric arteries from C57BL/6 and Ca_V3.1^−/− mice were loaded with Fluo-8, and rapid Ca²⁺ imaging was assessed by swept field confocal microscopy. Ca²⁺ waves in C57BL/6 mice were readily observed in 80% of smooth muscle cells (8 of 10 per vessel) at a frequency of 9 waves/cell/min, each with a duration of 3–4 s (Fig. 4A,B). Similar to rat vessels, nifedipine application had little discernible effect on Ca²⁺ wave generation²⁴. The deletion of Ca_V3.1 markedly reduced the number of firing smooth muscle cells (P = 0.0002) along with firing frequency (P < 0.0001) by 55% and 65%, respectively (Fig. 4A,B); the Ca²⁺ waves that remained were insensitive to nifedipine. Control experiments in C57BL/6 mesenteric arteries (Fig. 4C,D) subsequently confirmed that 2-APB, a blocker of IP₃Rs, notably attenuated the number of firing cells (P < 0.0001) and Ca²⁺ wave frequency (P = 0.0002) by 80% and 75%, respectively. Owing to the non-selective nature of 2-APB, and its reported inhibition of store-operated Ca²⁺ entry, the previous control experiments were repeated in the presence of xestospongin C, a selective IP₃R blocker. Similar to 2-APB, xestospongin C attenuated the number of firing cells (P = 0.011) and Ca²⁺ wave frequency (P = 0.002) (Fig. 4E,F). With this functional evidence indicating that Ca²⁺ flux through Ca_V3.1 triggers IP₃Rs and the induction of Ca²⁺ waves, the PLA was employed to assess whether these two proteins sat closely to one another. Consistent with Ca_V3.1 and IP₃R1 residing within 40 nm of one another, we observed red punctate labelling in smooth muscle cells isolated from C57BL/6 but not Ca_V3.1 mesenteric arteries (Fig. 5). Controls were performed on cells treated with anti-Ca_V3.1, anti-IP₃R1, or secondary antibodies alone, and revealed no evidence of nonspecific binding and false product amplification.

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.

Given the preceding observation, a final set of functional experiments were performed to address the contributory role of IP₃R-dependent Ca²⁺ waves to pressure-induced constriction. Using mesenteric arteries from C57BL/6 and Ca_V3.1^−/− mice, myogenic tone was examined over a full pressure range in the absence and presence of nifedipine (0.3 µM) ± 2-APB (50 µM). Of particular note, was the nifedipine-resistant tone that was present in C57BL/6 but not Ca_V3.1^−/− arteries, particularly at lower intravascular pressures (Fig. 6A,B,D,E). That tone per se was largely eliminated with the further application of 2-APB (20 mmHg: P = 0.013, 40 mmHg: P = 0.008) consistent with IP₃Rs and Ca²⁺ waves playing a role in its genesis (Fig. 6C,I). Note that IP₃R inhibition in Ca_V3.1^−/− arteries had no discernible effect on nifedipine insensitive tone at any pressure (Fig. 6D–F). In a set of control experiments, xestospongin C (3 µM, a more selective IP₃R antagonist) was used in place of 2-APB in C57BL/6 and it generated a contractile (Fig. 6G,H, 20 mmHg: P = 0.015, 40 mmHg: P = 0.001, 60 mmHg: P = 0.02, 80 mmHg: P = 0.013), and Ca²⁺ wave (Fig. 4E,F) phenotype akin to Ca_V3.1^−/− arteries. In a final set of controls, myogenic tone and Ca²⁺ waves were assessed in C57BL/6 vessels, with and without kurtoxin (Fig. 7) to block Ca_V3 channels. Kurtoxin reduced myogenic tone at 20 and 40 mmHg when place on top of nifedipine (20 mmHg: P = 0.04, 40 mmHg: P = 0.008). Also note, whereas nifedipine had no impact on Ca²⁺ wave generation (Fig. 4), kurtoxin + nifedipine had an inhibitory effect (Fig. 7: number of firing cells (P = 0.004) and firing frequency (P = 0.002)).

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.

Discussion

Bayliss first described the intrinsic ability of resistance arteries to constrict to a rise in intravascular pressure³. This foundational response is now known to set basal tone in key organs and stabilizes organ perfusion as blood pressure fluctuates. Further, this response has been intimately tied to arterial depolarization and the rise in [Ca²⁺]_i enabled by graded Ca²⁺ entry principally through L-type Ca²⁺ channels⁹. Vascular L-type Ca²⁺ channels are encoded by the Ca_V1.2 α₁ pore-forming subunit whose steady-state voltage-dependent properties are shifted rightward to more depolarized potentials²⁵. Recent work has revealed that L-type Ca²⁺ channels are not alone in vascular smooth muscle and that T-type Ca²⁺ channels are also expressed, with Ca_V3.1 being key to this investigation⁹. Its steady-state activation profile is hyperpolarized, and as such enables Ca²⁺ entry when L-type Ca²⁺ channels are deactivated. In theory, Ca²⁺ entry via T-type Ca²⁺ channels could impact tone development by directly contributing to the cytosolic Ca²⁺ pool or by acting locally and indirectly to trigger Ca²⁺ waves. Ca²⁺ waves are slow asynchronous events that spread from end to end and whose triggering is tied to the opening of sarcoplasmic reticulum IP₃Rs by IP₃ and Ca²⁺²⁴. Using a Ca_V3.1^−/− model, we tested whether Ca²⁺ entry through this particular T-type channel does indeed facilitate myogenic tone at hyperpolarized voltages and if this functional response is coupled to the governance of Ca²⁺ waves.

Our examination of Ca_V3.1 began with experiments to confirm the absence of Ca_V3.1 in mesenteric arterial smooth muscle cells from genetic deletion mice (Fig. 1). Three approaches were used, the first being PCR which confirmed Cacna1g gene (Ca_V3.1) modification in Ca_V3.1^−/− mice. Second, protein analysis using immunohistochemistry showed that surface expression of Ca_V3.1 was notably lacking in Ca_V3.1^−/− but not C57BL/6 cells. These observations aligned with the results from the third, functional approach (whole-cell electrophysiology), which revealed that the nifedipine/Ni²⁺ resistant Ba²⁺ current, previously ascribed to Ca_V3.1^21,22 was also absent in mesenteric arterial smooth muscle cells harvested from genetic deletion mice. The absence of this T-type Ca²⁺ channel coincided with a reduction in systolic and diastolic blood pressure, a finding consistent with a role in hemodynamic control. Past observations are limited, with one study reporting no difference in blood pressure, although values were unrealistically low for both Ca_V3.1^−/− and C57BL/6 mice²⁶. A second showed blood pressure trending lower in Ca_V3.1^−/− mice, along with a more substantive reduction in blood pressure variability²⁷. While the mechanism driving the blood pressure change is unclear, it’s reasonable to assert a role for diminished myogenic tone, an idea we tested in isolated mesenteric arteries across a full pressure range. Consistent with expectations, a reduction in myogenic tone was observed in Ca_V3.1^−/− arteries, particularly at lower pressure (Fig. 2) when vessels are hyperpolarized and T-type Ca²⁺ channels more active in the steady state²⁸. In considering these observations, prudent controls are key, the first being an assessment of an artery’s passive structural properties. In this regard, we observed no change in the arterial distensibility in vessels harvested from Ca_V3.1^−/− or C57BL/6 mice. Likewise, in a second set of controls, this study did not observe a change in arterial contractility to PE across a full concentration range in the absence or presence of nifedipine, an L-type Ca²⁺ channel blocker (Fig. 3). These results confirm that the molecular machinery mediating PE-induced constriction remains intact in Ca_V3.1^−/− animals, as does the signalling pathways downstream from the α₁-adrenoreceptor. Past studies have performed similar agonist controls and findings have been somewhat conflicted, with Ca_V3.1 deletion notably reducing mesenteric arterial responsiveness in one study²⁹, yet having the markedly opposite effect in another, presumptively increasing the Ca²⁺ sensitivity of the contractile apparatus²⁰.

In contextualizing the preceding observations, one should recognize past inferential work linking T-type Ca²⁺ channels to myogenic tone using pharmacology with known off-target effects. This approach typically entailed the probing of myogenic tone at rest and in the presence of an L-type Ca²⁺ channel blocker to isolate residual tone whose sensitivity to T-type Ca²⁺ channel inhibition was then tested^12,30,31. One should also consider past work with Ca_V3.1^−/− mice¹⁶ highlighting a role for Ca_V3.1 channels in tone development (at low pressure), although without defining mechanism²⁰. Finally, in using this genetic deletion model, acknowledgement of other cardiovascular effects is key, in particular bradycardia²⁶ and impaired blood pressure regulation through impaired NO formation³².

Ca²⁺ waves are slow-spreading, end-to-end events initiated by a stimulus that drives the release of Ca²⁺ from the sarcoplasmic reticulum^33,34. The initiation and spread of these asynchronous events are tied to IP₃R, Ca²⁺-permeable channels whose activation depends on IP₃ and Ca²⁺ binding to cytosolic sites²⁵. Past work in rat cerebral arteries has shown that Ca²⁺ waves are present at low intravascular pressure and that frequency rises as pressure is elevated into the lower physiological range²⁴. Pharmacological attenuation of Ca²⁺ waves through IP₃R blockade or impairment of store refilling results in diminished pressure-induced constriction particularly at low intravascular pressure when arteries are more hyperpolarized²⁴. Respectful of these results, it follows that low threshold Ca_V3.1 channels provide the Ca²⁺ needed to trigger Ca²⁺ waves and foster myogenic tone when L-type Ca²⁺ channels are decidedly less active. This concept was tested three ways, the first examining Ca²⁺ wave generation in Ca_V3.1^−/− arteries, the second ascertaining if Ca_V3.1 colocalized with IP₃Rs, and the final determining if Ca²⁺ wave inhibition in C57BL/6 mice results in a Ca_V3.1^−/− contractile phenotype. Findings in Fig. 4 first reveal that Ca²⁺ wave generation is robust in control mesenteric arteries as defined by the number of firing cells and the rate of Ca²⁺ waves per firing cell. Analogous to past work in rat cerebral arteries, nifedipine didn’t impact Ca²⁺ wave generation, consistent with L-type Ca²⁺ channels playing little role in initiating or maintaining these events²⁴. Ca²⁺ waves were significantly reduced in Ca_V3.1^−/− arteries and abolished in control arteries by 2-APB, and xestospongin C, IP₃R inhibitors, findings consistent with this T-type Ca²⁺ channel driving sarcoplasmic reticulum dependent events. These intriguing findings aligned with results from the PLA that note a close spatial association between Ca_V3.1 and IP₃R. In detail, this assay involves the binding of primary antibodies to two target proteins and then uses secondary antibodies with conjugated DNA strands which form a circular DNA template for amplification if proteins are < 40 nm apart¹⁰. The amplified product, detected as bright red puncta, is clearly visible in Fig. 5, thus, it is logical to conclude that Ca²⁺ flux via Ca_V3.1 should be sufficient to open IP₃Rs. In light of both results, final experiments assessed whether reduced Ca²⁺ wave production in C57BL/6 vessels generate a functional phenotype akin to Ca_V3.1^−/− arteries. In this regard, we monitored myogenic tone in mesenteric arteries (as a percentage; C57BL/6 and Ca_V3.1^−/−) at rest and following treatment with nifedipine alone or with 2-APB or xestospongin C (Fig. 6). We observed residual tone in nifedipine-treated C57BL/6 arteries but not Ca_V3.1^−/− arteries, a difference that could be abolished, particularly at low intravascular pressures (20–40 mmHg) through IP₃R blockade. This loss of tone parallels a similar loss in tone, and likewise Ca²⁺ waves in C57BL/6 mice when kurtoxin, a Ca_V3.x blocker was applied on top of nifedipine, an L-type Ca²⁺ channel blocker (Fig. 7). While some caution is warranted when drawing a relationship between Ca²⁺ waves and myogenic tone, the preceding interpretation does align with other published observations. They include: (1) 2-APB treatment having no impact on global [Ca²⁺] while reducing myogenic tone³⁵; (2) 2-APB only dilating arteries which prior to treatment were generating Ca²⁺ waves³⁶; and (3) Ca²⁺ waves abrogation corelating with reduced myosin light chain phosphorylation particularly at lower pressure²⁴.

Two final points in this study require further consideration. First, while differences in myogenic tone between Ca_V3.1^−/− and C57BL/6 arteries were evident at lower pressures (20–60 mmHg), the same trend was present at higher pressures, although without statistical significance. This finding is perhaps unsurprising as L-type Ca²⁺ channels rise to dominate [Ca²⁺]_i as arteries depolarize with pressurization. Second, while our work noted Ca²⁺ wave insensitivity to L-type Ca²⁺ channel blockade, like the cerebral vasculature²⁴, it lies in contrast to cremaster arterioles where nifedipine attenuated Ca²⁺ wave formation³⁷. This discrepancy suggests there may be mechanistic uniqueness among vascular beds, which to date is unappreciated. Alternatively, one could potentially argue the higher concentration of nifedipine (1 μM) used on cremaster arteries may be blocking Ca_V3.1 and consequently the triggering of IP₃R^38,39,40. This perspective is consistent with electrophysiology observations noting that T-type Ca²⁺ channels are partially blocked by low micromolar nifedipine^41,42.

Conclusion

This study presents three key findings, summarized in Fig. 8: First, Ca_V3.1^−/− mice have lower blood pressure, and mesenteric arteries display diminished myogenic tone compared to controls. Second, immunohistochemical analysis reveals that Ca_V3.1 lies within 40 nm of IP₃R1, and when this arrangement is genetically disrupted, arteries generate fewer Ca²⁺ waves. Third, a pharmacological blockade of IP₃Rs in C57BL/6 arteries produces a phenotype similar to Ca_V3.1^−/− vessels, that being diminished myogenic tone at lower intravascular pressure. By establishing a clear sequential relationship between Ca_V3.1, Ca²⁺ waves and myogenic tone, this study advances the understanding of vascular contractility and highlights a new target for therapeutic control. In this regard, one could provocatively suggest that development of selective Ca_V3.1 blockers could be of value in the management of hypertension.

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.