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 (CaV1.2) 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 (CaV3.1) using C57BL/6 wild type and CaV3.1−/− mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca2+ imaging defined the CaV3.1−/− phenotype relative to C57BL/6. CaV3.1−/− mice had absent CaV3.1 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/CaV3.1 close physical association. IP3R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a CaV3.1−/− contractile phenotype. Findings indicate that Ca2+ influx through CaV3.1 contributes to myogenic tone at hyperpolarized voltages through Ca2+-induced Ca2+ release tied to the sarcoplasmic reticulum. This study helps establish CaV3.1 as a potential therapeutic target to control blood pressure.
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Introduction
Smooth muscle cells in the arterial wall actively contract to intravascular pressure, maintaining organ blood flow under dynamic conditions1,2. 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 (CaV1–3), are encoded in the mammalian genome and each displays unique voltage-dependent properties5. In arterial smooth muscle, CaV1.2 (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 counterparts9,10. Two subtypes (CaV3.1 and CaV3.2) 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 CaV3.1 as to enabling myogenic tone at hyperpolarized voltages9,11,12, 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+ waves13,14,15.
This study explored whether and by what mechanisms CaV3.1 channels enable myogenic tone development in mouse mesenteric arteries. This work entailed the use of C57BL/6 wild type and CaV3.1−/− 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 CaV3.1 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 CaV3.1−/− arteries as this channel no longer resided near IP3R1 and that IP3R blockade in C57BL/6 arteries produced a CaV3.1−/− phenotype. These findings highlight a role for CaV3.1 in myogenic tone development and hemodynamic control through the triggering of sarcoplasmic reticulum Ca2+ waves. They additionally reveal the potential therapeutic value of CaV3.1 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 CaV3.1−/− (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 CaV3.1−/− 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 CaV3.1 primers: C57BL/6 F, 5ʹ-ATACGTGGTTCGAGCGAGTC-3ʹ; WT R, 5ʹ-CGAAGGCCTGACGTAGAAAG-3ʹ; CaV3.1−/− 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
CaV3.1 expression was assessed in mesenteric arterial smooth muscle cells isolated from C57BL/6 and CaV3.1−/− 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-CaV3.1 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 CaV3.1 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 CaV3.2 channels without affecting CaV3.1. 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 CaV3.1−/− 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 CaV3.1−/− 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 Research17,18. 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 (CaV3.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:
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:
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 CaV3.1 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-CaV3.1, 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 CaV3.1 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 CaV3.1−/− genotype and phenotype
Genetic deletion of CaV3.1 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 CaV3.1 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 CaV3.1−/− mice was seen at 385 bp (Fig. 1Aa). Figure 1Ab shows CaV3.1 protein expression is punctate in smooth muscle cells isolated from C57BL/6 but not CaV3.1−/− mesenteric arteries (n = 4 mice per group). This analysis aligned with whole-cell electrophysiology, which noted dampened CaV3.1 activity in smooth muscle cells from CaV3.1−/− mice relative to C57BL/6 controls (P = 0.013). Note, CaV3.1 activity was measured by first monitoring the total inward Ba2+ current, the collective sum of CaV1.2, CaV3.1, and CaV3.2 currents20. Based on past studies, nifedipine and Ni2+ were then applied to abolish CaV1.2 (L-type) and CaV3.2 (T-type) activities, respectively, and the residual current was then assigned to CaV3.121,22. The current–voltage relationship of each Ca2+ channel is illustrated in Fig. 1B,C (CaV3.1 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 CaV3.1−/− mice.
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 CaV3.1−/− mice (Table 1) in these parameters. However, CaV3.1−/− 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 CaV3.1−/− mice compared to C57BL/6 mice (Fig. 1E).
CaV3.1 channels contribute to the myogenic response
Mesenteric arteries from C57BL/6 and CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1−/− 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).
CaV3.1 enable myogenic tone by facilitating Ca2+ wave generation
To assess whether Ca2+ flux through CaV3.1 triggers Ca2+ wave generation, mesenteric arteries from C57BL/6 and CaV3.1−/− 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 CaV3.1 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 CaV3.1 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 CaV3.1 and IP3R1 residing within 40 nm of one another, we observed red punctate labelling in smooth muscle cells isolated from C57BL/6 but not CaV3.1 mesenteric arteries (Fig. 5). Controls were performed on cells treated with anti-CaV3.1, anti-IP3R1, or secondary antibodies alone, and revealed no evidence of nonspecific binding and false product amplification.
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 CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1−/− 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 CaV3 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)).
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 CaV1.2 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 CaV3.1 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 CaV3.1−/− 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 CaV3.1 began with experiments to confirm the absence of CaV3.1 in mesenteric arterial smooth muscle cells from genetic deletion mice (Fig. 1). Three approaches were used, the first being PCR which confirmed Cacna1g gene (CaV3.1) modification in CaV3.1−/− mice. Second, protein analysis using immunohistochemistry showed that surface expression of CaV3.1 was notably lacking in CaV3.1−/− 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 CaV3.121,22 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 CaV3.1−/− and C57BL/6 mice26. A second showed blood pressure trending lower in CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1 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 tested12,30,31. One should also consider past work with CaV3.1−/− mice16 highlighting a role for CaV3.1 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 reticulum33,34. 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 CaV3.1 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 CaV3.1−/− arteries, the second ascertaining if CaV3.1 colocalized with IP3Rs, and the final determining if Ca2+ wave inhibition in C57BL/6 mice results in a CaV3.1−/− 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 CaV3.1−/− 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 CaV3.1 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 CaV3.1 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 CaV3.1−/− arteries. In this regard, we monitored myogenic tone in mesenteric arteries (as a percentage; C57BL/6 and CaV3.1−/−) 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 CaV3.1−/− 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 CaV3.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 CaV3.1−/− 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 CaV3.1 and consequently the triggering of IP3R38,39,40. This perspective is consistent with electrophysiology observations noting that T-type Ca2+ channels are partially blocked by low micromolar nifedipine41,42.
Conclusion
This study presents three key findings, summarized in Fig. 8: First, CaV3.1−/− mice have lower blood pressure, and mesenteric arteries display diminished myogenic tone compared to controls. Second, immunohistochemical analysis reveals that CaV3.1 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 CaV3.1−/− vessels, that being diminished myogenic tone at lower intravascular pressure. By establishing a clear sequential relationship between CaV3.1, 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 CaV3.1 blockers could be of value in the management of hypertension.
Data availability
All data generated or analysed during this study are included in this published article.
Abbreviations
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- IP3 :
-
Inositol 1,4,5-trisphosphate
- PE:
-
Phenylephrine
- PLA:
-
Proximity ligation assay
- 2-APB:
-
2-Aminoethoxydiphenyl borate
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Acknowledgements
The authors would like to thank all the members of our research group who contributed to this study. Dr DG Welsh holds the Rorabeck Chair in Molecular Neuroscience and Vascular Biology.
Funding
This work was supported by the Natural Science and Engineering Research Council of Canada (NSERC, RGPIN/04659-2017), an Ontario Graduate Scholarship (M.A.E) and a CIHR doctoral award (M.A.E).
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M.A.E., N.H., N.A., A.M.H., G.Y.M., and M.S. performed experiments. M.A.E. and A.M.H. prepared the figures. N.H., prepared the graphical abstract. M.A.E. and D.G.W. interpreted the results of the experiments. R.G. provided expert supervision to the collection of whole animal measurements including metabolic caging and tail cuff. D.G.W. conceived and designed the research. M.A.E., and N.H. executed the first draft of the manuscript. All authors edited and revised the manuscript critically. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors qualify for authorship, and all those who qualify for authorship are listed.
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El-Lakany, M.A., Haghbin, N., Arora, N. et al. CaV3.1 channels facilitate calcium wave generation and myogenic tone development in mouse mesenteric arteries. Sci Rep 13, 20407 (2023). https://doi.org/10.1038/s41598-023-47715-3
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DOI: https://doi.org/10.1038/s41598-023-47715-3
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