The β3 Subunit Contributes to Vascular Calcium Channel Upregulation and Hypertension in Angiotensin II-infused C57BL/6 Mice

Abstract

Voltage-gated L-type Ca²⁺ (Ca_v1.2) channels in vascular smooth muscle cells (VSMCs) are a predominant Ca²⁺ influx pathway that mediates arterial tone. Channel biogenesis is accomplished when the pore-forming α_1C subunit co-assembles with regulatory Ca_vβ subunits intracellularly, and the multi-protein Ca_v1.2 complex translocates to the plasma membrane to form functional Ca²⁺ channels. We hypothesized that the main Ca_vβ isoform in VSMCs, Ca_vβ3, is required for the upregulation of arterial Ca_v1.2 channels during the development of hypertension, an event associated with abnormal Ca²⁺-dependent tone. Ca_v1.2 channel expression and function was compared between 2^nd order mesenteric arteries (MA) of C57BL/6 wild-type (WT) and Ca_vβ3^-/- mice infused with saline (control) or angiotensin II (Ang II) for 2 weeks to induce hypertension. The MA of Ang II-infused WT mice showed increased Ca_v1.2 channel expression and accentuated Ca²⁺-mediated contractions compared to saline-infused WT mice. In contrast, Ca_v1.2 channels failed to upregulate in MA of Ang II-infused Ca_vβ3^-/- mice and Ca²⁺-dependent reactivity was normal in these arteries. Basal systolic blood pressure (SBP) was not significantly different between WT and Ca_vβ3^-/- mice (98±2 mm Hg and 102±3 mm Hg, respectively), but the Ca_vβ3^-/- mice showed a blunted pressor response to Ang II infusion. Two weeks after the start of Ang II administration, the SBP of Ca_vβ3^-/- mice averaged 149±4 mm Hg compared to 180±5 mm Hg in WT mice. Thus, the Ca_vβ3 subunit is a critical regulatory protein required to upregulate arterial Ca_v1.2 channels and fully develop angiotensin II –dependent hypertension in C57BL/6 mice.

Introduction

During the development of hypertension, the L-type Ca²⁺ (Ca_v1.2) channel encoded by the Ca_v1.2 gene is upregulated in vascular smooth muscle cells (VSMCs).^1-3 An over-abundance of vascular Ca_v1.2 channels has been documented in rats with genetic, renal and salt-dependent forms of hypertension [for review, see 4]. At least one study suggests that even a short-term elevation of intravascular pressure increases expression of arterial Ca_v1.2 channels in vivo, implicating the upregulation of Ca_v1.2 channels as an early event in the pathogenesis of hypertension.¹ The increased expression of Ca_v1.2 channels enhances voltage-dependent Ca²⁺ influx resulting in abnormal vasoconstriction, which can be reversed by pharmacological antagonists of the Ca_v1.2 channel.^1,2,5-7 Correspondingly, an increased Ca_v1.2 channel –mediated arterial tone in hypertensive animals and humans with essential hypertension may partly explain their sensitivity to the antihypertensive effect of clinical Ca²⁺ channel antagonists that block Ca_v1.2 channels. The same drugs only mildly lower blood pressure in normotensive subjects.^8-11

The mechanisms that mediate the upregulation of arterial Ca_v1.2 channels during hypertension are unclear, but appear to primarily rely on post-transcriptional events since the increased expression of the Ca_v1.2 pore-forming α_1C subunit has not been linked to a corresponding increase of α_1C transcript.^2,3 In the present study, we hypothesized that the regulatory β3 subunit (Ca_vβ3) of the Ca_v1.2 channel, which acts post-transcriptionally to promote surface expression of the α_1C pore, may increase Ca_v1.2 channel abundance during hypertension. It is presumed that in VSMCs, similar to other cell types, the α_1C subunit co-assembles with smaller β and α₂δ subunits to form functional Ca²⁺ channels.^12-15 Four gene families (β1, β2, β3, β4) encode Ca_vβ subunits in a tissue-specific manner. Ca_vβ3 is the principal subtype in VSMCs,^16,17 where it is presumed to bind to α_1C in the endoplasmic reticulum (ER) to enhance its trafficking to the plasma membrane (PM). At the cell surface, the multi-protein Ca_v1.2 channel assumes its critical function of mediating voltage-dependent Ca²⁺ influx during membrane depolarization.

The precise domains that permit interaction between α_1C and Ca_vβ3 in the ER and the sequence of events that promote Ca_v1.2 expression on the surface of VSMCs remain elusive. However, Murakami et al^16,17 reported that the aortae of Ca_vβ3^-/- mice express fewer Ca_v1.2 channels compared to wild-type (WT) mice, implying that Ca_vβ3 contributes to the basal expression of Ca_v1.2 channels in VSMCs in vivo. Unexpectedly, despite the loss of arterial Ca_v1.2 channels, the Ca_vβ3^-/- mice exhibited normal systolic blood pressure (SBP).^16,17 This seeming dichotomy may indicate that blood pressure was sustained by compensatory mechanisms and/or indicate that Ca²⁺ influx through Ca_v1.2 channels contributes only minimally to basal blood pressure in normotensive subjects.^8-11

Here, we considered the hypothesis that the regulatory Ca_vβ3 subunit promotes vascular Ca_v1.2 channel expression in hypertension, a disease in which this channel is pathogenically over-expressed. Our studies utilized Ca_vβ3^-/- mice to explore if deletion of the Ca_vβ3 gene attenuates the upregulation of arterial Ca_v1.2 channels and the development of hypertension. The findings demonstrate that although Ca_vβ3^-/- mice show normal resting SBP, they fail to fully develop angiotensin II (Ang II)-dependent hypertension and this loss of function is associated with an inability to upregulate arterial Ca_v1.2 channels. Thus, Ca_vβ3 may be a previously unrecognized but critical protein that contributes to the vascular Ca_v1.2 channel abnormalities that elevate blood pressure.

Methods

Blood pressure measurement

Procedures using animals were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. SBP was recorded using tail-cuff plethysmography before and after implanting subcutaneous osmotic minipumps containing either saline or Ang II (infused at 2 ng/g/min).

Vessel perfusion assays

Second order mesenteric arteries (MA) were isolated from saline-infused (SAL) and Ang II-infused hypertensive (AHT) mice, and used to perform vessel perfusion assays.

Patch clamp studies

The VSMCs were enzymatically isolated from 2^nd order MA branches and whole-cell Ca²⁺ channel currents were recorded using Ba²⁺ (10 mmol/L) as a charge carrier with standard pulse protocols, solutions and a patch-clamp station described previously.¹

Quantitative real-time PCR

Whole tissue RNA was isolated from 2^nd order branches of MA pooled from two mice. Then qRT-PCR was performed using primers specific for α_1C, Ca_vβ3 and GAPDH.

Western blotting

Protein lysates were prepared by pooling MA from two mice.¹ Specific polyclonal antibodies directed against epitopes specific to α_1C and Ca_vβ3 were used as probes.

Statistics

Data represent mean ± standard error of the mean for the number (n) of animals indicated in parentheses. Student's t-test was used to compare two data sets and one-way ANOVA with Bonferroni's post-hoc test was used for multiple group comparisons. P ≤ 0.05 was considered significant.

Results

Ca_v1.2 channel α_1C and β3 subunits upregulate in MA of AHT mice

Initially we determined whether vascular Ca_v1.2 channels upregulate in Ang II-infused C57BL/6 mice, since previous studies demonstrating Ca_v1.2 upregulation were performed only in hypertensive rat models. Figure S1 shows that 14 days of subcutaneous Ang II infusion (2 ng/g/min) resulted in SBP values of 106±2, 155±5 and 172±8 mm Hg at 0, 7 and 14 days, respectively (n=10 each). The SBP values in SAL mice did not significantly differ from a basal value of 104±3 mm Hg. Subsequent studies were performed using mice infused with saline or Ang II for two weeks.

Next, we determined if the MA of AHT mice showed an increased expression of the α_1C and Ca_vβ3 subunits that co-assemble to form functional Ca_v1.2 channels. Adjacent lanes on Western blot (WB) were loaded with MA lysate from SAL or AHT mice. The α_1C protein was upregulated 2.4±0.4 fold in the MA of AHT mice compared to SAL mice (Fig. 1A,B). The abundance of the Ca_vβ3 subunit also increased during the development of hypertension. The immunodensity of the Ca_vβ3 band was 2.8±0.6 fold higher in MA lysate from AHT mice compared to SAL mice (Fig. 1A,C). Interestingly, qRT-PCR experiments using total RNA isolated from MA of SAL and AHT mice revealed that the transcript levels of both α_1C (ΔC_T values SAL 24.2±0.3, AHT 24.1±0.3) and Ca_vβ3 (ΔC_T values SAL 25.1±0.5, AHT 25.1±0.6) were similar between the two isolates (Fig. 1D). The C_T values were normalized to GAPDH, which was used as an amplification standard.

Figure 1.

A. WB showing adjacent lanes loaded with 40 μg of MA protein lysate pooled from either two SAL or AHT mice and probed with anti- α_1C and Ca_vβ3. GAPDH was a loading control. B, C. Densitometric analyses of α_1C and Ca_vβ3 immunoreactivity (n=6; * = p<0.05). D. qRT-PCR amplification corresponding to α_1C, Ca_vβ3 and GAPDH transcripts averaged from 4 MA samples from SAL and AHT mice.

Increased Ca_v1.2 current and vascular reactivity in AHT mice

We confirmed that the over-abundance of Ca_v1.2 channels in MA of AHT mice was associated with enhanced channel function. Patch-clamp studies revealed increased Ca_v1.2 channel -mediated current in mesenteric VSMCs of AHT compared to SAL animals (Fig. 2A, top traces), which were blocked by nifedipine (Fig. S2). Current-voltage relationships suggested a 1.9±0.2 fold increase in peak current density in the VSMCs of AHT mice (-10.7±1.0 pA/pF) compared to SAL mice (-5.6±0.7 pA/pF) (Fig. 2A, n=23 each). Voltage-sensitive activation and inactivation were not different between groups (Fig. S3), revealing no abnormalities of Ca_v1.2 channel properties in VSMCs of AHT mice. The MA from AHT mice also exhibited enhanced reactivity to the Ca_v1.2 channel opener, FPL 64176 (Fig. 2B) resulting in a 5.1 -fold lower EC₅₀ value of 224±37 nmol/L compared to 1144±331 nmol/L in arteries of SAL mice (Fig. 2C).

Figure 2.

A. Ca_v1.2 channel current elicited by 8 mV steps from -70 mV to +58 mV in mesenteric VSMCs of SAL and AHT mice (upper panel) and averaged I-V curves (n=23 each). B. On-line diameter responses of MA from SAL and AHT mice to increasing concentrations of FPL 64176 (1 nmol/L to 10 μmol/L, half-log units). C. Concentration-response relationships to FPL 64176 reveal a lower EC₅₀ value for MA from AHT mice compared to SAL mice (n=7 each). * = p<0.05.

Ca_vβ3^-/- mice show normal resting SBP

After linking an overabundance of arterial Ca_v1.2 channels in AHT mice to increased Ca_vβ3 expression for the first time, we directly tested if Ca_vβ3 subunits are required for Ca_v1.2 channel expression by employing WT and Ca_vβ3^-/- mice. We observed no difference in α_1C expression between MA from WT and Ca_vβ3^-/- mice (Fig. 3A-B, n=12 each). Resting SBP levels also were similar between WT mice (98±2 mm Hg) and Ca_vβ3^-/- mice (102±3 mm Hg) (Fig. S4A, n=10). The resting heart rate of 555±8 beats/min in WT mice (n=10) and 563±10 beats/min in Ca_vβ3^-/- mice (n=11) also was not different (Fig. S4B).

Figure 3.

A. WB showing adjacent lanes loaded with 40 μg of MA protein lysate each pooled from two WT or Ca_vβ3^-/- mice and probed with anti-α_1C or Ca_vβ3. GAPDH was a loading control. B. Average densitometric values for α_1C (n=12).

Ca_vβ3^-/- mice fail to fully develop hypertension

Subsequently, we explored if the Ca_vβ3 subunit contributes to the development of Ang II-dependent hypertension, which we postulated may partly rely on upregulation of Ca_v1.2 channels. WT and Ca_vβ3^-/- mice were infused with saline (Sal) or Ang II for two weeks, and SBP was recorded for 3 days before infusion (day 0) and on days 4, 7, 10 and 14 after infusion. As expected, WT and Ca_vβ3^-/- mice infused with saline maintained normal SBP that averaged 104±2 mm Hg and 102±5 mm Hg on day 14, respectively (Fig. 4, n=7 each). The WT mice infused with Ang II showed steadily rising SBP reaching an average value of 180±5 mm Hg on day 14 (Fig. 4, n=7). In contrast, the SBP response to Ang II infusion was significantly blunted in Ca_vβ3^-/- mice, averaging only 149±4 mm Hg on day 14 (Fig. 4, n=7).

Figure 4.

The SBP response to two weeks of saline (Sal) or Ang II infusion in WT and Ca_vβ3^-/- mice. Sal-infused WT and Ca_vβ3^-/- mice showed normal SBP during the infusion period. SBP progressively increased in Ang II-infused WT mice and reached 180±5 mm Hg at 14 days. Ca_vβ3^-/- mice showed a blunted blood pressure response to Ang II infusion and SBP at 14 days averaged only 149±4 mm Hg (n=7 each). * = p<0.05, significant difference between Ang II-infused WT and Ca_vβ3^-/-.

MA of Ca_vβ3^-/- mice fail to upregulate Ca_v1.2 channels

Next, we compared the expression level of the Ca_v1.2 channel α_1C subunit between MA of WT and Ca_vβ3^-/- mice infused with saline or Ang II. As expected, Ang II infusion was associated with an upregulation of the α_1C and Ca_vβ3 subunits in MA of WT mice (Fig. 5A, lanes 1 and 3), which correlated to a 1.90±0.1 fold increase in Ca_v1.2 α_1C (Fig. 5B, n=6) and a 2.65±0.7 fold increase in Ca_vβ3 immunosignal. In contrast, α_1C failed to upregulate in MA of Ang II –infused Ca_vβ3^-/- mice (Fig. 5A, lanes 2 and 4), showing only a 2.3% average increase compared to Sal-infused Ca_vβ3^-/- animals (Fig. 5B, n=6).

Figure 5.

A. WB probed with antibodies directed against α_1C and Ca_vβ3. Each lane was loaded with 40 μg of protein lysate pooled from the MA of two WT or Ca_vβ3^-/- mice infused with either saline (Sal) or Ang II for two weeks. GAPDH was a loading control. B. Densitometric analyses revealed that only MA of WT mice upregulated α_1C in response to Ang II (n=6). C, D. On-line picture and diameter recordings, respectively, of MA from Ang II-infused WT or Ca_vβ3^-/- mice. The contraction to FPL 64176 (0.3 μmol/L) was blunted in arteries of Ca_vβ3^-/- mice. E. Average diameter responses to FPL 64176 in MA from Ang II-infused WT and Ca_vβ3^-/- mice (n=7 each). *^{, #} = p<0.05, # indicates significant difference between Sal-infused mice and Ang II-infused WT mice. * indicates value is significantly different than Ang II -infused WT mice.

MA of Ca_vβ3^-/- mice fail to show abnormal Ca_v1.2 channel–mediated reactivity

Finally, we explored whether the failure of Ca_v1.2 channels to upregulate in MA of Ang II-infused Ca_vβ3^-/- mice also attenuated the heightened Ca²⁺-dependent reactivity observed in MA of Ang II-infused WT mice (Figs. 2B-C). To test this hypothesis, an ∼EC₅₀ of FPL 64176 (0.3 μmol/L) was used to activate Ca_v1.2 channels in isolated MA. The diameter response to FPL 64176 in MA from Ang II-infused Ca_vβ3^-/- of 34±11 μm was markedly less compared to a 72±12 μm reduction in MA from Ang II-infused WT mice (Fig. 5C-E; n=7).

Discussion

Our findings demonstrate that the MA of Ca_vβ3^-/- mice infused with Ang II fail to upregulate Ca_v1.2 channels or show enhanced Ca²⁺-dependent reactivity, which are abnormalities observed in MA of Ang II-infused WT mice. Correspondingly, Ca_vβ3^-/- mice fail to fully develop Ang II-dependent hypertension compared to WT mice. Collectively our findings draw attention to the Ca_vβ3 subunit in VSMCs as a critical protein necessary to upregulate Ca_v1.2 channels and elevate blood pressure in Ang II-dependent hypertension. The residual rise in SBP in response to Ang II infusion in Ca_vβ3^-/- mice infers that factors in addition to the upregulation of Ca_v1.2 channels contribute to the elevated blood pressure in this form of hypertension, reflecting its complex pathogenesis as reviewed by Hall.¹⁸

Many studies during the past decade have shown that hypertension is associated with an elevated Ca²⁺ influx through arterial Ca_v1.2 channels.¹⁴ At least three rat models of hypertension show an increased density of functional Ca_v1.2 channels in the VSMCs of the aorta and in vascular beds involved in blood pressure regulation.^1-3,5,6 In the renal circulation of aortic-banded rats, an increased Ca_v1.2 expression occurs by 48 hours after blood pressure elevation.¹ Thus, an upregulation of Ca_v1.2 channels, the opening of which may be driven by the depolarized membrane potential of VSMCs exposed to high intravascular pressure, appears to contribute to the elevated vascular tone of hypertension.^1,2 An increased Ca²⁺-dependent reactivity, and an enhanced vasodilator sensitivity to nifedipine, also has been reported in MA segments from hypertensive patients.⁷ These findings, combined with the observation that the blood pressure lowering effect of calcium channel blockers is exaggerated in hypertensive patients,^8,9 raise the possibility that Ca_v1.2 channel abnormalities extend to essential hypertension in humans.

Here, we extend earlier findings in rats to the Ang II –infused hypertensive mouse by confirming that MA from AHT mice show increased Ca_v1.2 expression and Ca²⁺-dependent reactivity compared to arteries from SAL mice with normal blood pressure. Similar to earlier studies in rats,^1-3 we failed to detect a major increase of α_1C transcript in arteries of AHT mice that could account for the marked over-abundance of the α_1C protein. Thus, the upregulation of arterial Ca_v1.2 channels in AHT mice appears to rely on post-transcriptional mechanisms that could include increases in translational efficiency and/or channel biogenesis, trafficking or surface stability.

In this regard, Ca_vβ subunits are known to increase the surface density of Ca²⁺ channels, although most studies have been performed in heterologous expression systems^19,20 or nonvascular cell types.^21,22 The Ca_vβ subunits co-assemble with pore-forming α-subunits during channel biogenesis to promote the trafficking of the Ca²⁺ channel complex from the ER to the PM.^14,19 Although Ca_vβ are postulated to mask an ER retention signal in α-subunits as a mechanism to target Ca²⁺ channels to the cell surface, no specific motif has been identified. Altier et al.²³ has alternatively demonstrated in HEK293 cells that the interaction between Ca_vβ and α_1C prevents ubiquitination of the α_1C subunit by ubiquitin ligase, thereby rescuing Ca_v1.2 channels from the ER-associated protein degradation (ERAD) complex that marks the channel for proteasomal degradation. Waithe et al.²⁴ reported a similar interaction between Ca_vβ and Ca_v2.2 channels in neurons and concluded that Ca_vβ increases Ca_v2.2 surface density by conferring protection from proteosomal degradation. Finally, by co-expressing Nedd4 ubiquitin ligases and Ca_v1.2 channels in tsA-201 cells, Rougier et al.²⁵ showed that Nedd4-1 ubiquitin ligase directly interferes with the Ca_vβ-mediated delivery of Ca_v1.2 channels to the PM.

The precise physiological role of Ca_vβ3 in VSMCs is unknown. Murakami et al.^16,17 reported that α_1C expression and Ca_v1.2 -mediated Ca²⁺ current were reduced in aortae from Ca_vβ3^-/- mice, although SBP was normal. Here, we also observed comparable basal SBP between WT and Ca_vβ3^-/- mice, and additionally, the MA of these mice exhibited similar basal Ca_v1.2 channel expression. However, MA of Ca_vβ3^-/- mice failed to upregulate Ca_v1.2 channels or show accentuated Ca²⁺-dependent reactivity in response to Ang II infusion, which were abnormalities consistently observed in arteries of hypertensive WT mice. The absence of Ca_v1.2 channel upregulation in MA of Ang II –infused Ca_vβ3^-/- mice was associated with a blunted blood pressure response to Ang II. Thus, our findings suggest an association between the Ca_vβ3 subunit, Ca_v1.2 upregulation and hypertension. Notably, the deletion of Ca_vβ3 was not limited to VSMCs in the Ca_vβ3^-/- mice of our study, and thus we cannot state with certainty that the failure of Ca_vβ3^-/- mice to fully develop Ang II –dependent hypertension relied on Ca_vβ3 deletion in VSMCs rather than nonvascular cell types. Although the Ca_vβ3^-/- mouse has no gross phenotype, the Ca_vβ3 subunit also is extensively expressed in the brain and may negatively regulate NMDA receptors and potentially affect other neuronal functions.²⁶ For this reason, the design of new Ca_vβ3^-/- mice showing conditional and SMC-specific Ca_vβ3 gene deletion will be required to directly test the hypothesis that arterial Ca_vβ3 subunits contribute to experimental hypertension.

Notably, Fan et al.²² designed mutant Ca_vβ subunits that interact with α_1C intracellularly, but lack the motifs required to target the Ca_v1.2 channel complex to the PM. These β subunit “decoys” represent potential therapeutics to reduce Ca_v1.2 surface expression in cardiovascular pathologies characterized by an overabundance of Ca_v1.2 channels.^22,27,28 It may be advantageous to ameliorate anomalous Ca²⁺ influx by preventing the upregulation of Ca_v1.2 channels rather than using pharmacological blockers to reduce the activity of over-expressed channels. Our findings provide proof-of-principle for this concept by showing that Ca_vβ3 deletion can normalize Ca_v1.2 channel expression in VSMCs of hypertensive mice in which Ca_v1.2 over-expression is a contributing defect.

Perspectives

To our knowledge, the present study provides the first evidence to implicate a distinct Ca²⁺ channel β subunit in the development of hypertension. It has been proposed that small molecule inhibitors of Ca_vβ subunits represent future therapies to lower Ca²⁺ channel abundance in conditions associated with excessive Ca²⁺ influx including hypertension. Our findings support the concept that strategies to knockdown or inhibit the Ca_vβ3 subunit in VSMCs and thereby reduce Ca_v1.2 channel expression in the arterial circulation may represent novel therapeutic approaches for lowering blood pressure in hypertensive subjects.

Supplementary Material

Figure S1. The SBP profiles of C57BL/6 mice infused with isotonic saline (vehicle, SAL) or Ang II (2 ng/g/min, AHT) respectively, for two weeks. Values of SBP at weeks 1 and 2 in SAL mice were not significantly different from baseline at week 0 (n=10). However, SBP was significantly increased at weeks 1 and 2 after the start of Ang II infusion in AHT mice (n=10). * = p<0.05.

Figure S2. Representative traces of families of Ca_v1.2 channel currents elicited by 8 mV steps from -70 mV to +58 mV in mesenteric VSMCs of SAL and AHT mice. The control currents (top traces) were blocked by 1 μmol/L nifedipine (lower traces).

Figure S3. Boltzmann fits to voltage-dependent activation and inactivation relationships. There was no difference in half-activation and inactivation voltages between SAL and AHT mice. The V_1/2 values for activation were 5.2±0.4 mV (SAL, n=11) and 4.0±0.4 mV (AHT, n=10). The V_1/2 values for inactivation were -2.4±0.7 mV (SAL, n=8) and -4.6±0.7 mV (AHT, n=7).

Figure S4. Resting SBP was similar between WT and Ca_vβ3^-/- mice (n=10 each). B. Resting heart rate also was not significantly different between WT and Ca_vβ^-/- mice (n=10, 11).

Novelty and Significance.

What is New?

• A β3 protein increases the number of voltage-gated Ca²⁺ channels in arteries during hypertension.

• Mice in which the β3 protein is deleted fail to fully develop hypertension.

What Is Relevant?

• The abnormal contraction of small arteries during hypertension is partly caused by the presence of too many Ca²⁺ channels.

• Here, we report that a β3 protein is required for Ca²⁺ channels to increase in arteries during hypertension and lack of this protein reduces the level of blood pressure elevation.

Summary.

The β3 subunit is a critical protein in arteries that contributes to the upregulation of voltage-gated Ca²⁺ channels and the development of hypertension.