Selective Down-regulation of K_V2.1 Function Contributes to Enhanced Arterial Tone during Diabetes

Background: K_V channels in vascular smooth muscle cells (VSMCs) regulate arterial tone.

Results: K_V2.1 in VSMCs is down-regulated via AKAP150-CaN-dependent NFATc3 signaling during diabetes.

Conclusion: Transcriptional suppression of K_V2.1 contributes to enhanced arterial tone in diabetes.

Significance: AKAP150-CaN-dependent activation of NFATc3 may be a general mechanism for transcriptional regulation of K⁺ channels, and a valuable target to prevent and treat diabetic vascular complications.

Abstract

Enhanced arterial tone is a leading cause of vascular complications during diabetes. Voltage-gated K⁺ (K_V) channels are key regulators of vascular smooth muscle cells (VSMCs) contractility and arterial tone. Whether impaired K_V channel function contributes to enhance arterial tone during diabetes is unclear. Here, we demonstrate a reduction in K_V-mediated currents (I_Kv) in VSMCs from a high fat diet (HFD) mouse model of type 2 diabetes. In particular, I_Kv sensitive to stromatoxin (ScTx), a potent K_V2 blocker, were selectively reduced in diabetic VSMCs. This was associated with decreased K_V2-mediated regulation of arterial tone and suppression of the K_V2.1 subunit mRNA and protein in VSMCs/arteries isolated from HFD mice. We identified protein kinase A anchoring protein 150 (AKAP150), via targeting of the phosphatase calcineurin (CaN), and the transcription factor nuclear factor of activated T-cells c3 (NFATc3) as required determinants of K_V2.1 suppression during diabetes. Interestingly, substantial reduction in transcript levels for K_V2.1 preceded down-regulation of large conductance Ca²⁺-activated K⁺ (BK_Ca) channel β1 subunits, which are ultimately suppressed in chronic hyperglycemia to a similar extent. Together, our study supports the concept that transcriptional suppression of K_V2.1 by activation of the AKAP150-CaN/NFATc3 signaling axis contributes to enhanced arterial tone during diabetes.

Introduction

Non-insulin-dependent type 2 diabetes is a devastating disease affecting millions worldwide due to an aging population, sedentary lifestyle, and overnutrition. Vascular dysfunction, including enhanced arterial tone, is a leading cause of cardiovascular complications contributing to morbidity and mortality in the diabetic population (1). Although endothelial dysfunction has long been recognized as a major factor contributing to vascular dysfunction and enhanced arterial tone during diabetes (2–5), abnormal VSMC function may also play a critical role, although the mechanisms for this remain poorly understood. Thus, advances in this area could prove valuable for the development of rational therapies to treat and prevent diabetic vascular complications.

The level of arterial tone is largely determined by vascular smooth muscle cell (VSMC)² membrane potential (V_M) and Ca²⁺ entry via voltage-gated, L-type Ca²⁺ channels (LTCCs) (6). A major regulator of V_M is the activity of K_V and BK_Ca channels (7–9). Physiological activation of these channels hyperpolarizes VSMCs, thereby decreasing LTCC open probability and Ca²⁺ influx leading to vasodilation, whereas their inhibition promotes vasoconstriction (10). Previous reports have shown that in vitro, short term exposure of coronary, cerebral, and mesenteric arteries to extracellular glucose concentrations that resemble diabetic hyperglycemia (e.g. 20 mm) inhibit K_V and BK_Ca channel activity in VSMCs (11–15). Thus, inhibition of these channels may contribute to enhanced arterial tone and vascular complications during diabetes. Consistent with this, our group and others have found that the activity of BK_Ca channels is suppressed in VSMCs of several mouse models of diabetes (14–17). However, whether impaired K_V channel activity in VSMCs contributes to enhanced arterial tone during diabetes is currently unclear.

We recently demonstrated that suppression of BK_Ca channel activity in a high fat diet (HFD) mouse model of type 2 diabetes proceeds through activation of the prohypertensive CaN/NFATc3 signaling pathway (14). Activation of NFATc3 required anchoring of CaN by the scaffolding protein AKAP150 (murine ortholog of human AKAP79) in diabetic cells (14). Considering that this transcription factor is also known to regulate the expression of K_V channels in VSMCs (18), we postulated that activation of this pathway may also modulate K_V function during diabetes.

In the present study, we tested the hypothesis that K_V channel function in VSMCs is impaired and contributes to increased arterial tone in a HFD mouse model of type 2 diabetes, and that the mechanism involves activation of the AKAP150-CaN/NFATc3 signaling pathway. Consistent with this hypothesis, we show that K_V channel function is decreased in VSMCs from wild type (WT) HFD mice due to a reduction in the expression of ScTx-sensitive K_V2.1, but not psora-4-sensitive K_V1.2 or K_V1.5 subunits. Genetic ablation of NFATc3, AKAP150, or disruption of the AKAP150-CaN interaction prevented down-regulation of the K_V2.1 subunit, suppression of ScTx-sensitive I_Kv, and enhanced arterial tone in HFD mice. Furthermore, our data indicate that down-regulation of K_V2.1 occurs at an earlier time point (i.e. 1 h) compared with BK_Ca β1 subunits under hyperglycemic conditions. These findings illuminate a critical role for AKAP150-anchored CaN, NFATc3, and K_V2.1 function in the regulation of arterial tone during diabetes.

MATERIALS AND METHODS

Animals

WT (C57Bl/6J), AKAP150^−/−, NFATc3^−/−, and knock-in mice expressing AKAP150 lacking its CaN binding site (ΔPIX) (19) were euthanized with a lethal dose of sodium pentobarbital (250 mg/kg; intraperitoneally), as approved by the University of California, Davis Institutional Animal Care and Use Committee. Mice were sustained on either a low fat (10% kcal; ct) or high fat (60% kcal) diet (Research Diets, New Brunswick, NJ) starting at 5 weeks of age for 24–26 weeks. The composition of these diets and the propensity of mice maintained on this HFD to develop type 2 diabetes and induce vascular dysfunction of small resistance arteries have been well documented in previous studies (14, 20, 21). Cerebral arteries were used for functional experiments (i.e. electrophysiology, arterial diameter, and immunofluorescence), whereas 3rd and 4th order mesenteric arteries were used for electrophysiology experiments in VSMCs from WT LFD and HFD mice and for molecular biology experiments requiring larger tissue samples (i.e. Western blots).

Isolation of VSMCs

VSMCs were dissociated from arteries using enzymatic digestion techniques as described previously (18, 22, 23). Middle and posterior cerebral arteries, as well as third and fourth order mesenteric arteries were dissected in ice-cold dissection buffer composed of (in mm): 140 NaCl, 5 KCl, 2 MgCl₂, 10 d-glucose, 10 HEPES, pH 7.4, with NaOH. Cerebral arteries were digested in a dissection solution supplemented with papain (1 mg/ml) and dithiothreitol (1 mg/ml) at 37 °C for 7 min, then incubated in dissection buffer supplemented with collagenase type H (0.3 mg/ml) and collagenase type F (0.7 mg/ml) at 37 °C for 7 min. Cells were then washed in ice-cold dissection buffer. Glass pipettes of decreasing diameters were used to gently triturate arteries and obtain single VSMCs. Isolated cells were maintained in ice-cold dissection buffer until use.

Electrophysiology

I_Kv from freshly dissociated VSMCs were measured using the conventional whole cell patch-clamp technique with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Currents were evoked by 0.6-s depolarizing pulses from a holding potential of −70 mV to +80 (for psora-4-sensitive currents) or to +40 mV (for ScTx-sensitive currents) in increments of +10 mV. A voltage error of 15 mV attributable to the liquid junction potential of the recording solutions used was corrected offline. I_Kv were recorded in the continuous presence of the BK_Ca channel blocker iberiotoxin (100 nm) to eliminate BK_Ca channel activity. Bath solution consisted of the following components (in mm): 120 NaCl, 3 NaHCO₃, 4.2 KCl, 1.2 KH₂PO₄, 2 MgCl₂, 0.1 CaCl₂, 10 d-glucose, and 10 HEPES (pH 7.4). Patch pipette solution was composed of (in mm): 110 K-gluconate, 30 KCl, 0.5 MgCl₂, 10 EGTA, 5 HEPES, 5 NaATP, and 1 GTP (pH 7.2 with KOH). Experiments were carried out at room temperature (22–25 °C). Currents were sampled at 20 kHz and low-pass filtered at 5 kHz. Electrophysiology recordings were analyzed using pCLAMP 10.

Quantitative Polymerase Chain Reaction

K_V transcript expression was analyzed in single isolated cells using the Power SYBR Green Cells-to-CT kit (Life Technologies). For these experiments, single VSMCs were collected after enzymatic isolation using a glass micropipette. RT product was used for quantitative PCR. Specific primers to detect K_V2.1 (NM_008420; reference number QT00285971), K_V1.2 (NM_008417; reference number QT00128100), and K_V1.5 (NM_145983; reference number QT00268387) were acquired from Qiagen (Valencia, CA). β-Actin was used as an internal control (GenBank^TM accession number V01217; sense nucleotide 2384–2404 and antisense nucleotide 3071–3091). Amplification was performed using a Power SYBR Green PCR cocktail (Life Technologies) and an Applied Biosystems real-time PCR instrument. Expression for each gene was normalized to β-actin and expressed as a percentage of LFD.

Western Blot Analysis

Cerebral and mesenteric arteries were homogenized in a Triton lysis buffer solution containing (in mm) 150 NaCl, 10 Na₂HPO₄, 1 EDTA with 1% deoxycholic acid, 0.1% sodium dodecyl sulfate and protease inhibitors (Complete Mini protease inhibitor cocktail Roche Applied Science), followed by sonication (20 min at 4 °C). Tissue debris and nuclear fragments were removed by centrifugation at 10,000 × g (10 min, 4 °C) and whole cell lysates were obtained as the supernatant. An equal amount of protein was loaded for each tissue lysate. Proteins were separated under reducing conditions on a 10% polyacrylamide gel (Bio-Rad) by electrophoresis at 100 V for 1.5 h. Proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane at 20 V (overnight, 4 °C). Membranes were washed in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and blocked with 10% nonfat milk in TBS-T (1 h, room temperature). Membranes were then incubated for 2 h at room temperature with subunit-specific antibodies from NeuroMab (University of California, Davis, Davis, CA). Antibodies and dilutions were as follows: mouse anti-K_V2.1 (73-014 clone K89/34; 1:2), anti-K_V1.2 (clone 14/16; 1:2), and K_V1.5 (clone K7/45; 1:2). Monoclonal antibody against β-actin (MA5–15739; 1:5,000) was from Pierce. Antibodies were prepared in TBS-T with 1% bovine serum albumin and 0.01% sodium azide. Membranes were then incubated (1 h, room temperature) with horseradish peroxidase-labeled goat anti-mouse (sc-2005; 1:5,000; Santa Cruz) in TBS-T containing 5% nonfat dried milk. Bands were identified by enhanced chemiluminescence and exposure to x-ray film. Densitometry for immunoreactive bands was performed with ImageJ software (National Institutes of Health). β-Actin was used as input control for normalization. Density was expressed as a percentage of control (LFD).

Immunofluorescence

Immunofluorescent labeling of freshly isolated VSMCs was performed as described previously (24) using a monoclonal antibody specific for K_V2.1 subunits (NeuroMab; 75-159; clone K39/25, University of California, Davis). The secondary antibody was an Alexa Fluor 488-conjugated donkey anti-mouse (5 mg/ml) from Molecular Probes. Cells were imaged (512 × 512 pixel images) using an Olympus FV1000 confocal microscope coupled with an Olympus ×60 oil immersion lens (NA = 1.4) and a zoom of 3.5 (pixel size = 0.1 μm). Images were collected at multiple optical planes (z axis step size = 0.5 μm). The specificity of the primary antibody was tested in negative control experiments in which the primary antibody was substituted with PBS. K_V2.1-associated fluorescence was undetected under this experimental condition. Cells for each group were imaged with the same laser power, gain settings, and pinhole for all the treatments.

Arterial Diameter Measurements

Freshly isolated posterior cerebral arteries were cannulated on glass micropipettes mounted in a 5-ml myograph chamber (Living Systems Instrumentation, St. Albans, VT) as described previously (14). Arteries were pressurized to 20 mm Hg and allowed to equilibrate while continuously superfused (37 °C, 30 min, 3–5 ml/min) with physiological saline solution consisting of (in mm): 119 NaCl, 4.7 KCl, 2 CaCl₂, 24 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, 0.023 EDTA, and 10 d-glucose aerated with 5% CO₂, 95% O₂. Bath pH was closely monitored and maintained at 7.35–7.40. Following the equilibration period, intravascular pressure was increased to 60 mm Hg and arteries were allowed to develop myogenic tone. Arteries not exhibiting stable myogenic tone after ∼1 h were discarded. To assess the contribution of K_V1.X and K_V2.1 channel function to regulation of arterial tone, the K_V1.X inhibitor psora-4 (500 nm) and K_V2.1 inhibitor ScTx (50 nm), respectively, were added to the superfusate. Arterial tone data are presented as a percent decrease in diameter relative to the maximum passive diameter at 60 mm Hg obtained at the end of each experiment using Ca²⁺-free saline solution containing nifedipine (1 μm).

Chemicals and Statistical Analyses

All chemical reagents were from Sigma unless otherwise stated. Iberiotoxin was from Peptides International and ScTx-1 was from Alomone Labs. Data are expressed as mean ± S.E. Data obtained using multiple vessels from the same animal were pooled for statistical analyses. Data were analyzed using GraphPad Prism software. Statistical significance was determined by Student's t test or one-way analysis of variance followed by Tukey multiple comparison test for comparison of multiple groups. p < 0.05 was considered statistically significant (denoted by * in figures).

RESULTS

Freshly isolated, small diameter (∼75–150 μm) cerebral and mesenteric arteries and VSMCs from age-matched mice fed ad libitum with either a low fat (LFD, 10% kcal) or high fat (HFD, 60% kcal) diet were used for this study (14, 20). This HFD model of type 2 diabetes was employed because it does not depend on genetic manipulations or chemical destruction of pancreatic β-cells. In addition, it recapitulates clinical features observed in patients with type 2 diabetes (20, 25), including enhanced arterial tone and increased blood pressure (14). WT mice on a HFD had significantly higher average body weight (42 ± 1.0 g) than WT LFD (30 ± 0.6 g; Table 1). Furthermore, non-fasting blood glucose levels were significantly increased in WT HFD (301 ± 12 mg/dl) compared with WT LFD mice (145 ± 4 mg/dl; Table 1).

TABLE 1.

Body weight and non-fasting blood glucose in wild type, AKAP150^−/−, ΔPIX, and NFATc3^−/− mice fed low and high fat diet

	Body weight	Blood glucose
	g	mg/dl
Wild-type
LFD	30 ± 0.6	145 ± 4
HFD	42 ± 1.0a	301 ± 12a
AKAP150−/−
LFD	25 ± 1.1	148 ± 4
HFD	36 ± 3.0a	264 ± 16a
ΔPIX
LFD	36 ± 2.0	169 ± 11
HFD	45 ± 1.0a	264 ± 24a
NFATc3−/−
LFD	32 ± 4.0	147 ± 8
HFD	44 ± 2.0a	241 ± 41a

^a Values are mean ± S.E. obtained at 24–26 weeks after start of diet at 5 weeks of age, p < 0.05.

I_Kv Function Is Suppressed in VSMCs from WT Diabetic Mice

Fig. 1 shows I_Kv evoked in cerebral VSMCs freshly dissociated from WT LFD and HFD mice in the presence of 100 nm iberiotoxin to inhibit BK_Ca channels. The average capacitance of cerebral WT LFD and HFD cells was 19.4 ± 1.2 and 19.0 ± 1.2 pF, respectively (p > 0.05). I_Kv were initially elicited by 600-ms depolarizing pulses from a holding potential of −85 mV to voltages ranging from −85 to +65 mV in 10-mV increments. We found that the amplitude of control I_Kv from WT HFD were significantly lower than that from WT LFD (Fig. 1Ai). Indeed, the current-voltage relationship of control I_Kv revealed that current densities were smaller in WT HFD than in WT LFD at most voltages examined (Fig. 1B, i; p < 0.05). At +65 mV, control I_Kv were ∼42% smaller in WT HFD (21.0 ± 1.8 pA/pF) when compared with WT LFD (36.4 ± 3.5 pA/pF; p < 0.05; Fig. 1B, i). Similar results were observed in mesenteric VSMCs under same experimental conditions (Fig. 1, B and D, panel i, insets). These results indicate impaired I_Kv in cerebral and mesenteric VSMCs during diabetes.

FIGURE 1.

Stromatoxin-sensitive I_Kv are suppressed in VSMCs from WT HFD mice. A and B, exemplar whole-cell I_Kv evoked by depolarizations from −85 to +65 mV and corresponding current-voltage relationships from cerebral and mesenteric (insets) LFD (n = 7 cerebral cells and 8 mesenteric cells from 5 mice) and HFD (n = 7 cerebral and 7 mesenteric cells from 5 mice) VSMCs before (i) and after (ii) psora-4 (500 nm) application and the resultant psora-4-sensitive component (iii). Average capacitance of mesenteric WT LFD and HFD cells is 18.8 ± 1.9 and 19.5 ± 1.5 pF, respectively. Average peak I_Kv at +65 mV in LFD and HFD mesenteric cells, respectively, were as follow: control, 37.4 ± 6.0 and 21.8 ± 3.2 pA/pF; +psora-4, 22.7 ± 3.0 and 11.9 ± 3.5 pA/pF; psora-4-sensitive, 17.2 ± 4.0 and 13.0 ± 2.4 pA/pF. C and D, representative I_Kv evoked by depolarization from −85 to +25 mV and the current-voltage relationship from cerebral and mesenteric (insets) LFD (n = 12 cerebral and 7 mesenteric cells from 7 mice) and HFD (n = 14 cerebral and 7 mesenteric cells from 5 mice) cells before (i) and after (ii) application of ScTx-1 (100 nm) and resultant ScTx-sensitive current (iii). Average capacitance of mesenteric WT LFD and HFD cells is 18.8 ± 1.9 and 19.5 ± 1.5 pF, respectively. Average peak I_Kv at +25 mV in LFD and HFD mesenteric cells, respectively, were as follows: control, 21.4 ± 3.3 and 11.5 ± 2.8 pA/pF; +ScTx, 5.0 ± 1.5 and 5.9 ± 2.0 pA/pF; ScTx-sensitive, 16.5 ± 2.5 and 5.9 ± 1.3 pA/pF. Psora-4- and ScTx-sensitive components were obtained by digital subtraction of currents in the presence of the specific inhibitor from the currents before application of the inhibitor (control). *, p < 0.05.

Impaired Stromatoxin Sensitivity in VSMCs and Arteries from WT Diabetic Mice

Previous studies indicate that K_V1.2, K_V1.5, and K_V2.1 subunits make predominant contributions to I_Kv in VSMCs from cerebral and mesenteric arteries (8, 9, 26–28). Thus, we examined whether impaired function of K_V1.X- and/or K_V2.1-containing channels contribute to suppressed I_Kv in VSMCs during diabetes. This was achieved by selectively inhibiting either K_V1 channels with psora-4 (500 nm) (29) or K_V2 channels with ScTx (100 nm) (8, 9). These agents have been used to evaluate the relative contribution of K_V1.X and K_V2 channels in the same VSMCs employed in the present study (8, 9, 30, 31). I_Kv were recorded from cerebral VSMCs as described above. The addition of psora-4 decreased I_Kv at +65 mV by ∼30% in both LFD and HFD VSMCs (Fig. 1A, ii). However, the amplitude and current-voltage relationship of the psora-4-sensitive I_Kv component was similar between these cells (Fig. 1, A and B, panels iii). Inhibition of K_V2 channels with ScTx becomes partial at positive potentials greater than +30 mV (32). Thus, K_V currents were evoked in the presence of ScTx by voltage steps from −85 to +25 mV. Application of ScTx reduced I_Kv at +25 mV by ∼70% in LFD and ∼50% in HFD cells (Fig. 1, C and D, panels ii). Unlike psora-4-senstive I_Kv, the amplitude of ScTx-sensitive I_Kv was significantly smaller across a range of voltages in WT HFD compared with those in WT LFD (5.8 ± 0.9 versus 14.2 ± 2.0 pA/pF at +25 mV, respectively; Fig. 1, C and D, panels iii; p < 0.05). Similar results were observed in mesenteric VSMCs (Fig. 1, B and D, insets). These data suggest that a reduction in I_Kv observed in cerebral and mesenteric VSMCs from HFD mice is due to reduced ScTx-sensitive I_Kv.

The functional significance of HFD-associated alterations in I_Kv function was assessed by measuring psora-4 and ScTx sensitivity of arterial tone in cerebral arteries from WT LFD and HFD mice. At 60 mm Hg intravascular pressure, WT HFD arteries were significantly more constricted than WT LFD (Fig. 2, A and B). Whereas application of psora-4 (500 nm) caused a significant constriction in both WT LFD (13 ± 2%) and HFD (17 ± 4%) arteries (Fig. 2, A and C), ScTx (50 nm) induced constriction only in WT LFD arteries (Fig. 2, B and D). Indeed, ScTx had little effect on arteries from WT HFD (3 ± 1%) as compared with LFD (11 ± 2%; p < 0.05) mice. Arteries from all groups responded with robust constriction to 60 mm extracellular K⁺ concentration (Fig. 2E) or phenylephrine (14), suggesting that altered ScTx response was not due to differences in the magnitude of tone development. These results are consistent with impaired ScTx-sensitive I_Kv contributing to enhanced arterial tone during diabetes.

FIGURE 2.

Impaired ScTx-induced constriction in arteries from WT HFD mice. Representative diameter recordings from pressurized (60 mm Hg) WT cerebral arteries from LFD and HFD mice before and after (A) psora-4 (500 nm) or (B) ScTx (50 nm). C, bar plot summarizing arterial tone in WT LFD (n = 5 arteries from 5 mice) and HFD (n = 5 arteries from 5 mice) arteries in the absence (−) and presence (+) of psora-4 (C). D, arterial tone in the absence (−) or presence (+) of ScTx in WT LFD (n = 7 arteries from 5 mice) and HFD (n = 7 arteries from 6 mice) arteries. E, amalgamated data of arterial tone from WT LFD (n = 8 arteries from 5 mice) and HFD (n = 10 arteries from 5 mice) at 60 mm Hg in response to 60 mm extracellular K⁺. *, p < 0.05.

Down-regulation of K_V2.1 Subunits in VSMCs during Diabetes

Impaired sensitivity to ScTx, but not psora-4, in diabetic cells and arteries could reflect selective down-regulation of K_V2.1 subunit expression. To test this, we evaluated K_V1.2, K_V1.5, and K_V2.1 transcript levels using quantitative PCR on single isolated cerebral and mesenteric VSMCs from WT LFD and HFD mice. We found that K_V2.1 transcript expression was ∼65% lower in WT HFD than in WT LFD cells, with no detectable change in the expression of K_V1.2 and K_V1.5 transcripts (Fig. 3A). Consistent with this, K_V2.1, but not K_V1.2 or K_V1.5, protein levels were significantly reduced (∼75%) in WT HFD arterial lysates (Fig. 3B).

FIGURE 3.

K_V2.1 subunit expression is down-regulated in VSMCs and arteries from WT HFD mice. A, bar plot summarizing quantitative real-time PCR data for K_V1.2, K_V1.5, K_V2.1, and KV9.3 transcript levels in WT VSMCs from LFD and HFD mice relative to control (normalized to β-actin; n ≈ 40–50 VSMCs from 7 mice per condition). B, representative blots of immunoreactive bands of expected molecular mass for K_V1.2 (∼64 kDa), K_V1.5 (∼66 kDa), K_V2.1 (∼110 kDa), and β-actin (∼43 kDa) (left), and corresponding (right) densitometry summary data (n = 4 lysates per condition). C, confocal images of K_V2.1-associated fluorescence in WT LFD and HFD VSMCs. Lower panels show DIC images for representative cells in the top panel (n = 14 cells from 3 mice per condition). *, p < 0.05.

Immunofluorescence was also used to determine changes in K_V2.1 subunit expression in isolated cerebral VSMCs from WT LFD and HFD with a K_V2.1-specific antibody. As illustrated in Fig. 3C, we observed a strong intensity of the K_V2.1-associated fluorescence along the sarcolemma of this WT LFD cell. No K_V2.1-associated fluorescence was observed when the primary or secondary antibodies were excluded from the preparation. Conversely, the intensity of the K_V2.1-associated fluorescence along the sarcolemma, under the same experimental conditions, was markedly lower in cells from WT HFD mice (Fig. 3C). Altogether, these data indicate that suppression of ScTx-sensitive I_Kv in VSMCs and impaired ScTx-induced vasoconstriction during diabetes results from down-regulation of K_V2.1 subunit expression.

AKAP150-anchored CaN and NFATc3 Activity Are Necessary for Impaired ScTx-sensitive I_Kv Function and Down-regulation of K_V2.1 Subunit Expression during Diabetes

CaN and NFATc3 are known to regulate transcription of K_V2.1 in VSMCs (18) and their activity is enhanced in diabetic arteries (14). Activation of CaN and CaN-mediated activation of NFATc3 in VSMCs of diabetic animals is dependent upon sarcolemmal phosphatase targeting by AKAP150 (18, 33). Thus, to test the involvement of this pathway in the suppression of K_V2.1 in HFD cells, we first examined the role of AKAP150. To do so, we fed AKAP150-null (AKAP150^−/−) mice either a LFD or HFD. Non-fasting blood glucose levels and body weight in AKAP150^−/− LFD and HFD mice were comparable with corresponding age-matched WT mice, and were elevated in HFD (Table 1).

We measured I_Kv in VSMCs from AKAP150^−/− LFD and HFD mice before and after ScTx using the voltage protocol described above. VSMCs from AKAP150^−/− LFD mice produced robust I_Kv (Fig. 4A) that were comparable with those observed in WT LFD cells, suggesting that AKAP150 does not influence basal I_Kv in VSMCs of control mice. The average capacitance of AKAP150^−/− LFD and HFD cells was 17.7 ± 1.2 and 16.4 ± 0.6 pF, respectively, which is similar to WT cells (p > 0.05). In contrast to diabetic WT cells, the amplitude of control and ScTx-sensitive I_Kv at +25 mV in AKAP150^−/− HFD cells (13.0 ± 2.0 pA/pF) was similar to the corresponding AKAP150^−/− LFD cells (14.0 ± 2.0 pA/pF; Fig. 4, A and B). Likewise, no differences in ScTx-insensitive components were observed. Consistent with the electrophysiological data, K_V2.1 transcript and protein levels (Fig. 4, D and E) were similar in cells and lysates, respectively, from AKAP150^−/− LFD and HFD mice. Ablation of AKAP150 did not alter basal expression of K_V2.1 subunits (Fig. 4E). These data indicate that AKAP150 is required for suppression of K_V2.1 subunit expression and function in diabetic VSMCs.

FIGURE 4.

AKAP150-anchored CaN is necessary for impaired K_V2.1 expression and function in HFD cells. A, representative whole cell K_V currents from VSMCs isolated from LFD and HFD AKAP150^−/− mice recorded before and after application of ScTx (100 nm), and the corresponding ScTx-sensitive component (n = 11 cells from 5 LFD mice and 9 cells from 5 HFD mice). Current-voltage relationships of I_Kv before and after ScTx and the corresponding ScTx-sensitive component in LFD and HFD (B) AKAP150^−/− and (C) ΔPIX cells. D, bar plot of K_V2.1 transcript levels in AKAP150^−/− and ΔPIX HFD cells relative to LFD normalized to β-actin (n ≈ 40–50 VSMCs from 3 mice per condition). E, Western blot of immunoreactive bands of the expected molecular masses for K_V2.1 (∼110 kDa) and β-actin (∼43 kDa) in control, LFD, and HFD arteries from AKAP150^−/− and ΔPIX mice (left) and corresponding (right) densitometry summary data (n = 4 lysates from 4 mice per condition). *, p < 0.05.

Next, we directly tested the role of AKAP150-CaN interactions in K_V2.1 suppression during diabetes. To do this, we used VSMCs from LFD and HFD knock-in mice expressing mutant AKAP150 that is unable to bind CaN (ΔPIX) (14, 19). Non-fasting glucose levels and body weight were significantly elevated in ΔPIX HFD compared with ΔPIX LFD mice (Table 1). Selective disruption of the AKAP150-CaN interaction abolished suppression of ScTx-sensitive I_Kv function and K_V2.1 subunit expression during diabetes (Fig. 4, C–E). These results indicate that subcellular targeting of CaN by AKAP150 is necessary for suppression of K_V2.1 expression and function in VSMCs during diabetes.

The above findings are consistent with the concept that AKAP150-targeted CaN signals K_V2.1 transcriptional suppression via downstream activation of NFATc3. Therefore, we investigated the role of NFATc3 in suppression of K_V2.1 expression and function during diabetes by using NFATc3 null (NFATc3^−/−) mice in LFD or HFD (Table 1). Fig. 5A demonstrated that NFATc3^−/− LFD and HFD myocytes produced robust I_Kv. Consistent with a role for NFATc3, ScTx-sensitive I_Kv function and K_V2.1 expression (Fig. 5, A–D) were similar in VSM and arteries from NFATc3^−/− LFD and HFD mice. Together, our data indicate that anchoring of CaN by AKAP150 mediates impairment of K_V2.1 expression and function during diabetes via downstream activation of NFATc3.

FIGURE 5.

NFATc3 is necessary for down-regulation of K_V2.1 expression and function in HFD cells. A, representative whole cell K_V currents from VSMCs isolated from LFD and HFD NFATc3^−/− mice recorded before and after application of ScTx (100 nm) and the corresponding ScTx-sensitive component (n = 6 cells from 3 LFD mice and 8 cells from 3 HFD mice). B, current-voltage relationships of I_Kv before and after ScTx and the corresponding ScTx-sensitive component in LFD and HFD NFATc3^−/− cells. C, bar plot of K_V2.1 transcript levels in NFATc3^−/− HFD cells relative to LFD normalized to β-actin (n ≈ 40–50 cells from 3 mice per condition). D, Western blot of immunoreactive bands of expected molecular masses for K_V2.1 (∼110 kDa) and β-actin (∼43 kDa) in LFD and HFD arteries from NFATc3^−/− mice (left) and corresponding (right) densitometry summary data (n = 3 lysates from 3 mice per condition). *, p < 0.05.

Loss of AKAP150, AKAP150-targeted CaN, or NFATc3 Prevents Enhanced Arterial Tone and Restores ScTx-sensitive Constriction in Diabetic Mice

We examined the functional relevance of the AKAP150-anchored CaN and NFATc3 signaling module on arterial tone and ScTx sensitivity. Diameter measurements at 60 mm Hg intravascular pressure were performed in cerebral arteries from AKAP150^−/−, ΔPIX, and NFATc3^−/− mice in LFD and HFD. Unlike ScTx observations in arteries from WT mice (see Fig. 2, B and D), arterial tone development and ScTx-induced constriction were similar in LFD and HFD vessels from AKAP150^−/−, ΔPIX, and NFATc3^−/− mice (Fig. 6, A–C). Altogether, these data indicate that disruption of the AKAP150-CaN interaction or ablation of NFATc3 restores normal K_V2.1 regulation of myogenic tone during diabetes.

FIGURE 6.

AKAP150-anchored CaN and NFATc3 are necessary for impaired arterial tone in HFD arteries. Representative diameter recordings (left panel) and summary arterial tone data (right panel) from pressurized (60 mm Hg) (A) AKAP150^−/− LFD (n = 7 arteries from 5 mice) and HFD mice (n = 6 arteries from 4 mice); B, ΔPIX LFD (n = 7 arteries from 3 mice) and HFD (n = 8 arteries from 3 mice), and C, NFATc3^−/− LFD (n = 6 arteries from 3 mice) and HFD (n = 6 arteries from 3 mice) cerebral arteries in the absence (−) and presence (+) of ScTx (50 nm). Maximum passive diameters for representative recordings are as follows: AKAP150^−/− LFD, 150 μm; AKAP150^−/− HFD, 145 μm; ΔPIX LFD, 161 μm, ΔPIX HFD, 154 μm; NFATc3^−/− LFD, 153 μm, NFATc3^−/− HFD, 155 μm. *, p < 0.05 as compared with a minus (−).

Differential Suppression of K_V2.1 and BK_Ca β1 Expression at Earlier Stages of Diabetic Hyperglycemia

We further examined the CaN/NFATc3 signaling axis in the suppression of K_V2.1 during diabetic hyperglycemia by determining K_V2.1 transcript expression in VSMCs from WT arteries incubated for 48 h in medium containing 10 mm d-glucose, 20 mm d-glucose, 20 mm d-glucose + 10 μm CaN inhibitory peptide (a specific CaN inhibitor), and 20 mm d-glucose + 5 μm VIVIT (a selective NFAT inhibitor). These d-glucose concentrations are within the range of observed non-fasting blood glucose levels reported for LFD (5–10 mm) and HFD (20 mm) mice. As expected, 20 mm d-glucose caused >60% reduction in the K_V2.1 transcript that was not observed when the non-metabolized l-glucose was substituted for d-glucose or when the CaN inhibitory peptide or VIVIT were present in cultured medium (Fig. 7A). These results suggest that activation of the CaN/NFATc3 axis in VSMCs is necessary for transcriptional suppression of K_V2.1 during diabetic hyperglycemia.

FIGURE 7.

Time course of K_V2.1 and BK_Ca β1 subunit mRNA expression during diabetic hyperglycemia, involvement of the AKAP150-CaN/NFAT axis, and model for AKAP150-anchored CaN-dependent suppression of K_V and BK_Ca subunits during diabetes. A, bar plot of K_V2.1 transcript levels in VSMCs from WT arteries incubated in medium supplemented with 10 mm d-glucose, 20 mm d-glucose, 5 mm d-glucose + 15 mm l-glucose, 20 mm d-glucose + 10 μm CaN inhibitory peptide, and 20 mm d-glucose + 5 μm VIVIT (n ≈ 40–50 cells from 3 mice per condition). Time course of K_V2.1 and BK_Ca β1 transcripts in WT (B) and ΔPIX VSMCs (C) following incubation of arteries in 20 mm d-glucose (n ≈ 40–50 cells from 3 mice per condition). D, proposed model for AKAP150-CaN/NFATc3-dependent suppression of K_V and BK_Ca channel function in VSMCs during diabetes. *, p < 0.05.

In a previous study, we found that the regulatory BK_Ca β1 subunit in VSMCs is down-regulated via activation of AKAP150-CaN/NFATc3 signaling in hyperglycemic animals on HFD leading to BK_Ca channel impairment (14). Considering a potential common mechanism of AKAP150-CaN/NFATc3-induced suppression of K_V and BK_Ca channels, an important question to be addressed is whether down-regulation of K_V2.1 and BK_Ca β1 occurs on a similar time scale in response to hyperglycemic stimuli. To test this possibility, we examined the time course of K_V2.1 versus BK_Ca β1 transcript suppression in VSMCs from WT arteries incubated at different time points in medium containing 10 or 20 mm extracellular d-glucose. We found that increasing the extracellular d-glucose concentration to 20 mm decreased K_V2.1 expression by ∼60% within the first hour of incubation, but had no effect on BK_Ca β1 (Fig. 7B). Transcript levels for both genes were subsequently down-regulated to a similar extent after 6, 12, 24, and 48 h in 20 mm d-glucose (Fig. 7B). Consistent with a role for AKAP150-anchored CaN in K_V2.1 and BK_Ca β1 suppression during diabetic hyperglycemia, 20 mm d-glucose had no effect on transcript levels for either gene in VSMCs from ΔPIX mice (p > 0.05; Fig. 7C). Together, these data suggest a distinct temporal profile of early K_V2.1 suppression during diabetic hyperglycemia, with subsequent concomitant down-regulation of both genes to a similar extent that is mediated by direct activation of the AKAP150-anchored CaN/NFAT signaling pathway.

DISCUSSION

In this study, we provide evidence implicating suppression of K_V channel function as a significant contributor of enhanced arterial tone during diabetes (Fig. 7D). We report the following novel findings. First, I_Kv in cerebral and mesenteric VSMCs are significantly suppressed during diabetes. Second, impaired I_Kv in these cells leading to enhanced arterial tone during diabetes occurs due to selective down-regulation of K_V2.1 expression. Third, AKAP150-anchored CaN and NFATc3 are obligatory components in the signaling pathway underlying suppression of I_Kv and K_V2.1 expression during diabetes. Fourth, although K_V2.1 and BK_Ca β1 transcripts are reduced to a similar extent during established diabetic hyperglycemia, our data suggest that K_V2.1 suppression may occur at an earlier stage. The implications of these findings are discussed below.

Considerable data attribute impairment of endothelium-dependent vasodilatory mechanisms as a significant contributor to vascular dysfunction and enhanced arterial tone during diabetes (2–5). However, abnormal VSM function may also critically contribute to enhancement of arterial tone during this pathological condition. For instance, enhanced arterial tone could result from a reduction in outward K⁺ conductance in VSMCs (34). Consistent with this idea, our initial experiments demonstrate that K_V channel function is suppressed in diabetic VSMCs (Fig. 1). This discovery is significant, as a reduction in K_V channel function will result in membrane depolarization, and increased activity of LTCCs, ultimately leading to elevated global [Ca²⁺]_i, and VSM contraction (6, 7). Although an increase in global cytosolic [Ca²⁺]_i could promote activation of BK_Ca channels to compensate for the loss of K_V channel function via negative feedback control of V_M depolarization, BK_Ca channel activity is also suppressed in WT HFD mice (14). Thus, synergistic impairment of K_V and BK_Ca activity in diabetic animals may substantially reduce feedback membrane potential hyperpolarization leading to VSM contraction and enhanced arterial tone during diabetes (Fig. 7D). Ultimately, this could significantly contribute to raise mean arterial blood pressure or limit blood flow in diabetic patients.

In this study, we found that psora-4, a selective K_V1.X inhibitor (29–31, 35), induced robust constriction in pressurized arteries from LFD and HFD mice. Yet, ScTx, which is a selective K_V2 inhibitor in VSM (8, 9), had a reduced effect on WT HFD vessels indicating impaired K_V2 channel function during diabetes. Interestingly, we observed a trend toward a greater psora-4-induced constriction in pressurized arteries from WT HFD as compared with WT LFD mice, perhaps reflecting compensatory activation of additional K_V1.X at more depolarized membrane potentials in pressurized arteries from diabetic mice. This difference, however, was not statistically significant. Note that vasoconstriction of cerebral vessels in response to K_V2 or K_V1 channel blockers is independent of functional endothelium (8, 13). Considering this, it follows that reduced endothelial vasodilatory function, as observed in several experimental models of diabetes, would not significantly impact the contribution of K_V2 channels to the regulation of arterial tone. Thus, altered response to ScTx during diabetes observed in this study likely reflects reduced expression of functional K_V2 channels. Consistent with this, ScTx-sensitive and psora-4-insensitive (presumably produced by K_V2.1 channels) current densities were significantly suppressed at most membrane potentials in HFD as compared with LFD cells. Conversely, the amplitude of the ScTx-insensitive currents, which presumably are predominantly mediated by K_V1.X, and psora-sensitive currents were not different in VSM isolated from LFD and HFD mice. These data indicate that K_V2.1, but not K_V1.X channel function is decreased in VSMCs from HFD mice. This conclusion is further supported at the molecular level upon demonstration of reduced K_V2.1, but not K_V1.2 or K_V1.5, transcript and proteins in HFD cells/arteries when compared with LFD cells/arteries. Altogether, these results are the first indication that selective K_V2.1 down-regulation underlies reduced I_Kv in VSM contributing to enhanced arterial tone during diabetes, and point to impairment of K_V function as a mediator of vascular dysfunction.

An interesting observation in this study is that K_V2.1 and BK_Ca β1 genes are differentially suppressed during diabetic hyperglycemia (Fig. 7B). Our data indicate that within the first hour of incubation in a hyperglycemic solution that resembles the levels of plasma glucose in HFD mice, K_V2.1, but not BK_Ca β1 expression, is significantly down-regulated. Subsequently, concomitant suppression of both genes occurs to a similar extent within 48 h of hyperglycemic exposure. These results suggest that K_V2.1 and BK_Ca β1 genes have different thresholds for hyperglycemia-induced transcriptional suppression. Mechanisms of early temporal differences in K_V2.1 and BK_Ca β1 expression during hyperglycemic stimuli are unclear, but may involve dissimilar thresholds for NFATc3-dependent suppression or differential mRNA degradation independent of the rate or magnitude of transcriptional suppression (36). Although future studies should further investigate this issue, it is intriguing to speculate that early initiation of K_V2.1 suppression could represent a key feature of pre-diabetic hyperglycemia, which drives pathophysiological engagement of the AKAP150-CaN/NFATc3 signaling pathway via membrane depolarization, LTCC activity, and Ca²⁺ influx (37, 38). Subsequently, further NFATc3 nuclear accumulation reaches levels sufficient for BK_Ca β1 down-regulation. Data demonstrating that disruption of the AKAP150-CaN interaction or specific inhibition of CaN and NFAT in VSMCs prevents the down-regulation of K_V2.1 and BK_Ca β1 expression (see also Ref. 14) and impaired arterial tone during diabetic hyperglycemia and in HFD gives credence to this hypothesis. However, future experiments should investigate the relationship between NFATc3 activity and K_V2.1 and BK_Ca β1 expression in further detail.

Our data support a mechanistic model whereby targeting of CaN by AKAP150 drives NFATc3 activation during diabetes. Once activated, NFATc3 translocates into the nucleus of VSMCs where it reduces K_V2.1 and subsequently BK_Ca β1 expression (Fig. 7D). Consistent with a critical role for AKAP150-anchored CaN, disruption of the interaction between these two proteins prevented NFATc3 dephosphorylation and nuclear accumulation during diabetes (14). This is correlated with restoration of K_V2.1 (Figs. 4, 6, and 7) and BK_Ca β1 subunit expression and function, and attenuation of blood pressure in diabetic AKAP150^−/− and ΔPIX mice (Fig. 7, B and C, and Ref. 14).

Although our data argue against a role for AKAP150 in basal regulation of K_V or BK_Ca channel function (see Figs. 1 and 4 and Ref. 14), this anchoring protein is known to interact with LTCCs (39). Hence, AKAP150 may also function to position CaN near LTCCs (23) to efficiently activate Ca²⁺-dependent CaN/NFATc3 signaling during diabetes. Indeed, we have previously found that NFATc3 is preferentially activated in VSMCs by LTCC-dependent Ca²⁺ microdomains (i.e. Ca²⁺ sparklets (38, 40, 41)), which are significantly elevated during diabetes (42), rather than by elevations in global [Ca²⁺]_i (40). Similar increases in LTCC-dependent Ca²⁺ microdomains have been observed in angiotensin II-induced hypertensive VSMCs (24, 40) and after activation of reactive oxygen species (43). Thus, increases in local LTCC activity leading to activation of the AKAP-CaN/NFATc3 signaling pathway may represent a wide ranging mechanism for development of vascular dysfunction in many pathological conditions. In the present model, NFATc3 nuclear accumulation and subsequent K_V2.1 and BK_Ca β1 down-regulation is a dynamic process highly dependent on NFAT nuclear import and export rate (44), as well as non-fasting plasma glucose levels in diabetic animals that could be sufficient for NFAT activation.

In addition to transcriptional suppression of K_V2.1 subunit expression, post-translational modification of this (and other K_V) subunit may also contribute to a reduction in I_Kv during diabetes (11–13, 45–47). Hence, multiple mechanisms may potentially synergize and contribute to impaired K_V function, and vascular complications during diabetes. Note, however, that activation of divergent mechanisms may vary between vessels and animal models of diabetes. Thus, the relative contribution of transcription-dependent and -independent pathways to altered K_V expression and function during diabetes warrants further investigation.

To summarize, our data demonstrate that suppression of K_V channel function via selective down-regulation of K_V2.1 contributes to enhanced arterial tone during diabetes. AKAP150-CaN/NFATc3 signaling is central to impaired K_V2.1 expression and function. Our findings also support the view that activation of this signaling pathway may be a general mechanism for transcriptional regulation of K⁺ channels and that with K_V2.1, may be novel therapeutic targets to prevent and/or treat vascular complications during diabetes, and perhaps other pathological conditions.