Regulation of Large Conductance Ca2+-activated K+ (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels*

Background: Impaired BK channel function in diabetic vessels is associated with decreased BK channel β1 subunit (BK-β1) expression. Results: Muscle RING finger protein 1 (MuRF1) physically interacts with BK-β1 and accelerates BK-β1 proteolysis. Conclusion: Increased MuRF1 expression is a novel mechanism underlying diabetic BK channelopathy and vasculopathy. Significance: MuRF1 is a potential therapeutic target of BK channel dysfunction and vascular complications in diabetes. The large conductance Ca2+-activated K+ (BK) channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK channelopathy and vasculopathy in diabetes.

Diabetes mellitus has become a global epidemic. In the United States, 25.8 million people have diabetes, and another 79 million Americans are prediabetic. These figures will more than double by 2030. Diabetes is an independent risk factor of vascular pathology and a leading cause of cardiovascular morbidity and mortality (1,2). Major causes of vascular dysfunction in diabetes involve both endothelium-dependent and endothelium-independent mechanisms. There is a large body of evidence showing the abnormality of vascular smooth muscle function in diabetes. However, the underlying molecular mechanisms remain unclear (3)(4)(5)(6)(7)(8)(9).
The large conductance Ca 2ϩ -activated K ϩ (BK) 3 channels, densely populated in vascular smooth muscle cells (SMCs), play a negative feedback role in the regulation of vascular tone. BK channels are activated by the increase of intracellular free Ca 2ϩ concentrations ([Ca 2ϩ ] i ) to generate spontaneous transient outward currents, which hyperpolarizes the membrane potential of vascular SMCs, inactivates voltage-dependent Ca 2ϩ channels, and leads to vascular relaxation (8, 10 -14). Functional vascular BK channels are octameric complexes with four BK-␣ and four BK-␤1 subunits. BK-␤1, encoded by the KCNMB1 gene, is a key determinant of BK channel electrophysiology, enhancing the BK-␣ sensitivity to Ca 2ϩ and voltage and allowing channel activation in the physiological ranges of [Ca 2ϩ ] i and membrane potentials (15)(16)(17)(18). Regulation of BK channel activity by many biological mediators, such as polyunsaturated fatty acids, thromboxane A2, and steroid hormones, are mediated through BK-␤1 stimulation, demonstrating the physiological importance of BK-␤1 (19 -22). The clinical relevance of BK-␤1 is underscored by the findings that down-regulation of BK-␤1 function contributes to hypertension and renal disease but that a gain-of-function mutation of BK-␤1 is associated with a low prevalence of diastolic hypertension in humans (23)(24)(25). However, vascular BK channel function is impaired in both type 1 and type 2 diabetes mellitus and is an important ionic mechanism leading to diabetic vascular dysfunction. Current experimental evidence indicates that BK channel malfunction in diabetic vessels is associated with a significant down-regulation of BK-␤1 protein expression, resulting in Ca 2ϩ sparks/spontaneous transient outward current uncoupling and loss of Ca 2ϩ -mediated channel activation even though the Ca 2ϩ spark amplitude and [Ca 2ϩ ] i were increased in vascular SMCs of diabetes (8, 26 -30). We have reported previously that ex vivo expression of the KCNMB1 gene in diabetic mouse coronary arteries rescued BK channel function and preserved normal coronary reactivity, suggesting that BK-␤1 is an important molecular target for the treatment of diabetic vascular diseases (31). However, the molecular mechanisms that produce BK-␤1 down-regulation have not been thoroughly delineated.
Protein homeostasis with a balanced regulation between syntheses and degradation is essential for normal cellular function. The ubiquitin proteasome system (UPS) is a major pathway of proteolysis, accounting for 80 -90% of intracellular protein degradation in mammalian cells (32). This process is facilitated by three distinct enzymatic steps that involve an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and an ubiquitin ligase (E3). E1 activates ubiquitin, E2 accepts the transfer of activated ubiquitin, and E3 selects proteins for ubiquitination. Finally, the polyubiquitinated target proteins are degraded to short peptides in the 26 S proteasome (32). Of these, the E3 reaction is critical for the system because it recognizes the targeted substrates for ubiquitination and confers specificity of protein turnover. There are 617 E3 ligases functionally annotated in the human genome (33). The muscle RING finger (MuRF) protein family, comprised of three members (MuRF1, MuRF2, and MuRF3), is a group of muscle-specific E3s. All three isoforms are specifically expressed in cardiac and skeletal muscles and contain four important structures: a RING finger domain, a MuRF family conserved region, a "B-box" domain, and multiple coiled-coil domains (34). MuRF1, in particular, has been implicated in many heart diseases, including myocardial hypertrophy, myocardial atrophy, ischemia, heart failure, myocarditis, and familial cardiomyopathy (35,36). Human genetic and functional studies have revealed the clinical significance of MuRF1, which confirmed that MuRF1 mutations cause hypertrophic cardiomyopathy in patients (37). However, the role of MuRF1 in vascular diseases is unclear.
The NF-B complex is composed of the p65 (RelA), RelB, c-Rel, and p105/p50 (NF-B1) or p100/p52 (NF-B2) subunits. In unstimulated cells, p65 is bound to an inhibitory subunit (IB) that keeps it sequestered in an inactive state in the cytoplasm. Phosphorylation of IB by IB kinase (IKK) leads to its dissociation from p65, which facilitates p65/p50 or p65/p52 dimeric complex nuclear translocation and promotes its tran-scriptional activity (38). In addition, the unbound phospho-IB is degraded through the UPS. MuRF1 is one of the target genes regulated by NF-B (39). In this study, we report that the MuRF1 physically interacted with BK-␤1 and down-regulated BK-␤1 expression through MuRF1-facilitated, UPS-dependent BK-␤1 proteolysis. MuRF1 expression was controlled by NF-B, which is up-regulated in diabetic vessels, leading to acceleration of BK-␤1 proteolysis. Most importantly, knockdown of MuRF1 or inhibition of proteasome and NF-B activities significantly enhanced BK-␤1 expression, restored vascular BK channel activity, and preserved coronary function in diabetes. Our results identified a fundamental mechanism that underlies diabetic BK channelopathy and vasculopathy. Therefore, MuRF1 is a potential novel target for the treatment of vascular complications in diabetes.

EXPERIMENTAL PROCEDURES
STZ-induced Diabetic Mice-Male mice (strain C57BL/CJ) 4 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were made diabetic by an injection of STZ (100 mg/kg body weight, intraperitoneally). Animals with blood glucose Ͼ300 mg/dl were considered diabetic and were used for experiments 8 weeks after developing hyperglycemia. All protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic, Rochester, MN.
Coronary Artery SMC Isolation-Mouse coronary SMCs were isolated enzymatically as described previously (40). In brief, the coronary arteries were carefully dissected in ice-cold dissociation buffer (145 mM NaCl, 4.0 mM KCl, 0.05 mM CaCl 2 , 1.0 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose (pH 7.2)). The vessels were placed in dissociation buffer containing 0.1% w/v BSA and incubated in a shaking water bath at 37°C for 3 min. The vessels were incubated with fresh 0.1% w/v BSA dissociation buffer containing 1.5 mg/ml papain and 1.0 mg/ml dithiothreitol in a shaking water bath at 37°C for another 3 min. This was followed by digestion in fresh 0.1% w/v BSA dissociation buffer containing 1.0 mg/ml collagenase and 1.0 mg/ml of trypsin inhibitor at 37°C for 3 min. The vessels were stored in 2 ml of dissociation buffer triturated gently with a fire-polished glass pipette until the cells were completely dissociated.
BK Channel Current Recordings-Single channel currents were elicited at ϩ60 mV from inside-out excised membrane patches using an Axopatch 200B (Axon Instruments, Inc.) (27,31,41). The output signals were filtered with an 8-pole Bessel filter (902 LPF, Frequency Devices, Inc.) at 5 kHz and digitized at 50 kHz. Patch pipettes had a typical tip resistance of 5-10 M⍀ when filled with the pipette solution, which contained 140 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM EGTA, and 10 mM HEPES (pH 7.4) with KOH. The bath solution contained 140 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 0.465 mM CaCl 2 (0.2 M free Ca 2ϩ , calculated using Chelator software), and 10 mM HEPES (pH 7.35) with KOH. The inside-out single BK currents were determined by its unitary current amplitude (i) and sensitivity to free Ca 2ϩ concentrations, as described previously (41). Normalized channel open probability (NP o ) was determined by the following equation (42):

Down-regulation of Vascular BK-␤1 Expression by MuRF1
where t represents the open time of a channel obtained at a distinct level (n), and T is the total recording time.
The effects of dehydrosoyasaponin-1 (DHS-1, a BK-␤1-specific activator) on BK channel open probability (P o ) were measured in the same membrane patch before and after drug application to the cytoplasmic surface. After the DHS-1 experiments were completed, the total number of channels in each membrane preparation was determined by exposure to 10 M Ca 2ϩ that activates all channels in the patch and produces the maximal BK channel P o in coronary SMCs (41). All patch clamp experiments were performed at room temperature (22-24°C). Data analysis was performed using Clampfit 10.2 software (Axon Instruments, Inc.).
Human BK-␤1 WT cDNA (amino acids 1ϳ191) (accession no. MN_004137.3) was subcloned into pIRES2-EGFP with two FLAG tags in the N-terminal of BK-␤1 (28). Three FLAG-BK-␤1 truncation mutants, the N-terminal mutant (amino acids 1ϳ39), the transmembrane domain mutant (amino acids 15ϳ179), and the C-terminal mutant (amino acids 155ϳ191) were also constructed. The orientations of the constructs and the correctness of the mutation were verified by DNA sequencing (DNA Facility Core of the Mayo Clinic, Rochester, MN). MuRF1 siRNA and control siRNA were obtained from Santa Cruz Biotechnology, Inc. HA-tagged human ubiquitin (HAubiquitin) in pcDNA3 was obtained from Addgene, Inc. cDNAs and siRNA transfection were conducted using a Lipofectamine 2000 transfection kit (Invitrogen). The aortas and coronary arteries of mice were transduced with Ad-MuRF1 and Ad-GFP at 1 ϫ 10 11 particles/ml for 12 h (28,31).
HEK293 cells were cultured in DMEM. Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 mM glucose (normal glucose, NG) or 22 mM glucose (high glucose, HG). All experiments were performed using cells between passages 4 and 5.
Videomicroscopy-Vasoreactivity was measured as described previously (31,44). Isolated mouse coronary arteries (1-2 mm in length and 80 -130 m in diameter) were mounted in a vessel chamber filled with Krebs solution containing 118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , and 11.1 mM glucose (pH 7.4) and were secured between two borosilicate glass micropipettes with a 10-O oph-thalmic suture. The lumen of each vessel was filled with Krebs solution through the micropipettes and maintained at a constant pressure (no flow) of 60 mm Hg, followed by equilibration for 60 min in oxygenated (20% O 2 ϩ 5% CO 2 balanced with N 2 , 37°C) Krebs solution. The vessels were deemed unacceptable for experiments if they demonstrated leaks or failed to produce more than a 50% constriction to graded doses of endothelin-1. The endothelium was denuded by slowly perfusing 3-5 ml of air through the lumen of unpressurized vessels. The effectiveness of endothelium denudation was verified by demonstrating that the vessel failed to dilate to 1 M acetylcholine, constricted normally to ET-1, and dilated normally to 100 M sodium nitroprusside. Concentration-response relationships of NS-1619 (10 Ϫ9 -10 Ϫ5 M) on vasodilatation were measured, and comparisons were made in mouse coronary arteries 12 h after transduction with Ad-GFP, Ad-MuRF1, or Ad-MuRF1 in the presence of 10 M MG132. At the end of each experiment, vessels were maximally dilated with a Ca 2ϩ -free solution, and the percentage of dilatation in response to NS-1619 was normalized to the maximal diameter.
Coimmunoprecipitation (Co-IP) and Western Blot Analysis-Co-IP was performed as described previously (40). Transfected HEK293 cells were washed with PBS three times. The aortas or collected cells were incubated with 200 l of radioimmune precipitation assay buffer (50 mM Tris, 150 mM NaCl, 1 mM NaF 2 , 1 mM EDTA, 1 mM EGTA, 1 mM NaVO 4 , and 1% Triton X-100 (pH 7.5)) and 1 l of protease inhibitor on ice for 30 min, homogenized, and then centrifuged at 8000 rpm at 4°C for 10 min. The supernatant (about 200 g in 200 l) was incubated with anti-HA (Sigma-Aldrich) or anti-FLAG (Cell Signaling Technology, Inc.) antibodies at a final concentration of 4 g/ml for each at 4°C overnight. The samples were then incubated with 20 l protein G Plus-agarose (Santa Cruz Biotechnology, Inc.) at 4°C for 2 h with rotation. After centrifugation at 1000 rpm for 7 min and washing twice with radioimmune precipitation assay/protease inhibitor buffer, the immunoprecipitates were collected and eluted from agarose with 30 l of SDS-PAGE loading buffer/tube. Size Exclusion Chromatography-Chromatographic separation of FLAG-BK-␤1(1-39) and Myc-MuRF1 mutant proteins was performed by the Mayo Clinic Proteomics Center (Rochester, MN) using a Superdex 75 10/300 column (GE Healthcare). Samples (350 l) were equilibrated in radioimmune precipitation assay buffer and run through the size exclusion column with a flow rate of 0.4 ml/min. 48 fractions (0.4 ml each) were collected. Standard proteins with known molecular weights (Gel Filtration Standards, Bio-Rad Laboratories) in phosphate-buffered saline were run under the same conditions. The presence of FLAG-BK-␤1 and Myc-MuRF1 proteins in each fraction (30 l) were measured using Western blot analyses, and the band intensities were plotted against standard protein peaks (bovine thyroglobulin, 670 kDa; bovine IgG, 158 kDa; chicken ovalbumin, 44 kDa; horse myoglobin, 17 kDa, and Vitamin B 12 , 1350 Da) to estimate the size of proteins.
Statistical Analysis-All data are expressed as mean Ϯ S.E. Student's t test was employed to compare data between two groups, and a paired t test was used to compare data before and after treatment. One-way analysis of variance followed by the Tukey test analysis was used to compare multiple groups using SigmaStat software (Jandel Scientific Software, Inc.). A statistically significant difference was defined as p Ͻ 0.05.

RESULTS
Type 1 Diabetic Mice-Male mice at 4 weeks of age received a single dose of STZ (100 mg/kg body weight, intraperitoneally), whereas controls received saline injections. Eight weeks after injection, the average body weights and blood glucose levels were 28.05 Ϯ 0.82 g (n ϭ 30) and 181.43 Ϯ 5.32 mg/dl (n ϭ 30) in control mice and 23.61 Ϯ 0.88 g (n ϭ 30, p Ͻ 0.05 versus control) and 466.22 Ϯ 18.05 mg/dl (n ϭ 30, p Ͻ 0.05 versus control) in diabetic mice, respectively. Hence, STZ-induced diabetic mice had a 15.8% decrease in body weight and a 157.0% increase in blood glucose level.
Diminished BK-␤1-mediated BK Channel Activation and Reduced BK-␤1 Protein Expression in Vascular SMCs from Diabetic Mice and in Human Coronary SMCs Cultured with HG-Inside-out single BK channel currents were recorded from the freshly isolated coronary SMCs of control and diabetic mice. BK channel NP o at baseline was 0.19 Ϯ 0.07 (n ϭ 11) in control SMCs and was reduced significantly in diabetic SMCs (0.01 Ϯ 0.004, n ϭ 11, p Ͻ 0.05 versus control). The BK-␤1mediated channel activation was determined by cytoplasmic exposure to 0.1 M DHS-1 (a BK-␤1-specific activator), which resulted in a 5.5-fold increase in NP o (1.10 Ϯ 0.30, n ϭ 11, p Ͻ 0.05 versus baseline) in control mice. In diabetic coronary SMCs, there was a small but significant increase in response to DHS-1 stimulation (0.05 Ϯ 0.01, n ϭ 11, p Ͻ 0.05 versus diabetic baseline) (Fig. 1A). Fig. 1B (left panel) represents a fulllane Western blot analysis of anti-ubiquitin antibody against the lysates of control and diabetic mouse aortas and shows an increase of total ubiquitinated proteins in diabetic aortas. To determine and compare the level of ubiquitinated BK-␤1 protein in control and diabetic vessels, we performed a pulldown assay of control and diabetic mouse aortas with anti-BK-␤1 antibodies, followed by Western blotting against anti-ubiquitin antibody. There was a 1.56 Ϯ 0.28-fold increase in BK-␤1 ubiquitination in diabetic aortas (n ϭ 4, p Ͻ 0.05 versus control) (Fig. 1B, right panel). Importantly, BK-␤1 expression was down-regulated by 0.51 Ϯ 0.13-fold (n ϭ 3, p Ͻ 0.05, versus control) in diabetic mouse aortas, accompanied by a 2.83 Ϯ 0.16-fold increase in MuRF1 expression (n ϭ 3, p Ͻ 0.05, versus control) (Fig. 1C). Similar results were observed in human coronary SMCs after a 2-week culture with HG, where BK-␤1 expression was 0.60 Ϯ 0.05-fold lower and MuRF1 expression was 1.84 Ϯ 0.21-fold higher than cells cultured with NG (n ϭ 3, p Ͻ 0.05 in both) (Fig. 1D). Note that the changes in protein expression profiles in HG culture conditions were not due to osmolality of culture mediums under our experimental conditions because human coronary SMCs cultured with 5 mM glucose plus 17 mM mannitol did not alter the protein levels of BK-␤1 and MuRF1 compared with those cultured with NG alone (data not shown). In addition, we have shown that the BK-␤1 mRNA level was not reduced in STZ-induced diabetic vessels or in HG-cultured human coronary SMCs (28). Hence, the impaired BK channel function in diabetes is most likely due to accelerated BK-␤1 degradation from increased MuRF1 expression.  (Fig. 2C).

Regulation of BK-␤1 Expression and Vascular BK Channel
The role of MuRF1 on the regulation of vascular BK channel activity and coronary vasoreactivity was further demonstrated by adenoviral-mediated overexpression of MuRF1 (Ad-MuRF1) in control mouse arteries. As shown in Fig. 3A, there was a 177.2 Ϯ 11.5% (n ϭ 3) increase of MuRF1 expression and a 66.8 Ϯ 15.6% decrease of BK-␤1 expression in mouse aortas 12 h after transduction with Ad-MuRF1 compared with control transduction with adenovirus carrying the GFP gene (Ad-GFP) (Fig. 3A). Patch clamp studies confirmed that BK channel activation by 0.1 M DHS-1 was diminished in mouse coronary SMCs 12 h after transduction with Ad-MuRF1, which was accompanied by impaired coronary vasodilation in response to NS1619 (a BK channel activator). The DHS-1-mediated BK channel activation and the NS1619-induced (10 Ϫ9 -10 Ϫ5 M) vasodilation were intact in coronary arteries with Ad-GFP transduction (Fig. 3, B and C). Moreover, the inhibitory effect of Ad-MuRF1 on coronary vasoreactivity was abrogated by a 12-h treatment with 10 M MG132 (a proteasomal inhibitor) (Fig.  3C). Our results confirmed that MuRF1 reduced BK-␤1-mediated channel activation and impaired coronary vasodilation through MuRF1-dependent BK-␤1 protein ubiquitination and protein turnover.
Functional Regions of BK-␤1 and MuRF1 Protein Complex Formation-To gain further understanding on interaction between BK-␤1 and MuRF1, we constructed a series of FLAG-BK-␤1 truncation mutants, as shown in Fig. 4A. We coexpressed Myc-MuRF1 WT with individual FLAG-BK-␤1 truncation mutants in HEK293 cells. The immunoprecipitates pulled down by anti-Myc antibody from the cell lysates were blotted against anti-FLAG antibody. As shown in Fig. 4A, the co-IP with MuRF1 was detected only when the N-terminal segment of BK-␤1(1-39) was present. Similarly, the domains in MuRF1 for BK-␤1 interaction were determined by coexpression of FLAG-BK-␤1 WT with individual Myc-MuRF1 mutants: Myc-MuRF1⌬R, Myc-MuRF1⌬MFC, Myc-MuRF1⌬B, and Myc-MuRF1⌬B⌬C (Fig. 4B). When the immunoprecipitates of anti-FLAG antibody were blotted against anti-Myc antibody, the co-IP with BK-␤1 was found in MuRF1 WT, MuRF1⌬B, and MuRF1⌬B⌬C mutants, indicating that the coiled-coil region of MuRF1 is essential for the interaction with BK-␤1 (Fig. 4B). Interestingly, BK-␤1 did not coimmunoprecipitate with MuRF1⌬R and MuRF1⌬MFC where the coiledcoil region is present, suggesting that the MuRF1-BK-␤1 interaction is complex and may be influenced by additional factors.
To confirm that the protein-protein interaction was not due to nonspecific protein aggregation, we further performed size exclusion chromatography separations of FLAG-BK-␤1(1-39) and Myc-MuRF1⌬B⌬C mutants. A 48-h after transfection with a plasmid carrying FLAG-BK-␤1(1-39) or Myc-MuRF1⌬B⌬C (160 -340) in HEK293 cells, respectively, the cell lysates were separated by a size exclusion column, and 48 fractions (0.4 ml each) were collected. Fig. 5A shows the protein expression of FLAG-BK-␤1(1-39) and Myc-MuRF1⌬B⌬C in the fractions of size exclusion chromatography. FLAG-BK-␤1(1-39) was detected only in fractions 23ϳ26 with a molecular size about 17 kDa (Fig. 5A, top panel). The Myc-MuRF1⌬B⌬C mutant was present in a fraction of 23ϳ25, with a molecular size about 27 kDa (Fig. 5A, bottom panel). The relative density of protein expression plotted against equivalent fractions is illustrated in   these results suggest that the proteins of FLAG-BK-␤1(1-39) and MuRF1⌬B⌬C were properly folded and that the pulldowns of BK-␤1(1-39) and MuRF1⌬B⌬C were not due to nonspecific protein aggregation.
We also performed size exclusion chromatography separation and immunoblot analysis in Myc-MuRF1⌬R and Myc-MuRF1⌬MFC mutants to determine whether the absence of FLAG-BK-␤1 interaction was caused by protein misfolding because of the deletions. Myc-MuRF1⌬R and Myc-MuRF1⌬MFC proteins were present in fractions 20ϳ22 (ϳ40 kDa) and 21ϳ 23 (ϳ34 kDa), respectively, similar to those we had predicted (data not shown). Hence, a lack of protein interaction between FLAG-BK-␤1 and Myc-MuRF1⌬R or Myc-MuRF1⌬MFC is unlikely because of a nonspecific interaction with other proteins.

DISCUSSION
In this study, we made several novel findings. First, we found that MuRF1 was abundantly expressed in vascular SMCs and that the expression of MuRF1 was significantly up-regulated in diabetic vessels and under HG culture conditions as a result of increased NF-B signaling. Second, MuRF1 interacted physically with BK-␤1 and facilitated BK-␤1 protein ubiquitination and degradation, leading to BK channelopathy and coronary vasculopathy in diabetes. Third, a co-IP assay demonstrated that the N terminus of BK-␤1 and the coiled-coil region of MuRF1 were required for the formation of protein complexes. Fourth, overexpression of MuRF1 in the coronary arteries of non-diabetic mice mimicked the dysfunction of BK channels and coronary arteries of diabetic mice. Most importantly, knockdown of MuRF1 expression and pharmacological suppression of proteasome and NF-B activities augmented BK-␤1 expression and preserved vascular BK channel function in dia-betes and hyperglycemia. Thus, we have provided compelling first evidence that impaired vascular BK channel function and coronary vasodilation in diabetes is associated with an increase of NF-B/MuRF1-dependent BK-␤1 degradation.
BK channel dysfunction promotes cardiovascular disorders, including hypertension, heart failure, myocardial infarction, stroke, retinopathy, and erectile dysfunction (45)(46)(47)(48). Cardiovascular diseases are the leading cause of death in individuals with diabetes. The mechanisms that underlie diabetic vascular disorders are multifactorial. Besides endothelium dysfunction, abnormal smooth muscle function also critically contributes to vascular pathology in diabetes, especially under the long-term conditions of glucotoxicity and lipotoxicity (49). A major cause of diabetic BK channel abnormality is attributed to the downregulation of BK-␤1 expression in vascular SMCs (26,27), resulting in impaired BK channel sensitivity to Ca 2ϩ and voltage activation (26,27). Many signaling pathways have been reported to be involved in the regulation of BK-␤1 protein expression. For instance, it has been shown that the activation of calcineurin/nuclear factor of activated T-cells, cytoplasmic 3 (NFATC3) signaling suppressed vascular BK-␤1 expression in angiotensin II-induced hypertensive mice (50). We have also reported previously that the down-regulation of BK-␤1 expression in diabetes and under HG culture conditions is ubiquitin/ proteasome-dependent (28). In our previous study, we have  found that atrogin-1, another E3, regulated BK-␤1 expression through interaction with the PDZ-binding motif in BK-␤1 that facilitated BK-␤1 protein ubiquitination (28). Moreover, the expression of atrogin-1 in diabetic vessels was up-regulated by oxidative stress-mediated up-regulation of forkhead box O transcription factor-3a (Foxo3a)-dependent atrogin-1 mRNA transcription (31). Foxo3a activity is dependent on its subcellular localization. Inhibition of Akt signaling by oxidative stress leads to Foxo3a nuclear translocation and facilitates Foxo biological function (51,52). In this study, we further demonstrated that MuRF1 expression was similarly increased in diabetic vessels and also contributed significantly to BK-␤1 dysfunction. Interestingly, MuRF1 mRNA and protein are only expressed in vascular SMCs, whereas those of atrogin-1 can be detected in both vascular SMCs and the vascular endothelium (data not shown). The PDZ motif of BK-␤1 is located at the extracellular loop, whereas the N terminus of BK-␤1 is intracellular. Hence, interaction between atrogin-1 and BK-␤1 must occur before BK-␤1 transportation to the sarcolemmal membrane. It is worth emphasizing that UPS is presented in the centrosomes and cytoskeletal networks as well as the outer surface of the endoplasmic reticulum and that UPS-associated proteolysis can take place at different cell organelles, dependent on the location of E3 recognizing motifs of substrates (53,54). An increase of both MuRF1 and atrogin-1 expression in diabetic vessels may synergistically accelerate BK-␤1 protein degradation at different stages of protein metabolism and exacerbate BK channel-mediated vascular dysfunction in diabetes.
MuRF1 transcription is controlled by NF-B (55). It is known that NF-B activation is a key event for the initiation of cardiovascular pathogenesis in diabetes as a result of increased oxidative stress (56,57). We found that the protein levels of the NF-B1/p50 and p65 subunits were augmented in diabetic vessels and in HG-cultured human coronary SMCs. Pretreatment with the IKK-2 inhibitor TPCA-1 significantly increased IB expression and reduced NF-B/p50 expression in both NGand HG-cultured human coronary SMCs, but the effects of TPCA-1 were more potent under HG culture conditions, which maintained BK-␤1 expression in HG-cultured cells to the level of NG-cells. Hence, NF-B/MuRF1/BK-␤1 should be considered a potential therapeutic target for diabetic coronary dysfunction (Fig. 8).
The specificity of substrate degradation by the proteasome is dependent on the E3 ligase. It has been confirmed that the E3 ligase Nedd-4 interacts with the PY motifs in the C terminus of the cardiac voltage-gated Na ϩ channel (Na v 1.5), the epithelial Na ϩ channel, and the voltage gated K ϩ channel and leads to channel ubiquitination and degradation (58 -60). We found that Nedd-4 did not regulate BK-␤1 expression even though there is a PY motif at the extracellular loop of BK-␤ (data not shown), suggesting that the three-dimensional protein folding is critical. Many regions of MuRF1 have been reported to be associated with substrate proteins. For instance, MuRF1 interacts with the hydrophobic amino acids of "Titin repeats" (61) and myosin heavy chain in skeletal muscles (62). The coiled-coil FIGURE 7. Regulation of MuRF1 and BK-␤1 expression by NF-B. A, protein expression of NF-B1/p105, NF-B1/p50, and RelA/p-65 in the aortas of control and STZ-induced diabetic mice. The levels of NF-B/p105, NF-B1/p50, and p-65 protein expression were significantly increased in diabetic mice compared with controls (Ctrl). B, 24-h incubation with 0.5 M TPCA-1 suppressed the levels of NF-B1/p105, NF-B1/p50, and MuRF1 expression but markedly increased that of IB-␤ in both NG-and HG-cultured human coronary SMCs. TPCA-1 treatment did not alter BK-␤1 expression in NG-cultured cells but significantly enhanced that in HG-cultured cells. Group data and statistical analysis are shown in the bar graphs. In diabetic vessels, increased NF-B/p50 and p65 expression stimulates MuRF1 mRNA transcription and protein expression. The coiled-coil domain of MuRF1 interacts with the N terminus of BK-␤1, which induces BK-␤1 protein ubiquitination and degradation through the UPS and leads to diminished BK channel activation and coronary vasodilation. TPCA-1 inhibits IKK-2 activity, which decreases IB phosphorylation and prevents its dissociation from NF-B/p65, thereby down-regulating MuRF1 expression. MG132 inhibits proteasomal activity and reduces BK-␤1 proteolysis.
region of MuRF1 is responsible for the binding of troponin I in cardiomyocytes (63). The MuRF family conserved region is required for the interaction with phospho-c-Jun in the heart (64). Using BK-␤1 and MuRF1 truncation mutants and a co-IP assay, we have identified the functional domains for physical interaction between BK-␤1 and MuRF1. The N terminus of BK-␤1 is the only region that interacts with MuRF1. The counterparts in MuRF1 are more complicated. We believe that the coiled-coil region of MuRF1 is the structure required for recruiting BK-␤1 to MuRF1 because BK-␤1 can be detected in the pulldown of the MuRF1 coiled-coil region alone, but a direct protein binding experiment is required to further confirm our findings. Our results are similar to previous observations that the coiled-coil region is sufficient for MuRF1 binding to sarcomeric M-line titin and thick filament (63). Interestingly, partially deleting the RING finger domain or the family conserved region of MuRF1 interferes with the physical association with BK-␤1. One explanation is that the RING finger domain and the MuRF1 family conserved region are also important for proper three-dimensional protein folding that allows the coiled-coil region to bind with the N terminus of BK-␤1. How the RING finger domain and the family conserved region influence the interaction between MuRF1 and BK-␤1 is currently unclear and needs further investigation using in vitro protein binding assays. Nevertheless, this is the first report that MuRF1 physically interacts with BK-␤1, facilitates BK-␤1 protein degradation, and impairs BK channel function in diabetic vessels. Inhibition of MuRF1 expression protects BK-␤1 expression and vascular BK channel function and preserves coronary vasoreactivity in diabetes.