Research grants from the NIH, the American Heart Association and the US Dept. of Veterans Affairs.
The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While
The multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is a ubiquitously expressed serine/threonine kinase that decodes intracellular Ca2+ oscillations into signaling events [
Recently, a role for CaMKII in vascular diseases such as hypertension and remodeling after injury has emerged [
These and further
The current study sought to overcome some of these obstacles by developing novel transgenic models of CaMKII inhibition in endothelium. Here, we used the approach of overexpressing CaMKII inhibitor peptides AC3-I [
Bradykinin, acetylcholine, L-NG-Nitroarginine methyl ester (L-NAME), L-NG-Nitroarginine (L-NNA), sodium nitroprusside (SNP), and Sepiapterin were purchased from Sigma Chemicals. KN-93 was purchased from EMD Milipore. The cell permeable NO donor, DDI guanyl cyclase activator and DAR-4M AM were purchased from Enzo Life Sciences. Anti-GFP tag antibody, pluronic acid F-127, Fura-2AM and Dynabeads Protein A were purchased from Thermo Fisher Scientific. Anti-CaMKII antibody used for western blots was purchased from EMD Millipore (#07–1496) and anti-CaMKII antibody used for immunofluorescence was from LifeSpan Biosciences (LS-C100735/5122). Comparable antigen detection with the two antibodies was confirmed in pilot experiments (
Human tissue samples from autopsies were procured from the University of Iowa Decedent Center in accordance with guidelines established by the University of Iowa Institutional Review Board. Upon submission of a full application, it was determined by the review board that the sample collection was exempt from federal regulations (University of Iowa IRB# 201210793). The exemption was granted on the basis that the research involved the collection or study of existing data and pathological specimens and that the information was recorded in such a manner that subjects could not be identified, directly or through identifiers linked to the subjects.
Autopsy samples were procured from subjects without a history of hypertension, diabetes, or cardiovascular disease. The absence of atherosclerosis was confirmed by gross pathology. Samples were fixed in formalin, processed and paraffin-embedded for immunohistochemistry. Some sections contained Ca2+ and required incubation in Decal prior to processing.
All animal care and housing requirements of the National Institutes of Health Committee on Care and Use of Laboratory Animals were followed. The protocols were reviewed and specifically approved by the Iowa City VA Healthcare System and the University of Iowa Animal Care and Use Committee. To study the role of endothelial CaMKII inhibition
The inducible model of CaMKII inhibition was generated as follows: Tet
BAEC and HUVEC were maintained in DMEM:F12 supplemented with 10% FBS, penicillin/streptomycin, sodium pyruvate, L-glutamine, and non-essential amino acids and fed every other day. They were used between passages 7 and 11. Cells were infected at 100 moi with adenovirus expressing CaMKIIN (Ad5.CMV.CaMKIIN.HA.IRESeGFP) or control adenovirus (Ad5.CMV.Empty. IRESeGFP), plated in 60 mm dishes at a density of 400,000 cells/dish and used for further experiments after 72–96 hr [
The CaMKII inhibitory peptide Autocamtide-2 (H-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu-OH, Santa Cruz Biotechnology, sc-3117) was transfected into HUVEC with the transfection reagent Chariot ™ (Active Motif, 30025) as recommended by the manufacturer. This approach provided CaMKII inhibition comparable to other methods as demonstrated by its effect on the downstream CaMKII signaling target eNOS (
Cells were lysed after treatments with buffer containing 50 mM TRIS/HCl, 150 mM NaCl, 0.012 M sodium deoxycholate, 0.1% SDS, and 1% NP-40 supplemented with protease (SantaCruz Biotechnology) and phosphatase inhibitors (Thermo Fisher Scientific). Equivalent amounts of cell lysate were separated by SDS/PAGE on 4–20% TRIS/Glycine precast gels (BioRad) and transferred to PVDF membranes (Millipore). Membranes were blocked in 3% BSA/1% gelatin and incubated overnight at 4°C with primary antibodies at a dilution of 1:500 to 1:1000. Blots were washed 3 times for 10 min each with 0.05% Tween-20 in TBS and incubated for 1 hour at room temperature with the respective secondary antibodies at a dilution of 1:2000 to 1:5000. After washing, blots were developed with ECL chemiluminescent substrate (Santa Cruz Biotechnology). Blots were scanned and analyzed using Image J software.
After treatment, cells were lysed with 50 mM TRIS, 150 mM NaCl and 1% NP-40 buffer supplemented with protease and phosphatase inhibitors and 250–300 μg of cell lysate were rotated overnight at 4°C with 5 μL of anti-eNOS rabbit antibody in a final volume of 200 μL. The next day, 10 μL of Dynabeads Protein A were added to the IgG-protein complex and incubated by rotation for 4 hr at 4°C. After magnetic separation, the IgG-protein complex was eluted from the beads by boiling for 1 minute with 2X Laemmli Sample Buffer. Immunoprecipitated proteins were separated by SDS/PAGE on 4–20% TRIS/Glycine gels, transferred to PVDF membranes, and immunoblotted for eNOS and calmodulin.
Mouse thoracic aorta was fixed by immersion in 4% paraformaldehyde/PBS. After 48 hr, the aorta was cryopreserved at -80°C in Tissue Freezing Medium. Ten-μm frozen sections were cut, post-fixed on Superfrost Plus glass slides and washed in PBS. They were pre-incubated for 10 min in egg substitute, washed and blocked for 2 hr at room temperature in 5% non-fat dry milk. After washing with PBS for 10 min, the sections were incubated overnight with primary antibodies (anti-eGFP or anti-CaMKIIN) diluted 1:200 in PBS. After washes in PBS for 15 min, sections were incubated for 2 hr with biotinylated secondary antibodies diluted 1:250 in PBS. Sections were then washed with PBS for 15 min and incubated with Alexa 568-conjugated secondary. After washing in PBS, sections were counterstained with ToPro-3 iodide to visualize nuclei and mounted in Vectashield. Ten μm sections of fixed and paraffin-embedded human aorta, carotid and coronary vessels were deparaffinized and subjected to immunohistochemistry using anti-von Willebrand and CaMKII antibodies as described above. Negative controls without primary antibody were performed in every experiment. Images were captured with Zeiss LSM 710m Laser scanning microscope using the following parameters: DAPI was imaged with an excitation at 405 nm and emission filter set from 410 nm to 484 nm. CaMKII was imaged with an excitation at 488 nm and emission path from 494 nm to 572 nm and von Willebrand factor imaged with an excitation at 561 nm and emission filter set from 574 nm to 712 nm. A Plan-Apochromat 63x/1.40 Oil DIC M27 lens was used and the resolution was set to 1024 pixels in x an 1024 pixels in y, using a 8-bit encoding with a line averaging of 2. The pinhole was set to 51 μm and the dwell time to 1.58 msec. The imaging parameters were set based on the negative control samples and all images were obtained with the same parameters.
NO generation in endothelial cells was measured using the cell permeable NO-sensitive dye, DAR-4M AM as described previously [
Vasoreactivity measurements in the thoracic aorta and third-order mesenteric arteries were performed in TekCre and endo-CaMKIIN mice described previously [
Additional experiments were performed in aortic rings after transduction with adenovirus expressing CaMKIIN or control adenovirus [
Aortic rings were placed overnight (16–20 hr) in EGM2 medium (Lonza, Walkersville, MD USA) in the presence of Ad5.CMV.Empty.IRESeGFP or Ad5.CMV.CaMKIIN.HA.IRESeGFP with 20x107 particles per aortic ring. In pilot experiments, we determined the virus concentration that resulted in strong CaMKIIN expression in the endothelium. Transduction efficiency was assessed by eGFP fluorescence in 10-μm frozen sections of the descending thoracic aorta. Images were taken as described under “Immunohistochemistry”.
The next day, the vessels were suspended between two wire stirrups (150 mm) in a four-chamber myograph system (DMT Instruments) in 6 ml Krebs-Ringer (95% O2-5% CO2, pH 7.4, 37°C). The mechanical force signal was amplified, digitalized, and recorded (PowerLab 8/30). Cumulative concentration-response curves to acetylcholine (ACh, 10−9–10−5 M), or sodium nitroprusside (SNP, 10−9–10−5 M) were obtained in aortic rings after pre-contraction with phenylephrine (10−6 M). To estimate NO-dependent vasoreactivity, vessel segments were pre-incubated with 100 μM L-NAME for 10 min. Vasorelaxation evoked by ACh and SNP was expressed as percent relaxation, determined by calculating percentage of inhibition to the pre-constricted tension. Additional aortic rings were used for immunoblots.
Genomic DNA was isolated from mouse tails using DirectAmp tissue genomic DNA amplification kit (Denville Scientific, Holliston, MA). Tek-Cre genotyping was performed with the following primers: Tek-Cre forward (
DNA-free total RNA was isolated from mouse arteries using the Micro RNeasy kit (Qiagen), and reverse transcribed using RTIII (Invitrogen). cDNA was amplified with mRNA-specific primers for CaMKIIN, AC3-I/eGFP and acidic ribosomal phosphoprotein (ARP) in Sybr Green PCR master mix (ABI) in a qPCR reaction using an ABI real-time PCR machine. CaMKIIN forward primer was
Blood pressure was measured by the tail cuff method in TekCre and endo-CaMKIIN mice. The mice were age- and sex-matched and 11–22 weeks old. One week prior to the start of the experiment, mice were trained on tail cuff blood pressure equipment (BP-2000 Blood Pressure Analysis System, Visitech Systems Inc.). Thereafter, blood pressure was recorded daily for 20 min for 5 days.
To detect subtle differences, blood pressure was also monitored by radiotelemetry (PA-C10; Data Science International) in conscious, unrestrained eCdh5-tTA and endo-AC3-I mice. Mice were age- and sex-matched and 10–23 weeks old. Under ketamine (80–100 mg/kg) and xylazine (10 mg/kg) anesthesia, radiotelemetric catheters were implanted into the left common carotid artery through an anterior neck incision. The radiotelemeter transmitter was implanted subcutaneously into the left flank. After surgery, pain control was provided with flunixin meglumine and meloxicam. Six days after surgery, arterial blood pressure, heart rate and activity levels were recorded at 500 Hz for 10-sec intervals every 10 min over a total period of 48 hr. The following day, mice were switched from doxycycline-containing chow to normal chow to induce endothelial AC3-I expression. After 14 days on the normal chow, arterial blood pressure, heart rate, and activity levels were recorded as above. The averages of 48-hr recordings were calculated in each mouse.
Blood from the left ventricle of eCdh5-tTA and endo-AC3-I mice that had been on regular chow for at least two weeks was collected in BD Microtainer tubes. Plasma was separated following centrifugation at 9300 x g for 5 min and immediately frozen at -80°C. Total reducible NO content (NOx) in thawed samples of plasma was determined using a Sievers NOA 280i Nitric Oxide Analyzer (GE Analytical Instruments, Boulder, CO) and includes NO released from nitrosothiols, nitrite and nitrate [
Data are expressed as mean SEM and analyzed by with GraphPad Prism 7.0 software using 2-tailed Unpaired Student’s t-test (
We first examined the expression of CaMKII in the human and murine arterial wall by immunofluorescence. In a variety of vascular beds, CaMKII was strongly detected in endothelium and, as expected, in medial smooth muscle cells (
Immunofluorescence of CaMKII. Red: von Willebrand factor (endothelium); green: CaMKII; yellow: merged von Willebrand factor and CaMKII; blue: DAPI (nuclei). Arrows denote CaMKII expression. NC: negative control without primary antibody. Immunofluorescence in three arterial beds in human autopsy samples (Scale bars: 25 μm coronary and carotid artery, 5 μm aorta) and in three arterial beds in murine samples. (Scale bars: 50 μm).
In a constitutive model of endothelial CaMKII inhibition with CaMKIIN (
Since chronic CaMKII inhibition may induce compensatory mechanisms that counteract effects of the inhibitor, and transgene expression in Tek-driven Cre models also occurs in bone-marrow derived cells, we opted to engineer a second model of inducible CaMKII inhibition. In this model, the inhibitor peptide AC3-I fused to GFP is expressed under the control of the cadherin-5 (eCdh5) promoter upon doxycycline withdrawal (
Since NO production is a key mechanism by which the endothelium impacts vascular function and previous reports have yielded contradictory results on CaMKII-dependent eNOS activation [
Next, we tested whether CaMKII inhibition affects serum NO in the inducible model of CaMKII inhibition (endo-AC3-I). As implied by the results of the blood pressure measurements, no significant differences in plasma NO, as determined by measuring total reducible NO (nitrites and nitrates) were detected with a Sievers Nitric Oxide Analyzer (
We next assessed CaMKII activity in the arterial wall at baseline conditions by immunoblots for CaMKII autophosphorylation at Thr-287. In aortic lysates from WT mice, we detected low levels of active CaMKII (
Previous evidence suggests that CaMKII modulates vasodilation secondary to inducing eNOS activation [
To confirm these findings, we performed vasoreactivity studies in aortic rings from C57Bl/6 mice that were incubated ex vivo with adenovirus that expresses the inhibitor peptide CaMKIIN. Despite robust transgene expression, no differences in vasodilation to ACh or SNP were detected (
Previous studies in cultured endothelial cells
Because of previous reports on CaMKII as regulator of cytosolic Ca2+ in endothelial cells [
eNOS can be activated by both Ca2+-dependent as well as -independent mechanisms [
To understand the net effect of these findings, we tested whether CaMKII inhibition affects NO production in bradykinin-stimulated endothelial cells infected with either empty or CaMKIIN virus. As expected, bradykinin significantly induced NO production in control cells but little to no response to bradykinin was detected in CaMKIIN-expressing cells (
CaMKII has been extensively studied in excitable cells such as neuronal cells and cardiac myocytes. However, its role in the vasculature is less well investigated. Our current understanding is limited to its actions in the smooth muscle cell layer of the vascular wall [
NO is well accepted to control vascular tone and blood pressure
While total body deletion of eNOS in mice impairs vasodilation to ACh and increases basal mean blood pressure by 30–50 mmHg [
Our study is not the first to suggest that the
Constitutive inhibition of endothelial CaMKII
Many agonists, such as bradykinin, induce eNOS activation by increasing Ca2+ influx [
In summary, our data identify CaMKII as a regulator of eNOS phosphorylation and NO production in endothelium
Immunoblots for total CaMKII protein in lysates from aortas of C57Bl/6 mice, BAEC and HEK cells infected with an adenovirus expressing CaMKIIδ for 72 hr and blotted with an anti-CaMKII antibody from EMD Millipore used for immunoblots in Figs
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BAEC were treated with KN-93 for 2 hr or transfected with the CaMKII inhibitory protein Autocamtide-2 using the the transfection reagent Chariot for 48 hr. Treatment with bradykinin was performed after serum starvation for 24 hr. Representative immunoblots for eNOS pSer-1179 and eNOS. These approaches resulted in eNOS inhibition comparable to inhibition with CaMKIIN as shown in
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(A) Immunoblots for total CaMKII protein in aortas of eCdh5-tTA control and endo-AC3-I mice (lysates from one mouse per lane). (B) Quantiifcation of data in (A). Mean±SEM, n = 10 mice/group.
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NO-sensitive DAR-4M FM fluorescence in bovine aortic endothelial cells infected with control adenovirus Ad5.CMV.IRES.eGFP.Empty (control adenovirus expresses eGFP). Treatment with 1μM bradykinin. Additional samples were pretreated with 100μM L-NAME for 30 minutes. Green: eGFP; red: DAR-4M FM. B. Densitometric analyses of the DAR-4M FM signal. Data are the average of 3 independent experiments. * p<0.05 vs.—BK.
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We thank Kristina W. Thiel in preparation of the manuscript, the Central Microscopy Research Facility, University of Iowa for assistance with immunofluorescence detection of CaMKII, and the Electron Spin Resonance Facility, University of Iowa for assistance with plasma NO detection.