Deficiency in the anti‐aging gene Klotho promotes aortic valve fibrosis through AMPKα‐mediated activation of RUNX2

Summary Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti‐aging Klotho gene (KL) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho‐deficient (KL +/−) mice and WT littermates were fed with a high‐fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKα activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL +/− mice than of WT mice, indicating that Klotho deficiency promotes HFD‐induced aortic valve fibrosis (AVF). AMPKα activity (pAMPKα) was decreased, while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL +/− mice fed with a HFD. Treatment with AICAR markedly attenuated HFD‐induced AVF in KL +/− mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL +/− mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKα by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD‐induced fibrosis in aortic valves, likely through the AMPKα–RUNX2 pathway.


Animal studies
Briefly, all mice were housed in cages at room temperature (25±1°C) and provided with laboratory chow (cat. no. 5053,PicoLab) and tap water ad libitum throughout the experiment.
This study was carried out according to the guidelines of the National Institutes of Health (NIH) on the Care and Use of Laboratory Animals. Heterozygous Klotho-deficient (KL +/-) mice and WT littermates at the age of 6 months were used. Mice were fed with a HFD (Harlan Teklad) containing 21.2% fat (wt/wt) and 1% cholesterol (wt/wt) or a normal diet containing 5% fat and 141 ppm cholesterol. At the end of the 13 th week of HFD feeding, AICAR (500 mg/Kg, BIoVision Inc., Milpitas, CA, USA), which is an AMP analog and activator of AMPK (Sriwijitkamol & Musi 2008), was injected (IP) daily for 2 weeks.

Tissue collection
After 2 weeks of AICAR treatment, animals were euthanized with an overdose of ketamine (180 mg/kg body weight) and xylazine (20 mg/kg body weight), and blood was collected with heparin as an anticoagulant. Following blood collection, animals were perfused transcardiacally using heparinized saline. The upper part of the heart along with the ascending aorta was placed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 24 hours and then embedded in paraffin.

Morphological Analysis
A series of 5-µm cross-sections of the aortic valve were cut and stained using the Masson Trichrome Staining Kit (EMS, Hatfield, PA). Immunohistochemical (IHC) procedures were performed as described in our previous studies (Crosswhite et al. 2014;Chen et al. 2015;Lin & Sun 2015a;Lin & Sun 2015b;Zhou et al. 2015). Briefly, IHC staining against AMPKα, phospho-AMPKα (cat. no. SAB4503754, Sigma-Aldrich), RUNX2 (cat. no. ab23981, Abcam), and collagen I (cat. no. ab34710, Abcam) was performed using the rabbit ABC staining system (Santa Cruz Biotechnology). Images of aortic valves were collected at the same exposure conditions under a Nikon Eclipse Ti microscope (10x, 20x, and 40x objective). The fractional areas of collagen fibrosis components (blue, trichrome staining; brown, IHC staining) in the aortic valve region were obtained using image J software (NIH).

Isolation and Culture of Primary Porcine Aortic Valve Interstitial Cells
Primary porcine aortic valve interstitial cells (PAVICs) were collected from the hearts of female pigs as described (Johnson et al. 1987;Butcher & Nerem 2004). Briefly, intact porcine heart with ascending aortas were obtained from a local slaughterhouse (Edmond, OK). The aortic valve was cut from the ascending aorta above the sinuses, and the leaflets were then excised onethird of the distance from the base of the cusp. The leaflets were then serially rinsed in cold PBS with 1% penicillin/streptomycin and 1% amphotericin B. The surfaces of the leaflets were then partially digested with collagenase type II (1000 U/ml; cat. no. 17101015 Life Technologies) for 5 min, after which the endothelium was denuded with gentle scraping. The remaining portions of the leaflets were then minced and digested overnight in fresh collagenase. Undigested tissue pieces were removed with a 70-μM cell strainer, and the cells were seeded into 6-well plates after washing. PAVICs were cultured in DMEM (1 g/L glucose, Invitrogen), supplemented with

Immunofluorescence Analysis
Cells were fixed with 4% PFA for 10 minutes and then permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. After blocking with 1% BSA, 0.3 M glycine in PBST for 1 hour, cells were incubated with a specific primary antibody at 4⁰C overnight. After washing with PBST, the cells were then incubated with Alexa Fluor secondary antibodies (cat. no. A-11008, Invitrogen, Carlsbad, CA) at dilutions of 1:500 for 1 hour at room temperature in the dark. The slides were mounted with UltraCruz mounting medium containing 4',6-diamidino-2-phenylindole (cat. no. sc24941, DAPI, Santa Cruz Biotechnology, TX). Fluorescent images were taken with an IX73 microscope (Olympus, Tokyo, Japan).

Serum Klotho 65KDa
Ponceau Stain Supplemental Figure 4. Cardiac output, stroke distance, mean velocity, and mean acceleration of KL +/mice fed with a HFD and treated with AICAR.

Heart Function Data Collected Using Pulsed Doppler
Aortic flow was measured using a Doppler Signal Processing Workstation (DSPW, Indus Instruments, Houston, TX, USA) (Reddy et al. 2005). Briefly, all mice were anesthetized with ketamine (90 mg/kg body weight) and xylazine (10 mg/kg body weight) via IP injection. Each mouse was taped supine to electrocardiogram (ECG) electrodes on a heated procedure board with a constant temperature of 37 °C (Indus Instruments). A 2-mm diameter, 10-MHz Doppler probe was used to measure aortic flow. Heart function data was calculated using the manufacturer's DSPW software.