Cardiovascular Research Advance Access published January 29, 2016 1

Cardiac hypertrophy is exacerbated in aged mice lacking the osteoprotegerin gene

Yilin Hao1,*, Toshihiro Tsuruda1,*, Yoko Sekita-Hatakeyama1, Syuji Kurogi2, Keishi Kubo1, Sumiharu Sakamoto1, Midori Nakamura3, Nobuyuki Udagawa4, Tomohisa Sekimoto2, Kinta Hatakeyama5, Etsuo Chosa2, Yujiro Asada6, Kazuo Kitamura1

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Department of Internal Medicine, Circulatory and Body Fluid Regulation, University of Miyazaki,

Miyazaki, 889-1692, Japan Division of Orthopedic Surgery, Department of Sensory and Motor Organs, University of

Miyazaki, Miyazaki, 889-1692, Japan 3

Department of Pediatric Dentistry, Matsumoto Dental University, Nagano, 399-0781, Japan

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Department of Biochemistry, Matsumoto Dental University, Nagano, 399-0781, Japan

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Department of Diagnostic Pathology, Nara Medical University, Nara, 634-0813, Japan

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Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692,

Japan

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: [email protected].

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Corresponding author: Toshihiro Tsuruda, MD, PhD, Department of Internal Medicine, Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan, Tel: +81-985-85-0872, Fax: +81-985-85-6596, E-mail: [email protected] *

These 2 authors equally contribute to this work

Abstract Aims: Osteoprotegerin (OPG) may play a role in the progression of cardiac hypertrophy and heart

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aging remains to be elucidated. Methods and results: We conducted experiments using 2.5- and 12-month-old OPG-/- mice and age-matched wild type mice and compared the morphology and function of the left ventricle (LV). Both 2.5- and 12-month-old OPG-/- mice showed a higher systolic blood pressure than did age-matched wild type mice, as well as a greater heart weight/body weight ratio. Twelve-month-old OPG-/- mice had a significantly larger LV chamber and reduced wall thickness compared to age-matched wild type mice and contractile function was decreased. The morphological differences were accompanied by increase in numbers of apoptotic cells, activation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in the LV of 12-month-old OPG-/- mice. Correspondingly, OPG siRNA induced the expressions of TRAIL and cleaved caspase-3 in cultured cardiac myocytes. In addition, these mice revealed a decrease in interstitial fibrosis, and activations

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failure. However, their pathophysiological role in changes in cardiac structure and function with

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of matrix metalloproteinase (MMP)-2, tissue inhibitors of MMP-1, -2 with the inactivation of procollagen 1 synthesis. Moreover, intraperitoneal administration of recombinant OPG to either 2.5- or 12-month-old OPG-/- mice for 28 days led to partial improvement of LV structure and function without affecting systolic blood pressure. Conclusion: These results suggest that OPG plays a role in preserving myocardial structure and function with aging through reduced apoptosis and preserved matrix structure. In addition, this appears to be independent of effects on the vasculature.

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Key words: age, hypertrophy, heart failure, osteoporosis

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1. Introduction The proportion of the elderly in the population is increasing rapidly, and heart failure is one of the major public health problems in industrialized countries1. Hypertension is the most prevalent risk factors for heart failure in the elderly2-4, and despite the recent advances in medical and mechanical devices, the prognosis for heart failure remains poor5, 6. Elderly patients with heart failure often have co-morbidities, such as anemia7, chronic obstructive pulmonary disease8, and chronic kidney disease9. Furthermore, recent epidemiological studies have suggested that osteoporosis is prevalent in patients with heart failure10-13. These findings have raised a question

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pathogenesis of heart failure. Tumor necrosis factor (TNF) superfamily comprises over 20 different ligands such as receptor activator of nuclear factor-b ligand (RANKL) and TNF-related apoptosis inducing ligand (TRAIL) that mediate their cellular responses through more than 30 receptors constituting the TNF receptor super family14. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor is a secretory glycoprotein belonging to the TNF receptor superfamily15, 16. OPG inhibits the RANKL to bind to its membrane-bound receptor, RANK17. OPG deficient mice exhibit osteoporosis at an early stage of life18, suggesting a role for OPG in regulating bone remodeling. RANKL is produced primarily in bone19, whereas OPG is widely distributed throughout the body, including in the heart and vasculature20-22. The plasma concentration of OPG increases with age in a healthy aged population23 and is elevated in patients with cardiac hypertrophy, heart failure and myocardial infarction21, 24-26.

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regarding whether these conditions are not merely co-occurring but might contribute to the

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However, the pathophysiological roles of OPG/RANK/RANKL axis in the development of cardiac hypertrophy and heart failure remain to be elucidated. We have reported that OPG is secreted into the coronary circulation in humans and that the magnitude was associated with concentric hypertrophy but less with eccentric hypertrophy27. Based on these findings, we hypothesized that OPG might play important roles in maintaining the structural integrity of the heart. This study sought to uncover the role of endogenous OPG in age-related alterations in the morphology and function of the LV, using mice genetically lacking the OPG gene and overexpressing the RANKL

2. Methods An expanded Methods section can be found in the Online Supplement. All protocols for the present experiments were reviewed and approved by the University of Miyazaki Institutional Animal Care and Use Committee (approval number 2012-506-3) and Genetic Modification Safety Committee of Miyazaki University (approval number 403). This investigation also conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th edition, 2011).

2.1 Animals Male OPG-/- and RANKL-transgenic mice were housed in a temperature and light-controlled room (25±1 °C; 12/12-h light/dark cycle) with free access to normal chow and water, and we used them at

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gene.

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2.5- and 12-months old in the experiment. In another setting of experiment, we administered 10 mg/kg/day of human recombinant (hr) OPG intraperitoneally every 2 days for 28 days to either 2.5or 12-month-old OPG-/- and wild type mice.

2.2 Blood pressure measurement We measured blood pressure and heart rate in conscious mice at least 3 times by tail-cuff plethysmography, and averaged these measurements.

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We performed transthoracic echocardiography on conscious mice using a 15-MHz high-frequency linear transducer connected to a SONOS 5500 system28. We obtained LV posterior free wall (LVPWd), LV diastolic internal dimension (LVIDd) and LV systolic internal dimension (LVIDs) from M-mode tracings. The % fractional shortening was calculated from M-mode-derived LV dimensions using the following formula: (LVDd-LVDs)/LVDd x100% at each measurement. All LV dimensions and % fractional shortening values are presented as the average of 5 independent measurements.

2.4 Sample collection We collected blood samples from the right ventricle under anesthesia by the intraperitoneal administration of 50 mg/kg pentobarbital sodium. The blood samples were centrifuged at 3,000 rpm

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2.3 Echocardiography

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at 4 °C for 10 minutes, and the plasma was stored at –80 °C until use. We removed the whole heart and lungs from the body and weighed the whole heart. Then, we removed both atria and the right ventricle. Thereafter, we weighed the LV, fixed the middle portion of the LV in 4% paraformaldehyde, and froze the rest of the LV in liquid nitrogen. Furthermore, we took whole aorta and the right lower extremity of the mouse body after sacrificing.

2.5 Cell culture We isolated cardiac myocytes from the ventricles of 1-day-old Wistar rats29. We transfected OPG

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cells were harvested for quantitative RT-PCR and Western blotting. Multiple sets of Stealth Select siRNA were designed to define RNA targeting nucleic acid and the following set was chosen: sense 5’-AACACUCAGCCAAUUCGGUAUAAUC-3’ and antisense: 5’-GAUUAUACCGAAUUGGCUGAGUGUU-3’. Control siRNA was a scrambled sequence to minimize the effect on baseline or inducible expression levels of OPG/18S ribosomal (r)RNA. The oligonucleotide sequences of probe and primers of rat OPG are listed in Supplementary Table 1.

2.6 RNA isolation and real-time quantitative RT-PCR We extracted total RNA for synthesizing complementary DNA with SuperScript reverse transcriptase. Then, complementary DNA was amplified using oligonucleotide primers and probes labeled with 6-carboxy-fluorescein as reporter fluorescence and with

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small interfering RNA (siRNA) or control siRNA to cardiac myocytes with Lipofectamine. The

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6-carboxy-tetramethyl-rhodamine as quencher fluorescence by real-time quantitative RT-PCR. The oligonucleotide sequences of probes and primers used are listed in Supplementary Table 1.

2.7 Western blotting We subjected equal amounts of denatured protein to SDS-PAGE. Then, the separated proteins were electrically transferred onto polyvinylidene difluoride (PVDF) membranes. After incubating the PVDF membranes in 5% skim milk overnight at 4 °C, we incubated the membranes with primary antibodies directed against extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK,

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temperature, followed by incubation with horseradish peroxidase-conjugated secondary antibody.

2.8 Picrosirius red staining We stained the slide-mounted LV tissue sections with 0.1% picrosirius red dissolved in saturated picric acid for 10 minutes. We assessed collagen volume fraction in the interstitial space under polarized light.

2.9 Calcifications in the aortic valve and aortic root We cut the removed whole aorta cross-sectionally into 6 parts at the levels of aortic valve, proximal ascending aorta, aortic arch, thoracic descending aorta, abdominal aorta and bifurcation of common iliac arteries. We stained them with the von Kossa or Alizarin red in order to detect the calcium

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c-jun N-terminal kinase (JNK), p-JNK, TRAIL, cleaved caspase-3 and -tubulin for 1 hour at room

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salts.

2.10 In situ detection of apoptosis We assessed the number of apoptotic cells in the LV cross-sections by in situ detection of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining.

2.11 RANKL concentration

2.12 Micro computed tomography We performed micro computed tomography scans. For bone morphometry, the measurement area was set to the secondary trabecular area at the proximal metaphysis of the tibia (2.0 mm trimming). We analyzed trabecular bone volume/tissue volume using a 3D image analyzing system.

2.13 Statistical analysis The data are presented as dot plots with mean±standard deviation. Statistical analyses were performed using GraphPad Prism 6 (La Jolla, CA, USA). We assessed comparison of the two variables with Student’s unpaired or paired t-test, and multiple comparisons with one-way, two-way (genotype and age effect) or repeated measures (drug dose- and time- dependent effect) Analysis of Variance (ANOVA), followed by Bonferroni post hoc test. In addition, two-way ANOVA with

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We measured the plasma concentration of RANKL in duplicate using a Quantikine ELISA kit.

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Bonferroni correction for multiple comparisons was used to evaluate the morphometric measurements with or without hrOPG treatment in each genotype. The respective method for statistical test was described in the Figure legends. P

Cardiac hypertrophy is exacerbated in aged mice lacking the osteoprotegerin gene.

Osteoprotegerin (OPG) may play a role in the progression of cardiac hypertrophy and heart failure. However, its pathophysiological role in changes in ...
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