Animal study design

Twenty-four-month-old Fischer 344/Brown Norway F1 male rats from the National Institute of Health were separated into RLX (400 µg/ml/day, n = 6) and control (Na+ acetate, n = 5) groups and treated for 14 days via subcutaneous osmotic mini-pumps (Alzet® Cupertino, CA; model 2ML2). Relaxin was the generous gift of the RRCA Foundation (Foundation for Research on Relaxin in Cardiovascular and Other Diseases, Drs. Mario Bigazzi and Daniele Bani, Prosperius Institute, Florence, IT). Studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Plasma relaxin concentration

Circulating levels of RLX in the aged rats were determined using a pre-packaged Quantikine ELISA kit (R&D Systems, DRL200) according to manufacturer instructions.

Optical mapping

The optical mapping apparatus was described in detail in several reports5,32. Briefly, hearts were excised and perfused on a Langendorff apparatus with Tyrode’s solution containing (in mM): NaCl (130), KCl (4.5), KH 2 PO 4 (0.6), Na 2 HP0 4 (0.6), MgSO 4 (1.2), HEPES (10), NaHCO 3 (24), Glucose (50), gassed with 95% O 2 and 5% CO 2 , pH 7.0 at 37 °C. Hearts were placed in a custom-designed chamber to abate motion artifacts and blebbistatin (5–7 µM) briefly added to the perfusate to minimize movement artifacts. Bolus injections of voltage (RH 237 50 µl of 1 mg/mL dimethyl sulfoxide (DMSO)) and Ca2+-indicator dye (Rhod-2/AM, 80 µl of 1 mg/mL DMSO) were made in the air-trap above the aortic cannula. Fluorescence from the epicardium was collected with a camera lens, split with a 570 nm dichroic mirror and focused on two CMOS cameras (Sci-Media UltimaOne) capturing at the fluorescence emission at 570–595 nm for cytosolic Ca2+ and 610–750 nm wavelengths for voltage. The hearts were paced at various cycle lengths (CL) and with programmed stimulation, starting with a baseline CL for 10 beats at S1-S1 = 250 ms followed by a single S2 pulse delivered at decreasing S1-S2 intervals. Pacing protocols were used to measure CV at various stable CLs (S1-S1) and CV restitution kinetics was measured through the CV of the premature impulse delivered at decreasing S1-S2, until the premature impulse failed to capture or triggered an arrhythmia.

Immunohistochemistry

Tissue sections (7 μm) were treated with 0.1% Triton X-100 followed by block with 2% BSA. Primary antibody was added for 1 hour at room temperature. Guinea Pig and Rabbit anti-Nav1.5 (AGP-008, Alomone Labs, 1:200 in tissue and ab56240, Abcam, 1:200 in cells), rabbit anti-β-catenin (ab32572, Abcam, 1:250), mouse anti-Cx43 (sc-13558, Santa Cruz Biotechnology, Inc, 1:100), rabbit anti-collagen I (ab34710, Abcam, 1:200), mouse anti-Wnt1 (10C8, Thermo Fisher, 1:200) antibodies were used to measure protein expression. Secondary antibodies were applied for 1 hour at room temperature. Immunofluorescence imaging was performed using an Olympus Fluoview 1000 confocal microscope or an Olympus Provis light microscope. Isolated cardiomyocytes and fibroblasts were stained in a similar manner after fixation with 4% paraformaldehyde. In order to ensure quantitative reliability of the results, all samples being compared were immunostained simultaneously with the same antibody preparation, and identical confocal microscope settings were utilized for the recording of all data. NIH ImageJ was used for image analysis. Quantitation of fluorescence labeling was done after subtracting backgrounds using the built-in background subtraction function of ImageJ. To measure the fluorescence intensity of proteins associated to the intercalated disk, the cell-cell contact regions of adjacent cardiomyocytes were identified in the microscopic image, selected, and the intensities measured and recorded using ImageJ tools.

Myocyte isolation

Excised hearts were placed on a Langendorff apparatus and perfused for 4 minutes with Perfusion Buffer containing (in mM): NaCl (130), KCl (14.7), KH2PO4 (0.6), Na2HP04 (0.6), MgSO4 (1.2), HEPES (10), NaHCO3 (4.6), Taurine (30), BDM (10), Glucose (5.5), pH 7.0. Heart was perfused for 11–14 minutes with digestion buffer containing: 50 mL Perfusion Buffer with 2 mg/mL Collagenase Type II. Heart was placed in small glass beaker with 3 mL digestion buffer and minced into small pieces with surgical scissors. Additional mincing was conducted by cutting plastic transfer pipettes at 45 degrees at largest diameter, slowing reducing pipette diameter size as tissue becomes solubilized. Stopping buffer (10 mL) was added containing: 45 mL Perfusion Buffer, 5 mL of 10% FBS and 12.5 µM CaCl 2 . Myocytes were transferred to a 50-mL tube through a cell strainer primed with stopping buffer and allowed to pellet (~20 minutes). Myocytes were washed with Calcium re-introduction solutions (100 µM, 400 µM and 900 µM: diluted in stopping buffer) with pelleting allowed between washes. Cells were mixed with plating medium containing: 10% FBS, Blebbistatin (25 µM), HEPEPS (10 mM), ATP (2 mM) and PrimocinTM (diluted 50 mg to 500 mL MEM, Invivogen, San Diego, CA. USA). Cells were placed on laminin coated coverslips, in 24 well plates and incubated at 37 °C for 2 hours. Plating medium was replaced by culture medium containing: 0.1% BSA, Blebbistatin (25 uM), HEPES (10 mM), ITS (250 uL of 100x stock) and PrimocinTM.

Picro-sirius red stain

Left ventricular sections (7 μm) were washed in Xylene followed by washes in 100% and 95% EtOH. Tissue was placed in Hematoxylin, followed by tap water wash and Picro-Sirius Red application. This was followed by washes with acidic water, 100% EtOH and Xylene. Finally, coverslips were mounted onto slides with Permount. Imaging was performed using an Olympus Provis Light Microscope at 10x magnification. Data is reported as collagen: tissue ratio.

Fibroblast culture

Primary cardiac fibroblasts isolated from the male F-344 rat model were grown to 80–90% confluence and washed with HEPES BSS (100 μL/cm2), followed by Trypsin/EDTA (100 μL/cm2) for less than 5 minutes, and Trypsin Neutralization Solution (100 μL/cm2). Solution was then spun down for 3 minutes at 220 × g followed by resuspension in pre-warmed growth media. Cells were counted and stored in Cryo-SFM media (PromoCell, C-29910) at −80 °C for slow freezing for 24 hours and moved to −120 °C for long term storage. Frozen fibroblasts were thawed at 37 °C until no ice remained in the vial (~2 minutes). Fibroblasts were seeded between 5,000–10,000 cell/cm2. Media (PromoCell, C-23130) was replaced after 24 hours and every 2 days thereafter until 90% confluency was reached. Fibroblasts were treated with RLX (25 nM), TGFβ (2 ng/mL), Dkk (0.1 μg/mL), and Wnt1 (0.1 μg/mL).

RT-PCR analysis

RNA was isolated (RNAEasy, Qiagen) and copied to cDNA (High Capacity Reverse Transcription kit, Applied Biosystems) according to manufacturer protocols. A Syber-green-based formulation (Absolute Sybr-Green, Thermo Fischer Scientific, Waltham, MA) was utilized for fluorescence-based kinetic real-time PCR using an Applied Biosystems model 7000 detection system (Applied Biosystems Inc., Foster City, CA). Expression levels of RNAs of interest were normalized to that of GAPDH using the ΔΔCt method, and reported relative to the mean of the WTV group. Primer pair sequences (forward and reverse for each target, listed 5′ to 3′) used for RT-PCR are as follows:

MMP-2: gcaccaccgaggattatgac, cacccacagtggacatagca;

MMP-9: cctctgcatgaagacgacataa, ggtcaggtttagagccacga;

αSMA: tggctgatggagtact-tc, gatagagaagccaggatg;

Wnt1: cctgcacctgcgactacag, ggttcatgaggaagcgtagg

GAPDH: agctggtcatcaatgggaa, atttgatgttagcgggatc.

Western blot

Protein from aged rat lysates ± RLX (n = 3/group) was separated using pre-cast Mini-PROTEAN TGX 7.5% polyacrylamide gels and transferred to a PVDF membrane. Membranes were then probed for mouse anti-DKK1 (1:500, Santa Cruz sc374574) and rabbit anti-α-tubulin (1:1000, Abcam, ab4047). Membranes were developed using Pierce ECL Western Blotting Substrate (ThermoScientific, #32109) according to manufacturer instructions and developed by autoradiography. Quantification of the optical density of DKK1 or tubulin bands were performed using ImageJ software and DKK1 expression was normalized to α-tubulin.

Statistical analysis

Comparisons between two groups were done using an unpaired, 2-tailed t-test, or the Mann-Whitney U test. Comparisons of three or more groups were done using ANOVA or Kruskal-Wallis tests. Nuclear β-catenin was compared using Chi-squared. All data is presented as mean ± SEM, unless otherwise noted. Statistical comparisons were performed using Graphpad Prism software. A value of P < 0.05 was considered to be statistically significant.

Ethical approval and informed consent

1. An approval of experimental protocols is stated in the Methods section from the University of Pittsburgh. 2. All methods were carried out in accordance with NIH guidelines and the animal usage committee of the University of Pittsburgh. 3. There was no informed consent as this did not involve patients or human tissues.