Experimental animals

All studies were performed at Cedars-Sinai Medical Center in accordance with the Institutional Animal Care and Use Committee guidelines.

Intravenous TY1 pharmacokinetics

In vivo, experimental protocols were performed on 10- to 12-week-old male C57BL76J mice (Jackson Laboratory). Mice were housed under pathogen-free conditions in a temperature-controlled room with a 12-h photoperiod. TY1 (0.15 mg/kg) or PBS in Dharmafect (Horizon) was resuspended in 50 μl of saline and injected into the retro-bulbar space of healthy mice anesthetized by isofluorane. At multiple time-points (15 min, 30 min, 60 min, 3 h, 24 h, 48 h) mice were sacrificed and tissues (heart, lung, liver, kidney, spleen, thymus, white adipose tissue, soleus muscle, tibialis anterior muscle, Peyer’s patches, inguinal lymph nodes, axillary lymph nodes, mesenteric lymph nodes, brain) were removed (without prior perfusion), washed in PBS, and flash frozen in liquid nitrogen until further processing was performed. Femurs and tibias were isolated and flushed with PBS to collect cells in the bone marrow. Cells were filtered with 70 µm cell strainer (431,751, Corning), treated with ACK Lysing Buffer (A1049201, ThermoFisher Scientific) for 30 s, neutralized with excess PBS, centrifuged 500 g 10 min at 4 °C, then resuspended in PBS and frozen at -80 °C until further processing was performed. Blood was collected from the heart (using K2 EDTA tubes) and PBMCs and plasma were isolated using SepMate PBMC Isolation Tubes (85,415, StemCell Technologies) and Ficoll-Paque PLUS density gradient media (17,144,002, Cytiva) according to SepMate manufacturer’s protocol. Plasma was frozen at -80 °C and PBMCs were used for fractionation or resuspended in Qiazol (Qiagen) and frozen at -80 °C until further processing was performed.

CD3ε and CD11B fractionation

PBMCs were resuspended in MACS buffer from a 1:20 dilution of MACS BSA Stock Solution (130–091-376, Miltenyi Biotec) in autoMACS Rinsing Solution (130-091-222, Miltenyi Biotec) according to manufacturer’s protocol. CD3ε cells were isolated from PBMCs using CD3ε MicroBead Kit (130-094-973, Miltenyi Biotec) with the OctoMACS Separator (130-042-109, Miltenyi Biotec) and LD Columns (130-042-901, Miltenyi Biotec) according to manufacturer’s protocol. CD11B cells were isolated from the CD3ε- fraction using CD11B MicroBeads UltraPure (130-126-725, Miltenyi Biotec) with the OctoMACS Separator and MS Columns (130-042-201, Miltenyi Biotec) according to manufacturer’s protocol. CD3ε + fraction, CD11B + fraction, and double negative fraction were resuspended in Qiazol and frozen at -80 °C until further processing was performed.

Tissue RNA isolation and TY1 qPCR assay

Tissues were homogenized in Qiazol using Bead Ruptor 12 (OMNI 194 International) with RNase free steel beads. Total RNA was extracted from mouse cells and tissue or plasma using miRNeasy mini kit (217,004, QIAGEN) or miRNeasy Serum/Plasma kit (217,184, QIAGEN) respectively according to the manufacturer’s protocol. RNA was resuspended in 35 µl RNase free water for the organs and bone marrow cells, 25 µl RNase free water for PBMCs or fractionated cells, and 20 µl RNase free water for plasma samples; RNA concentration and purity were determined using a NanoDrop Spectrophotometer (Thermo Scientific). cDNA was synthesized from RNA using miRCURY LNA RT Kit (339,340, QIAGEN) using UniSp6 RNA spike-in in each reaction, according to the manufacturer’s protocol. cDNA was diluted 1:60 and Real-time PCR was performed in duplicate using the following kits and primers: miRCURY SYBR Green PCR Kit (339,345, QIAGEN), miRCURY LNA miRNA PCR Assay (339,306, QIAGEN), miRCURY LNA miRNA Custom PCR Assay (339,317, QIAGEN). qPCR was done on QuantStudio 12 K Flex or QuantStudio 6 Flex system (Applied Biosystems) using the following protocol: initial activation step 2 min 95 °C, 2-step cycling (Denaturation 10 s 95 °C, Annealing 1 min 56 °C) for 40 cycles, then melt curve formation (Melting 15 s 95 °C, Annealing 1 min 60 °C, Deactivation 15 s 95 °C). Analysis was performed by calculating fold change as 2(−ΔΔCT) for TY1, normalizing TY1 values to UniSp6 values. Analysis for plasma was performed by normalizing TY1 values to U6 values. Results are presented in Log2FC.

Mouse two-hit model of heart failure with preserved ejection fraction

Eight to ten-week-old male C57BL/6 mice were obtained from Charles River laboratories. Mice were housed under controlled with a 12:12-h light–dark cycle and had unrestricted access to food (2916, Teklad for Control groups and D12492, Research Diet for High Fat Diet groups) and water. L-NAME (Nω-Nitro-L-arginine methyl ester hydrochloride, N5751, Millipore sigma 0.5 g/l) was added to drinking water after adjusting the pH to 7.4.

TY1 synthesis and quality control

Research-grade TY1 is synthesized commercially by Integrated DNA Technologies (IDT, Inc.) using a proprietary solid-state synthesis method followed by standard desalting. IDT performs quality control on all custom-manufactured RNA products using electrospray-ionization mass spectrometry, optical density (OD260), and melting temperature. Below is a table of taken from the specification sheets of various batches of synthesized RNA TY1 oligos.

Order ID

Extinction coefficient (L/mole•cm)

OD260

Tm (°C)

nmoles

mg

17,818,277

240,800

18.7

63.0

77.6

0.61

17,009,804

241,500

19.1

63.0

79.3

0.62

16,961,714

246,100

18.4

63.2

75

0.58

For transfection and IV formulation, the RNA oligo is mixed with Dharmafect (Perkin Elmer), a proprietary cationic lipid for the transfection of small RNAs. For oral formulation, the TY1-Dharmafect complex is further encased in a casein-chitosan micelle. Chitosan, casein, and acetic acid are all sourced from Sigma Aldrich at research grade.

Formulations of TY1 for intravenous and oral delivery

As described, TY1 was formulated using lipid transfection by admixture with DharmaFect® per manufacturer’s instructions (Perkin Elmer). This formulation sufficed for IV use (0.15 mg/Kg, retroorbitally once every two weeks, once a week, or twice a week). In some experiments as indicated, DharmaFect® was omitted to determine if it was required for IV efficacy. For oral administration, as described previously, TY1-DharmaFect® (at a dose of 0.2 mg/kg) is mixed with a casein solution. Micelles are formed by the addition of chitosan solution under acidic conditions. The suspension was incubated at room temperature one hour prior to administration by oral gavage (given twice weekly).

Echocardiography

Cardiac function and morphology were assessed under general anesthesia by transthoracic echocardiography using Vevo 3100 (VisualSonics). Apical four-chamber views were performed for diastolic function measurements using pulsed-wave and tissue Doppler imaging at the level of the mitral valve. During echocardiography, body temperature of mice was controlled, and isoflurane was reduced to under 1.0% and adjusted to maintain a heart rate in the range of 420–470 bpm.

Western blot

Protein extracts from mouse tissue were prepared by lysis in RIPA buffer (89,900, Thermo Scientific) containing protease and phosphatase inhibitor (78,442, Thermo Scientific). Protein samples (normalized value between 10 and 30 μg) were separated for gel electrophoresis (NUPAGE 4%-12% Bis–Tris gel, NP0336 Thermo Fisher Scientific) and transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System, Bio-Rad. Proteins were detected with the following primary antibodies: p21 (ab109199, abcam) and GAPDH (3683S, Cell Signaling technology).

RNA isolation and quantitative PCR

Total RNA was extracted from mouse tissue using RNeasy plus kit (74,136, QIAGEN) and Maxtract High density (129,056, QIAGEN). cDNA was synthesized from RNA using High capacity cDNA reverse transcription kit (4,368,813, Applied Biosystems) according to the manufacturer’s protocol. Real-time PCR (QuantStudio 12 K Flex Real-Time PCR system; Thermo Fisher Scientific) was performed in triplicate using the following TaqMan Gene Expression Assay probes (Cat# 4,331,182; with corresponding Assay IDs);

Species

Gene

Assay ID

Mouse

P21

Mm04205640_g1

Differential gene expression analysis was done using the ddCt method.

RNA sequencing

Cell and tissue RNA samples were sequenced at the Cedars-Sinai Genomics Core as described previously[11]. Total RNA was analyzed using an Illumina NextSeq 500 platform.

Library preparation and sequencing

Total RNA samples were assessed for concentration using a Qubit fluorometer (ThermoFisher Scientific, Waltham, MA) and for quality using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Library construction was performed using the QIASeq Stranded RNA Library kit (Qiagen, Hilden, Germany) with QIAseq FastSelect—rRNA HMR Kit (Qiagen) for ribosomal RNA depletion. Library concentration was measured with a Qubit fluorometer and library size on a Bioanalyzer. Libraries were multiplexed and sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) using 75 bp single-end sequencing. On average, approximately 50 million reads were generated from each sample.

Data analysis

Raw sequencing data were demultiplexed and converted to fastq format by using bcl2fastq v2.20 (Illumina, San Diego, California). Then reads were aligned to the GRCm38 reference genome (http://www.gencodegenes.org) using STAR (version 2.6.1)4 with default parameters. Gene expression was quantified by RSEM (version 1.2.28)5 to generate a raw count expression matrix with gene identities as rows and samples as columns. DESeq2 (version 1.26.0)6 was used to normalize the raw count expression and correct the batch effect.

Statistical analysis

Statistical parameters including the number of samples (n), descriptive statistics (mean and standard deviation), and significance are reported in the figures and figure legends. Differences between groups were examined for statistical significance using the Student’s t-tests or analysis of variance with Tukey’s post hoc test. Differences with p values < 0.05 were regarded as significant.