DOI: 10.25338/b82p9d
title: Multi-omic, histopathologic, and clinicopathologic effects of once-weekly oral rapamycin in a naturally occurring feline model of hypertrophic cardiomyopathy: A pilot study

description: Hypertrophic cardiomyopathy (HCM) remains the single most common
 cardiomyopathy in cats, with a staggering prevalence as high as 15%. To
 date, little to no direct therapeutical intervention for HCM exists for
 veterinary patients. A previous study aimed to evaluate the effects of
 delayed-release (DR) rapamycin dosing in a client-owned population of
 subclinical, non-obstructive, HCM-affected cats and reported that the drug
 was well tolerated and resulted in beneficial LV remodeling. However, the
 precise effects of rapamycin in the hypertrophied myocardium remain
 unknown. Using a feline research colony with naturally occurring
 hereditary HCM (n = 9), we embarked on the first-ever pilot study to
 examine the tissue-, urine-, and plasma-level proteomic and tissue-level
 transcriptomic effects of an intermittent low dose (0.15 mg/kg) and high
 dose (0.30 mg/kg) of DR oral rapamycin once weekly. Rapamycin remained
 safe and well tolerated in cats receiving both doses for eight weeks.
 Following repeated weekly dosing, transcriptomic differences between the
 low- and high-dose groups support dose-responsive suppressive effects on
 myocardial hypertrophy and stimulatory effects on autophagy. Differences
 in the myocardial proteome between treated and control cats suggest
 potential anti-coagulant/-thrombotic, cellular remodeling, and metabolic
 effects of the drug. The results of this study closely recapitulate what
 is observed in the human literature, and the use of rapamycin in the
 clinical setting as the first therapeutic agent with disease-modifying
 effects on HCM remains promising. The results of this study establish the
 need for future validation efforts that investigate the fine-scale
 relationship between rapamycin treatment and the most compelling gene
 expression and protein abundance differences reported here.
description: See publication
description: # Multi-omic, histopathologic, and clinicopathologic effects of
 once-weekly oral rapamycin in a naturally occurring feline model of
 hypertrophic cardiomyopathy: A pilot study
 [https://doi.org/10.25338/B82P9D](https://doi.org/10.25338/B82P9D) ##
 Description of the data and file structure Project overview: Raw tissue
 transcriptomics (LV & IVS) and proteomics (LV, IVS, plasma, and
 urine) samples are provided. Nine colony cats affected with hypertrophic
 cardiomyopathy (HCM) were included in this study: three cats did not
 receive treatment, whereas, six cats were equally randomized and subject
 to either low- (0.15mg/kg) or high-dose (0.30mg/kg) rapamycin (sirolimus)
 treatment. Pre- and post-treatment plasma and urine proteomic files are
 provided for rapamycin-treated cats. A supporting table decoding raw
 proteomic file names is provided as ProteomicsFileNameKey.xlsx. Refer to
 publication for expanded study outlines and methods. Transcriptomic
 methods: Tissues of the LV posterior wall and IVS were collected for RNA
 extraction. Mature RNA transcripts were selected using the polyA selection
 method. High-quality mature RNA (RNA integrity number [RIN] = 7–10) was
 converted to cDNA and NEBNext Ultra II stranded library preparations were
 made. Samples were later used for paired-end 150 bp RNA-seq on the
 Illumina HiSeq platform with a targeted read depth of 50 million reads per
 sample (Azenta Life Sciences, South Plainfield, NJ, USA). The resulting
 raw RNA-sequencing files are provided here. Proteomic methods: LV and IVS
 tissues were cut into 0.30 cm pieces and placed in 1 mL of solubilization
 buffer (5% SDS, 50 mM triethyl ammonium bicarbonate, complete protease
 inhibitor cocktail [Roche], pH 7.5) inside MagNA Lyser tubes containing
 ceramic beads (Roche) and were later homogenized with a MagNA Lyser
 instrument (Roche) to achieve full mechanical disruption. Tissue
 homogenates, plasma, and precipitated urine protein samples were subjected
 to proteolysis by using suspension-trap (ProtiFi) devices. All operations
 of reduction, alkylation, tryptic digestion, and peptide clean-up were
 carried out within these devices. The eluted tryptic peptides were dried
 in a vacuum centrifuge and re-constituted in 0.1% trifluoroacetic acid.
 Peptides were injected into an LCMS system, where they were first trapped
 on a Thermo PepMap trap for desalting and separated on PepSep 100 μm × 25
 cm analytical columns. Chromatography was performed with a Dionex Ultimate
 3000 nUPLC at 600 nL/min. Peptides were eluted directly into a Tribrid
 Lumos Mass Spectrometer (ThermoFisher, Waltham, MA, USA) using a gradient
 mix of 0.1% formic acid in water and 0.1% formic acid in acetonitrile over
 a 60 min period. The samples were run in data-independent analysis (DIA)
 mode; mass spectra were acquired using a collision energy of 35, M1
 resolution of 120 k, and MS2 of 30 k, using overlapped isolation windows
 of 47 Da in a 360–1080 m/z range and 3 s cycle time. The resulting raw
 LCMS files are provided here. HCM-affected low-dose group: 5183-Sarabi
 5232-Mikey 5189-Cow HCM-affected high-dose group: 5148-Sokka
 5194-Buttercup 5272-Sam HCM-affected untreated: 479-Juan 482-Rockstar
 717-Dali