Monosomy X in female mice influences the regional formation and augments the severity of angiotensin II-induced aortopathies

Objective: Turner Syndrome women (monosomy X) have high risk of aortopathies consistent with a role for sex chromosomes in disease development. We demonstrated that sex chromosomes influence regional development of angiotensin II (AngII)-induced aortopathies in mice. In this study, we determined if the number of X chromosomes regulates regional development of AngII-induced aortopathies. Approach and Results: We used females with varying numbers of X chromosomes (XXF or XOF) on an C57BL/6J (ascending aortopathies) or Ldlr-/- background (descending and abdominal aortopathies) compared to XY males (XYM). To induce aortopathies, mice were infused with AngII. XOF (C57BL/6J) exhibited larger percent increases in ascending aortic lumen diameters than AngII-infused XXF or XYM. AngII-infused XOF (Ldlr-/-) exhibited similar incidences of thoracic (XOF, 50%; XYM, 71%) and abdominal aortopathies (XOF, 83%; XYM, 71%) as XYM, which were greater than XXF (XXF, 0%). Abdominal aortic lumen diameters and maximal external diameters were similar between XOF and XYM but greater than XXF, and these effects persisted with extended AngII infusions. Larger aortic lumen diameters, abdominal aortopathy incidence (XXF, 20%; XOF, 75%), and maximal aneurysm diameters (XXF, 1.02 ± 0.17; XOF, 1.96 ± 0.32 mm; P=0.027) persisted in ovariectomized AngII-infused XOF mice. Data from RNA seq demonstrated that X chromosome genes that escape X-inactivation (histone lysine demethylases Kdm5c and Kdm6a), exhibited lower mRNA abundance in aortas of XOF than XXF (P=0.033 and 0.024 respectively). Conversely, DNA methylation was higher in aortas of XOF than XXF (P=0.038). Conclusion: The absence of a second X chromosome promotes diffuse AngII-induced aortopathies in females. Female C57BL/6J Ldlr-/- mice at 14-18 wks of age (n = 5 XX genotype, n =5 X0 genotype) were fed a Western (high fat- TD88137, Envigo) diet for 1 wk, and euthanized under ketamine/xylazine anesthesia (100:10mg/Kg). RNA was extracted from the thoracic aorta (defined as the segment extending from the aortic root to the diaphragm) and abdominal aorta (defined as the segment extending from the diaphragm to the iliac bifurcation) using a Maxwell Rapid Sample Concentrator (RSC) simply RNA Tissue kit (REF#AS1340, Promega, Madison, WI). Samples were provided to UK’s Genomics Core, who prepared the samples and performed sequencing. Briefly, RNA Integrity Number [RIN]: 8.97 ± 0.04 were of sufficient quality. Poly-A enriched mRNA underwent whole transcriptome sequencing (TruSeq library kit in an Illumina HiSeq 2500 High Output, single-end 100 bp reads). Pre-alignment quality control showed an average read depth of 37.4 ± 2.2 million reads per sample at a read length of 99.5 ± 0.1/ 100 base pairs, with an average read quality of 36.21 ± 0.01 phred (between 99.9 and 99.99% base identification accuracy). Reads were aligned to Genome Reference Consortium Mouse Build 38 (mm10) using the Spliced Transcripts Alignment to a Reference [STAR] (2.6.1) sequence aligning algorithm [1]. Post-alignment assembled results (Partek Flow, v 8.0) retained 30.9 ± 1.9 million unique reads per sample with a read quality of 36.30 ± 0.01 phred. The resulting counts were normalized to abundance measures with the trimmed-mean of M values (TMM) algorithm [2] in WebMev [3]. 19,296 rows of data were filtered to retain protein encoding genes for which at least 2 samples had TMM signal ≥ 1.5, resulting in 13,270 rows of prestatistically filtered data. The filtered data set underwent statistical analysis using LIMMA [4]. The False Discovery Rate (FDR) procedure [5], as modified by Storey [6] was used to estimate the error of multiple testing. Functional categorization was determined with the prestatistically filtered gene list as a background using DAVID bioinformatic tools’ functional overrepresentation clustering algorithm output [7] on the Gene Ontology annotation set [8, 9].