Natriuretic Peptide Receptor 2 Locus Contributes to Carotid Remodeling.

Background Carotid artery intima/media thickness (IMT) is a hallmark trait associated with future cardiovascular events. The goal of this study was to map new genes that regulate carotid IMT by genome-wide association. Methods and Results We induced IMT by ligation procedure of the left carotid artery in 30 inbred mouse strains. Histologic reconstruction revealed significant variation in left carotid artery intima, media, adventitia, external elastic lamina volumes, intima-to-media ratio, and (intima+media)/external elastic lamina percent ratio in inbred mice. The carotid remodeling trait was regulated by distinct genomic signatures with a dozen common single-nucleotide polymorphisms associated with left carotid artery intima volume, intima-to-media ratio, and (intima+media)/external elastic lamina percent ratio. Among genetic loci on mouse chromosomes 1, 4, and 12, there was natriuretic peptide receptor 2 (Npr2), a strong candidate gene. We observed that only male, not female, mice heterozygous for a targeted Npr2 deletion (Npr2+/-) exhibited defective carotid artery remodeling compared with Npr2 wild-type (Npr2+/+) littermates. Fibrosis in carotid IMT was significantly increased in Npr2+/- males compared with Npr2+/- females or Npr2+/+ mice. We also detected decreased Npr2 expression in human atherosclerotic plaques, similar to that seen in studies in Npr2+/- mice. Conclusions We found that components of carotid IMT were regulated by distinct genetic factors. We also showed a critical role for Npr2 in genetic regulation of vascular fibrosis associated with defective carotid remodeling.

CRISPR-Cas9-Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin-Brief Report.

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.