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. Author manuscript; available in PMC: 2020 Apr 23.
Published in final edited form as: Circulation. 2019 Apr 23;139(17):2073–2076. doi: 10.1161/CIRCULATIONAHA.118.037939

Figure 1. Interrogation of the MTSS1 locus.

Figure 1.

A, association of minor allele (T) at rs12541595 with MTSS1 expression in human LV (P=6×10–23 across all samples using an additive genetic model adjusting for presence/absence of heart failure; n=313). B, cis associations with LV MTSS1 expression (–log10 P-values using same model as in A; n=313). Variants within a putative enhancer (yellow) in linkage disequilibrium with rs12541595 shows strong associations. C, the enhancer region was cloned upstream of a green fluorescent protein reporter and transfected into zebrafish embryos to determine anatomical site of activity (green). Activity is specific to cardiac-muscle (white arrow) and skeletal-muscle (dorsal axis) 72 hour post fertilization. D, analysis of cardiac-restricted enhancer activity within excised zebrafish hearts demonstrates strong activity (green) in the ventricle (V) and limited activity in the atrium (A) and outflow tract (OFT). Hearts are counterstained for sarcomeric architecture (F-actin, red) and nuclei (blue). E, CRISPR-Cas9 enhancer deletion abolishes MTSS1 expression in WM266–4 cells (n=6 per group; Mann-Whitney U test P-value). F, Relative activity of major and minor enhancer haplotypes defined by rs12541595, rs35006907, and rs34866937. The minor enhancer haplotype, marked by the rs12541595 minor allele, shows reduced activity (n=6 per group; Mann-Whitney U test P-values). G, CRISPR interference targeting enhancer variants shows reduced activity with dCas9 positioned at rs35006907 using two different guide RNAs (n=3 per group; Mann-Whitney U test P-values). H, association of the MTSS1 rs12541595 minor allele (T) with cardiac traits in human populations (P-values by additive genetic models). I, immunoblot of murine myocardial protein extracts verifying successful knock-out of Mtss1. J, LV structure and function in Mtss1+/+ (n= 23; 8 female, 15 male) and Mtss1–/– (n=16; 10 female, 6 male) mice by echocardiography under isoflurane anesthesia (gender adjusted P-values). LVIDd, LV internal dimension in diastole; LVIDs, LV internal dimension in systole; FS, fractional shortening; LVM/BM, LV mass normalized to body mass; HR, heart rate.