Epigenetic alteration of smooth muscle cells regulates endothelin-dependent blood pressure and hypertensive arterial remodeling
Kevin D. Mangum, Qinmengge Li, Katherine Hartmann, Tyler M. Bauer, Sonya J. Wolf, James Shadiow, Jadie Y. Moon, Emily C. Barrett, Amrita D. Joshi, Gabriela Saldana de Jimenez, Zara Ahmed, Rachael Wasikowski, Kylie Boyer, Andrea T. Obi, Frank M. Davis, Lin Chang, Lam C. Tsoi, Johann Gudjonsson, Scott M. Damrauer, Katherine A. Gallagher
Kevin D. Mangum, Qinmengge Li, Katherine Hartmann, Tyler M. Bauer, Sonya J. Wolf, James Shadiow, Jadie Y. Moon, Emily C. Barrett, Amrita D. Joshi, Gabriela Saldana de Jimenez, Zara Ahmed, Rachael Wasikowski, Kylie Boyer, Andrea T. Obi, Frank M. Davis, Lin Chang, Lam C. Tsoi, Johann Gudjonsson, Scott M. Damrauer, Katherine A. Gallagher
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Research Article Cardiology Genetics

Epigenetic alteration of smooth muscle cells regulates endothelin-dependent blood pressure and hypertensive arterial remodeling

Abstract

Long-standing hypertension (HTN) affects multiple organs and leads to pathologic arterial remodeling, which is driven by smooth muscle cell (SMC) plasticity. To identify relevant genes regulating SMC function in HTN, we considered Genome Wide Association Studies (GWAS) of blood pressure, focusing on genes encoding epigenetic enzymes, which control SMC fate in cardiovascular disease. Using statistical fine mapping of the KDM6 Jumonji domain-containing protein D3 (JMJD3) locus, we found that rs62059712 is the most likely casual variant, with each major T allele copy associated with a 0.47 mmHg increase in systolic blood pressure. We show that the T allele decreased JMJD3 transcription in SMCs via decreased SP1 binding to the JMJD3 promoter. Using our unique SMC-specific Jmjd3-deficient murine model (Jmjd3fl/flMyh11CreERT), we show that loss of Jmjd3 in SMCs results in HTN due to decreased endothelin receptor B (EDNRB) expression and increased endothelin receptor A (EDNRA) expression. Importantly, the EDNRA antagonist BQ-123 reversed HTN after Jmjd3 deletion in vivo. Additionally, single-cell RNA-Seq (scRNA-Seq) of human arteries revealed a strong correlation between JMJD3 and EDNRB in SMCs. Further, JMJD3 is required for SMC-specific gene expression, and loss of JMJD3 in SMCs increased HTN-induced arterial remodeling. Our findings link a HTN-associated human DNA variant with regulation of SMC plasticity, revealing targets that may be used in personalized management of HTN.

Authors

Kevin D. Mangum, Qinmengge Li, Katherine Hartmann, Tyler M. Bauer, Sonya J. Wolf, James Shadiow, Jadie Y. Moon, Emily C. Barrett, Amrita D. Joshi, Gabriela Saldana de Jimenez, Zara Ahmed, Rachael Wasikowski, Kylie Boyer, Andrea T. Obi, Frank M. Davis, Lin Chang, Lam C. Tsoi, Johann Gudjonsson, Scott M. Damrauer, Katherine A. Gallagher

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Figure 1

rs62059712 minor C allele increases JMJD3 transcription via enhanced SP1 binding.

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rs62059712 minor C allele increases JMJD3 transcription via enhanced SP1...
(A) Schematic of the human JMJD3 gene, including gene structure, H3K27Ac, DHS, BP-associated SNPs, cloned fragments for luciferase assays, and vertebrate conservation (UCSC genome browser). (B) Luciferase results of DHS1 rs62059712 (T-major vs. C-minor) and (C) DHS2 rs74480102 (G-major vs. A-minor) in cultured primary human bronchial smooth muscle cells (HuBrSMCs). (D) qPCR for JMJD3 in HuSMCs with CRISPR/Cas9-mediated deletion of a 450 bp region encompassing rs62059712 within DHS1 (ΔDHS1) compared with unedited (WT) HuSMCs. (E) Sequence of rs62059712 T-major and C-minor sequences with SP1 consensus binding site underlined. (F) Western blot for SP1 after affinity purification using T versus C probes corresponding to rs62059712 SNP region incubated with human aortic smooth muscle cell (HuAoSMC) nuclear lysate. (G) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs compared with IgG negative control. (H) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs serum starved or treated with TGF-β. (I) Luciferase assays in mAoSMCs treated with siNTC and siSp1 siRNAs, then transfected with pGL3-DHS1-T and -C. (J) Luciferase assays in HuSMCs transfected with DHS1-C construct and then treated with TGF-β (20 ng/ml). (K) qPCR for Jmjd3 after NTC versus Sp1 knockdown in mAoSMCs serum starved for 16 hours. Data are represented as mean ± SEM. Results are representative of data from SMCs from 4–6 mice per group. n = 3 independent experiments. Two-tailed Student’s t test, *P < 0.05; **P < 0.01.