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CRISPR/Cas9 creation of a mouse lacking a unique motif on alpha globin demonstrates its critical role in regulating nitric oxide in vivo
Author(s) -
Filho Gilson Bras Broseghini,
Butcher Joshua T,
Keller Steven TC,
Poonam Sharma,
Kwangseok Hong,
DeLalio Leon J,
Wenhao Xu,
Padilha Alessandra Simão,
Columbus Linda,
Sonkusare Swapnil,
Isakson Brant E
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.685.3
Subject(s) - enos , in vivo , globin , biology , nitric oxide , medicine , microbiology and biotechnology , chemistry , endocrinology , hemoglobin , nitric oxide synthase , biochemistry , genetics
Alpha globin and eNOS form molecular complexes in microvascular endothelial cells that can regulate nitric oxide (NO) availability. On the alpha globin protein we have identified the amino acids 35–44 (FASFPTTKTY) as being essential for eNOS interaction. Using in silico prediction, we identified the 37–40 amino acid motif (SFPT) as being especially crucial for eNOS interaction, which we confirmed with purified protein binding assays. To confirm the importance of this motif in vivo, we used CRISPR/Cas9 to create a mouse with alpha globin missing amino acids 37–40 in the Hba1 gene. Whole genome sequencing confirmed the mutation and demonstrated a lack of any off‐target effects. We found that homozygous Hba1 Δ37–40 mice were not viable (0 mice born from 8 different heterozygous pairs; 16 litters) and were re‐absorbed in utero by approximately day E12. The lethal homozygous Hba1 Δ37–40 embryos had significantly dilated arteries and cardiac ventricles. For this reason we utilized Hba1 WT/Δ37–40 heterozygous animals; these mice had no difference in total alpha globin protein expression in arterioles and were viable. These mice had decreased blood pressure and significantly increased heart rates. Angiotensin II infusion only had a mild effect on blood pressure that was significantly lower than controls with Angiotensin II. Blood NO X levels were significantly higher than controls. In line with this observation, third‐order mesenteric arterioles from heterozygous Hba1 WT/Δ37–40 mice had significant increases in NO release when stimulated with the endothelial muscarinic agonists carbachol. We also detected a significant decrease in the amount of cGC present in smooth muscle cells. Our results demonstrate the importance of discreet motifs on alpha globin in regulating NO in endothelium in a unique CRISPR‐derived in vivo mouse model. Support or Funding Information Funding: HL088554,HL120840.