Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist-induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

Prime editing in mice with an engineered pegRNA.

CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3'-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells. Here, we show the inability of three-component CRISPR or conventional PE to incorporate a nonsynonymous substitution in the Capn2 gene, expected to disrupt a phosphorylation site (S50A) in CAPN2. In contrast, an epegRNA with the same protospacer correctly installed the desired edit in two founder mice, as evidenced by robust genotyping assays for the detection of subtle nucleotide substitutions. Long-read sequencing demonstrated sequence fidelity around the edited site as well as top-ranked distal off-target sites. Western blotting and histological analysis of lipopolysaccharide-treated lung tissue revealed a decrease in phosphorylation of CAPN2 and notable alleviation of inflammation, respectively. These results demonstrate the first successful use of an epegRNA for germline transmission in an animal model and provide a solution to targeting essential developmental genes that otherwise may be challenging to edit.

Generation and Comparative Analysis of an Itga8-CreER T2 Mouse with Preferential Activity in Vascular Smooth Muscle Cells.

All current smooth muscle cell (SMC) Cre mice similarly recombine floxed alleles in vascular and visceral SMCs. Here, we present an Itga8-CreER T2 knock-in mouse and compare its activity with a Myh11-CreER T2 mouse. Both Cre drivers demonstrate equivalent recombination in vascular SMCs. However, Myh11-CreER T2 mice, but not Itga8-CreER T2 mice, display high activity in visceral SMC-containing tissues such as intestine, show early tamoxifen-independent activity, and produce high levels of CreERT2 protein. Whereas Myh11-CreER T2 -mediated knockout of serum response factor (Srf) causes a lethal intestinal phenotype precluding analysis of the vasculature, loss of Srf with Itga8-CreER T2 (Srf Itga8 ) yields viable mice with no evidence of intestinal pathology. Male and female Srf Itga8 mice exhibit vascular contractile incompetence, and angiotensin II causes elevated blood pressure in wild type, but not Srf Itga8 , male mice. These findings establish the Itga8-CreER T2 mouse as an alternative to existing SMC Cre mice for unfettered phenotyping of vascular SMCs following selective gene loss.