Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in MiceNovelty and Significance

Mark C. Blaser, Kuiru Wei, Rachel L.E. Adams, Yu-Qing Zhou, Laura-lee Caruso, Zahra Mirzaei, Alan Y.-L. Lam, Richard K.K. Tam, Hangjun Zhang, Scott P. Heximer, R. Mark Henkelman, Craig A. Simmons
2017 Circulation Research  
Detailed Methods Unless otherwise noted, all materials were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). Mice All animal procedures were approved by the Animal Care Committees of the University of Toronto and The Centre for Phenogenomics, in accordance with guidelines of the Canadian Council on Animal Care. Heterozygous Npr2+/-(JAX STOCK Npr2 tm1Gar /J; #007658) and homozygous Ldlr-/-(JAX B6.129S7-Ldlr tm1Her /J; #002207) mutant mice were obtained from The Jackson Laboratory (JAX;
more » ... ar Harbor, ME) and housed in a pathogen-free facility. Npr2+/mice were obtained from JAX on a mixed 129/B6 background 1 at N2 to C57Bl/6 and were subsequently backcrossed with C57Bl/6J mice (JAX #000664) to N3 prior to experimental use. Npr2+/mice (N3 to C57Bl/6) were then bred to Ldlr-/mice to generate Npr2+/-;Ldlr-/mice (Npr2 tm1Gar ;Ldlr tm1Her ; N4 to C57Bl/6 at experimental use). Due to well-defined mortality and infertility in homozygous Npr2 mutants, 1 Npr2 mice were maintained via heterozygous sibling breeding, while Npr2;Ldlr mice were maintained by breeding Npr2+/-;Ldlr-/siblings together. Mice were genotyped for Npr2 by the standard polymerase chain reaction (PCR) genotyping protocol for stock number 007658 (version 1.0, The Jackson Laboratory). In brief, DNA was isolated from neonatal tail clips by alkaline lysis with 25 mM NaOH/0.2 mM EDTA at 98°C for 1 hour followed by neutralization with 40 mM Tris HCl. PCR was performed with JAX primers 9950 (common forward): 5' -CGGCTATCAGGCTCAGTTTT -3", 9951 (wild-type reverse): 5' -CAGCATTCTGGAGGCTAAGG -3", and oIMR7415 (mutant reverse): 5' -GCCAGAGGCCACTTGTGTAG -3". Bands were produced by the wild type allele (Npr2 + ) at 490 bp and by the mutant allele (Npr2 tm1Gar ) at 234 bp. Mice were genotyped for Ldlr by PCR using DNA from tail clips as above, with primers P15 (wild-type forward): 5' -AAGACGTGCTCCCAGGATGACTTC -3", P16 (common reverse): 5' -GTGCTCCTCATCTGACTTGTCCTTG -3", and P17 (mutant forward): 5' -CGCATTGTCTGAGTAGGTGTCATTC -3". Bands were produced by the wild type allele (Ldlr + ) at 377 bp and by the mutant allele (Ldlr tm1Her ) at 274 bp. Quantitative Real Time PCR Neonatal mouse hearts or lungs (postnatal day 4-5) were dissected and stored in RNAlater (Qiagen, Valencia, CA) at 4°C overnight. RNA was extracted using the RNeasy Fibrous Tissue Mini Kit, quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Mississauga, Canada), with reverse transcription using the Superscript III kit (Thermo Fisher Scientific). Transcript expression of wild type (Npr2 + ) mRNA (targeting within exons 3-7 of accession number: NM_173788.3, forward
doi:10.1161/circresaha.117.311194 pmid:29273600 fatcat:aursswkcffa5bcohjjsfrzz2ka