Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome–associated PTPN11 mutation
Talita M. Marin, … , Benjamin G. Neel, Maria I. Kontaridis
Talita M. Marin, … , Benjamin G. Neel, Maria I. Kontaridis
Published February 21, 2011
Citation Information: J Clin Invest. 2011;121(3):1026-1043. https://doi.org/10.1172/JCI44972.
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Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome–associated PTPN11 mutation

Abstract

LEOPARD syndrome (LS) is an autosomal dominant “RASopathy” that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11Y279C/+ (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.

Authors

Talita M. Marin, Kimberly Keith, Benjamin Davies, David A. Conner, Prajna Guha, Demetrios Kalaitzidis, Xue Wu, Jessica Lauriol, Bo Wang, Michael Bauer, Roderick Bronson, Kleber G. Franchini, Benjamin G. Neel, Maria I. Kontaridis

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Figure 1

Generation of mice expressing an inducible Ptpn11 Y279C allele.

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Generation of mice expressing an inducible Ptpn11 Y279C allele. (A) ...
(A) Structure of the Ptpn11 targeting locus and targeting construct region. Protein coding regions, LoxP sites, and Frt sites are shown. The arrows highlight the target positions of PCR primers used to identify homologous recombinants. Note that primers PLF and PRR are outside the region of homology. The expected amplicons are indicated with brackets and are specific to homologously recombined templates. LoxP sites are shown as black triangles; mutant LoxP511 sites are shown as white triangles. Their relative orientation is shown by the orientation of the triangles. FRT sites are shown as white ovals. Exons are indicated by rectangles. (B) Structure of locus after Flp-mediated recombination. (C) Structure of locus after Cre-mediated recombination. (D) Confirmation of correct targeting of ES cell clone 1F10 by PCR, revealing a 4.6-kb fragment on each correctly targeted side of the allele. M indicates molecular weight markers. (E) Germline transmission in F1 mice, as depicted by PCR, for the presence of the neo gene. (F) PCR detection strategy using LSGenofwd and LSR primers for detection of WT and LS/+ mice. (G) Restriction digest (HpyCH4V) of Ptpn11 exon 7 RT-PCR–derived fragment confirming expression of the mutant allele (full-length = 185 bp; fragment sizes = 100, 75 bp, 58 bp, 42 bp, and 10 bp [LS]; 100 bp, 75 bp, and 10 bp [WT]). Note that the 10-bp fragment probably comigrates with primer alone and/or contaminating RNA. PCR primers used are 721 forward/900 reverse. See Supplemental Table 1 for details on primers used throughout this figure. Fwd, forward; Rev, reverse.