Using a unique conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2) in post-migratory neural crest (NC) via our Periostin-Cre line, we generated a novel fully penetrant mouse model of diminished sympathetic cardiac innervation and neonatal death. These mice are indistinguishable from control littermates at birth and exhibit no structural cardiac anomalies observable by histology. However, 12 days after birth, cKOs develop sinus bradycardia as observed via electrocardiogram (ECG) and 100% of cKOs die by 3 weeks. The sinus bradycardia and prolonged PR intervals observed by ECG can predispose to arrhythmogenesis which may lead to the neonatal death observed in our Shp2 cKO mouse model. Shp2 is known to exert effects through many different signaling cascades and therefore plays diverse roles in many human diseases. Thus, we propose two aims to examine the role that Shp2 and downstream mediators play in our sympathetic innervation phenotype.
Aim 1 will test our hypothesis that disruption within the NGF-SHP2- ERK signaling pathway is directly responsible for the diminished sympathetic innervation of the heart during development and define the functional consequences of that defective innervation as it relates to lethality. We will accomplish this aim via combined functional, molecular, and histological evaluation designed to characterize the Shp2 cKO phenotype from in utero stages to death at 3 weeks of age. Since no gross phenotype is evident until the first week of life, we expect that our longitudinal study will allowus to sequentially describe the events leading up to death and identify the role(s) that Shp2 and downstream targets play in this dynamic process. Our preliminary data as well as previously published works indicate a prominent role for pERK as a key downstream element involved in establishing sympathetic innervation. Therefore, Aim 2 will examine the direct role of pERK in mediating the Shp2 cKO phenotype.
Aim 2 tests the hypothesis that lineage-specific restoration of Erk1/2 signaling will rescue the diminished sympathetic innervation phenotype. This will be accomplished by bypassing Shp2 to directly activate the downstream Erk1/2 pathway by expressing a constitutively active MEK1 (directly upstream of ERK) to replenish diminished pERK levels within the Shp2 lineage restricted cKO cells. Significantly, preliminary data demonstrates that Peri-Cre;caMEK1 mice on a wildtype background are hyperinnervated and induce both pERK and TH (sympathetic nerve marker) upregulation in vivo. Thus, we expect CAG-MEK1;Peri-Cre;Shp2f/f neonates will exhibit rescue of sympathetic innervation in the heart as assessed molecularly by TH levels via IHC and Western as well as functionally by HR measurements made by ECG and echocardiogram.

Public Health Relevance

Shp2 signaling plays an important role in a variety of human diseases, and yet the specific contributions of individual downstream targets have not been established in many cases. Our preliminary data reveal that when we knockout Shp2 in post-migratory neural crest (NC) lineages the resulting phenotype is diminished sympathetic innervation of the postnatal heart. We propose to elucidate the specific roles of downstream targets of Shp2 such as ERK1/2 in causing this severe phenotype to better understand the complex nature of Shp2 signaling as well as the pathogenesis of aberrant cardiac innervation and its contribution to neonatal lethality.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
4F30HL116106-03
Application #
8970716
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Meadows, Tawanna
Project Start
2013-12-01
Project End
2016-05-31
Budget Start
2015-12-01
Budget End
2016-05-31
Support Year
3
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Lajiness, Jacquelyn D; Conway, Simon J (2012) The dynamic role of cardiac fibroblasts in development and disease. J Cardiovasc Transl Res 5:739-48