Double p52Shc/p46Shc Rat Knockout Demonstrates Severe Gait Abnormalities Accompanied by Dilated Cardiomyopathy

Abstract

The ubiquitously expressed adaptor protein Shc exists in three isoforms p46Shc, p52Shc, and p66Shc, which execute distinctly different actions in cells. The role of p46Shc is insufficiently studied, and the purpose of this study was to further investigate its functional significance. We developed unique rat mutants lacking p52Shc and p46Shc isoforms (p52Shc/46Shc-KO) and carried out histological analysis of skeletal and cardiac muscle of parental and genetically modified rats with impaired gait. p52Shc/46Shc-KO rats demonstrate severe functional abnormalities associated with impaired gait. Our analysis of p52Shc/46Shc-KO rat axons and myelin sheets in cross-sections of the sciatic nerve revealed the presence of significant anomalies. Based on the lack of skeletal muscle fiber atrophy and the presence of sciatic nerve abnormalities, we suggest that the impaired gait in p52Shc/46Shc-KO rats might be due to the sensory feedback from active muscle to the brain locomotor centers. The lack of dystrophin in some heart muscle fibers reflects damage due to dilated cardiomyopathy. Since rats with only p52Shc knockout do not display the phenotype of p52Shc/p46Shc-KO, abnormal locomotion is likely to be caused by p46Shc deletion. Our data suggest a previously unknown role of 46Shc actions and signaling in regulation of gait.

Keywords: Shc signaling; dystrophin; sciatic nerve.

Figure 1

Generation and characterization of rats deficient in Shc isoform expression. (A) Introduced deletions into Shc1 gene. The position of p52Shc start codon is indicated. (B) Protein expression of Shc isoforms in wild type (WT) and genetically modified rats. Expression of three Shc isoforms in renal tissues of wild type (+/+), heterozygote (+/−) and p52Shc/p46Shc knockout (−/−) rats is shown by Western blot with anti-Shc antibodies following SDS-PAGE. The position of Shc isoforms is indicated. Equal loading was verified by anti-β-actin antibodies.

Figure 2

Gait analysis (A) Representative examples of gait profile were recorded for p52Shc/p46Shc-KO, two left lanes, and WT, two right lanes. In WT, a consistent step and stride pattern of paw prints was established by the end of the trace, whereas, in knockout animals, lower abdomen and/or hind paw drag created smeared traces between shorter, more irregular front paw prints. (B) Summary analysis of total paw print area. Het, rats heterozygous for both p52Shc/p46Shc-KO. Individual data points are shown, with horizontal lines designating mean ± standard deviation; * p = 0.007.

Figure 3

Skeletal muscle mass of p52Shc/p46Shc-KO rats and WT littermates. Absolute unadjusted and total body weight adjusted weight of GTN, PLN, EDL, and Sol muscles is presented. *: indicates a significant difference from the WT definition.

Figure 4

Comparison of GTN muscle and diaphragm morphology and fiber type composition of p52Shc/p46Shc-KO rats and WT littermates. (A) Cross-sections of white (glycolytic) and red (oxidative) areas of GTN muscle stained with WGA-fluorescein (green staining) are presented. (B) Cross-sections of red (oxidative) areas of GTN muscle and diaphragm stained with anti-slow MHC antibody (red staining) and WGA-fluorescein (green staining) are presented. DAPI (blue staining) was used to visualize nuclei.

Figure 5

Analysis of sciatic nerve cross-sections of p52Shc/p46Shc-KO rats and WT littermates. (A) Cross-sections of sciatic nerve were stained with anti-NCAM antibody (red staining) and WGA-fluorescein (green staining). DAPI (blue staining) was used to visualize nuclei. White arrowheads indicate large myelin sheets that are missing axons in the cross-sections of p52Shc/p46Shc-KO rats. (B) Cross-sections of sciatic nerve were stained with anti-PMCA antibody (red staining) and WGA-fluorescein (green staining). White arrows indicate large myelin sheets that are missing axons in the cross sections of p52Shc/p46Shc-KO rats.

Figure 6

Analysis of cardiac muscle of p52Shc/p46Shc-KO rats and WT littermates. Cross-sections of heart were stained with anti-dystrophin antibody (red staining) and wheat germ agglutinin (WGA)-fluorescein (green staining). DAPI (blue staining) was used to visualize nuclei. Combined images have dystrophin, WGA-fluorescein and DAPI staining. Note large areas that are missing dystrophin staining in the heart section of p52Shc/p46Shc-KO rat.