Targeted Sprouty1 overexpression in cardiac myocytes does not alter myocardial remodeling or function

Abstract

The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (alpha-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male alpha-MHC-Spry1 mice compared to control mice. Ventricular function of alpha-MHC-Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.

Fig. 1

Characterization of the Sprouty1 transgenic mouse model. a The mouse construct design for cardiac-specific Spry1 overexpression as driven by the α-MHC promoter. b Southern blot analysis using a transgene-specific probe (solid bar in part (a)) confirmed control (CL) from transgenic (TG) animals. c PCR-based genotyping using transgene-specific primers (arrows in Fig. 1a denote primer location). d Immunoblot for Sprouty1 protein showed greater expression in transgenic (TG) mice versus control (CL) animals (n = 4)

Fig. 2

Effects of Spry1 overexpression on ERK1/2 phosphorylation in the heart. a Representative immunoblot of phospho-ERK1/2 and total-ERK1/2 in control (CL) and transgenic (TG) animals. b Densitometric analysis revealed no significant differences in the degree of phospho-ERK1 relative to total-ERK1 or phospho-ERK2 relative to total-ERK2 in control (CL) (mean ± SD, n = 6) versus transgenic (TG) (mean ± SD, n = 5) animals

Fig. 3

Effects of Spry1 overexpression on cardiac morphometry and cardiac myocyte morphology. a Control (CL) and transgenic (TG) mouse hearts showed no differences in gross cardiac morphometry (n = 3). b There were no significant differences in normalized heart mass of control (CL) versus transgenic (TG) animals (mean ± SD, n = 6). c–e Histologic analysis revealed no deviation in control (CL) and transgenic (TG) hearts with respect to: c cardiac myocyte morphology by H&E staining (×400) (n = 5), d cardiac myocyte size or cardiac vessel number (asterisks) by WGA-TRITC staining (×400) (n = 5), or e cardiac fibrosis by tri-chrome staining (×200) (n = 5)

Fig. 4

Effects of Spry1 overexpression on cardiac structure. a, b Control (CL) and transgenic (TG) hearts revealed no gross differences in chamber dimensions or wall thicknesses by H&E staining in either: a cross-section (n = 5) or b longitudinal-section (n = 5)