Abstract
Background: S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and furthermore examine the functional effects of S100A4 levels in a 3D model of engineered heart tissue (EHT).Methods and Results: S100A4 mRNA and protein levels were analyzed in different models of cardiac pathology via quantitative RT-PCR and Western blot, showing a higher S100A4 steady-state protein concentration in hearts of Mybpc3-knock-in (KI) hypertrophic cardiomyopathy (HCM) mice. COS-7 cells co-transfected with plasmids encoding mutant (MUT) Asb2β lacking the E3 ligase activity in combination with V5-tagged S100A4 plasmid presented higher S100A4-V5 protein steady-state concentrations than cells co-transfected with the Asb2β wild type (WT) plasmid. This effect was blunted by treatment with the specific proteasome inhibitor epoxomicin. Adeno-associated virus serotype 6 (AAV6)-mediated S100A4 overexpression in a 3D model of EHT did not affect contractile parameters. Immunofluorescence analysis showed a cytosolic and partly nuclear expression pattern of S100A4. Gene expression analysis in EHTs overexpressing S100A4-V5 showed markedly lower steady-state concentrations of genes involved in cardiac fibrosis and pathological cardiac hypertrophy.Conclusion: We showed that S100A4 protein level is higher in cardiac tissue of Mybpc3-KI HCM mice probably as a result of a lower degradation by the E3 ligase Asb2β. While an overexpression of S100A4 did not alter contractile parameters in EHTs, downstream gene expression analysis points toward modulation of signaling cascades involved in fibrosis and hypertrophy.
Highlights
S100 proteins are a group of small EF-hand calcium-binding proteins that do not have an enzymatic activity of their own but can influence a variety of fundamental molecular processes such as proliferation, migration, cell differentiation, and cardiac contractility (Donato, 2001; Zimmer et al, 2003; Donato et al, 2013)
S100A4 mRNA level was elevated in different animal models of cardiac diseases such as acute afterload enhancement and acute myocardial infarction in rats (Schneider et al, 2007) and mice (Schneider et al, 2007; Tamaki et al, 2013; Doroudgar et al, 2016)
Corresponding to previous findings of increased S100A4 mRNA in diseased heart tissue, we found significantly higher levels of S100A4 mRNA in hearts of 1-year-old LMNA K32mice compared to corresponding wild type (WT) controls (Figure 1A)
Summary
S100 proteins are a group of small EF-hand calcium-binding proteins that do not have an enzymatic activity of their own but can influence a variety of fundamental molecular processes such as proliferation, migration, cell differentiation, and cardiac contractility (Donato, 2001; Zimmer et al, 2003; Donato et al, 2013). Higher S100A4 protein levels were detected in human samples of hypertrophic cardiomyopathy (HCM) (Qi et al, 2016) and acute myocardial infarction (Gong et al, 2015) It has yet been unclear whether S100A4 is expressed within cardiac myocytes or instead taken up in a paracrine fashion, adding to the uncertainty regarding the role of S100A4 in the heart (Schneider et al, 2007). The goal of the current study was to analyze S100A4 expression in different models of cardiac pathology, investigate its degradation and gain a better understanding of its functional role after overexpression in a 3D model of engineered heart tissue (EHT). S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and examine the functional effects of S100A4 levels in a 3D model of engineered heart tissue (EHT)
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