RESUMEN
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) Asb2ß 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 Asb2ß 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 Asb2ß. 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.
RESUMEN
Background: Hypertrophic cardiomyopathy (HCM) patients often present with diastolic dysfunction and a normal to supranormal systolic function. To counteract this hypercontractility, guideline therapies advocate treatment with beta-adrenoceptor and Ca2+ channel blockers. One well established pathomechanism for the hypercontractile phenotype frequently observed in HCM patients and several HCM mouse models is an increased myofilament Ca2+ sensitivity. Nebivolol, a commonly used beta-adrenoceptor antagonist, has been reported to lower maximal force development and myofilament Ca2+ sensitivity in rabbit and human heart tissues. The aim of this study was to evaluate the effect of nebivolol in cardiac muscle strips of an established HCM Mybpc3 mouse model. Furthermore, we investigated actions of nebivolol and epigallocatechin-gallate, which has been shown to desensitize myofilaments for Ca2+ in mouse and human HCM models, in cardiac strips of HCM patients with a mutation in the most frequently mutated HCM gene MYBPC3. Methods and Results: Nebivolol effects were tested on contractile parameters and force-Ca2+ relationship of skinned ventricular muscle strips isolated from Mybpc3-targeted knock-in (KI), wild-type (WT) mice and cardiac strips of three HCM patients with MYBPC3 mutations. At baseline, KI strips showed no difference in maximal force development compared to WT mouse heart strips. Neither 1 nor 10 µM nebivolol had an effect on maximal force development in both genotypes. 10 µM nebivolol induced myofilament Ca2+ desensitization in WT strips and to a greater extent in KI strips. Neither 1 nor 10 µM nebivolol had an effect on Ca2+ sensitivity in cardiac muscle strips of three HCM patients with MYBPC3 mutations, whereas epigallocatechin-gallate induced a right shift in the force-Ca2+ curve. Conclusion: Nebivolol induced a myofilament Ca2+ desensitization in both WT and KI strips, which was more pronounced in KI muscle strips. In human cardiac muscle strips of three HCM patients nebivolol had no effect on myofilament Ca2+ sensitivity.