S100A4 as a Target of the E3-Ligase Asb2β and Its Effect on Engineered Heart Tissue

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

The atypical kinase RIOK1 promotes tumor growth and invasive behavior

Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers

I-1-deficiency negatively impacts survival in a cardiomyopathy mouse model

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis. Current treatment is based on beta-adrenoceptor (AR) and calcium channel blockers. Since mice deficient of protein phosphatase-1 inhibitor-1 (I-1), an amplifier in beta-AR signalling, were protected from pathological adrenergic stimulation in vivo, we hypothesized that I-1 ablation could result in an improved outcome in a HCM mouse model. We crossed mice deficient of I-1 with homozygous myosin-binding protein C knock-out (Mybpc3 KO) mice exhibiting cardiac dilatation and reduced survival. Unexpectedly, survival time was shorter in double I-1/Mybpc3 KO than in single Mybpc3 KO mice. Longitudinal echocardiographic assessment revealed lower fractional area change, and higher diastolic left ventricular inner dimensions and end-diastolic volumes in Mybpc3 KO than in WT mice. In comparison to Mybpc3 KO, double I-1/Mybpc3 KO presented higher left ventricular end-diastolic volumes, inner dimensions and ventricular surface areas with increasing differences over time. Phosphorylation levels of PKA-downstream targets and mRNA levels of hypertrophic markers did not differ between I-1/Mybpc3 KO and single Mybpc3 KO mice, except a trend towards higher beta-myosin heavy chain levels in double I-1/Mybpc3 KO. The data indicate that interference with beta-AR signalling has no long-term benefit in this severe MYBPC3-related cardiomyopathy mouse model

S100A4 as a Target of the E3-Ligase Asb2β and Its Effect on Engineered Heart Tissue

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) Asb2 beta 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 Asb2 beta 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 Asb2 beta. 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

Ranolazine antagonizes catecholamine-induced dysfunction in isolated cardiomyocytes, but lacks long-term therapeutic effectsin vivoin a mouse model of hypertrophic cardiomyopathy

Aims Hypertrophic cardiomyopathy (HCM) is often accompanied by increased myofilament Ca2+ sensitivity and diastolic dysfunction. Recent findings indicate increased late Na+ current density in human HCM cardiomyocytes. Since ranolazine has the potential to decrease myofilament Ca2+ sensitivity and late Na+ current, we investigated its effects in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Methods and results Unloaded sarcomere shortening and Ca2+ transients were measured in KI and wild-type (WT) cardiomyocytes. Measurements were performed at baseline (1 Hz) and under increased workload (30 nM isoprenaline (ISO), 5 Hz) in the absence or presence of 10 mu M ranolazine. KI myocytes showed shorter diastolic sarcomere length at baseline, stronger inotropic response to ISO, and drastic drop of diastolic sarcomere length under increased workload. Ranolazine attenuated ISO responses in WT and KI cells and prevented workload-induced diastolic failure in KI. Late Na+ current density was diminished and insensitive to ranolazine in KI cardiomyocytes. Ca2+ sensitivity of skinned KI trabeculae was slightly decreased by ranolazine. Phosphorylation analysis of cAMP-dependent protein kinase A-target proteins and ISO concentration-response measurements on muscle strips indicated antagonism at beta-adrenoceptors with 10 mu M ranolazine shifting the ISO response by 0.6 log units. Six-month treatment with ranolazine (plasma level > 20 mu M) demonstrated a beta-blocking effect, but did not reverse cardiac hypertrophy or dysfunction in KI mice. Conclusion Ranolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na+ current, but by antagonizing beta-adrenergic stimulation and slightly desensitizing myofilaments to Ca2+. This effect did not translate in therapeutic efficacy in vivo

Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3 -targeted knock-in mice

International audienceExon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5–6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5þ6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5þ6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM

FHL2 expression and variants in hypertrophic cardiomyopathy

International audienceBased on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomy-opathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding den-sity were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynony-mous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes mark-edly down-regulated a-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal condi-tions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in Electronic supplementary material The online version of this article (T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine-or endo-thelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM

Evidence for FHL1 as a novel disease gene for isolated hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in>40 % of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known diseas