GATA4-targeted compound exhibits cardioprotective actions against doxorubicin-induced toxicity in vitro and in vivo : establishment of a chronic cardiotoxicity model using human iPSC-derived cardiomyocytes

Doxorubicin is a widely used anticancer drug that causes dose-related cardiotoxicity. The exact mechanisms of doxorubicin toxicity are still unclear, partly because most in vitro studies have evaluated the effects of short-term high-dose doxorubicin treatments. Here, we developed an in vitro model of long-term low-dose administration of doxorubicin utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Moreover, given that current strategies for prevention and management of doxorubicin-induced cardiotoxicity fail to prevent cancer patients developing heart failure, we also investigated whether the GATA4-targeted compound 3i-1000 has cardioprotective potential against doxorubicin toxicity both in vitro and in vivo. The final doxorubicin concentration used in the chronic toxicity model in vitro was chosen based on cell viability data evaluation. Exposure to doxorubicin at the concentrations of 1-3 mu M markedly reduced (60%) hiPSC-CM viability already within 48 h, while a 14-day treatment with 100 nM doxorubicin concentration induced only a modest 26% reduction in hiPCS-CM viability. Doxorubicin treatment also decreased DNA content in hiPSC-CMs. Interestingly, the compound 3i-1000 attenuated doxorubicin-induced increase in pro-B-type natriuretic peptide (proBNP) expression and caspase-3/7 activation in hiPSC-CMs. Moreover, treatment with 3i-1000 for 2 weeks (30 mg/kg/day, i.p.) inhibited doxorubicin cardiotoxicity by restoring left ventricular ejection fraction and fractional shortening in chronic in vivo rat model. In conclusion, the results demonstrate that long-term exposure of hiPSC-CMs can be utilized as an in vitro model of delayed doxorubicin-induced toxicity and provide in vitro and in vivo evidence that targeting GATA4 may be an effective strategy to counteract doxorubicin-induced cardiotoxicity.Peer reviewe

Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

Aims Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. Methods and Results A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. Conclusions The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.Peer reviewe

Discovery of Small Molecules Targeting the Synergy of Cardiac Transcription Factors GATA4 and NKX2-5

Transcription factors are pivotal regulators of gene transcription, and many diseases are associated with the deregulation of transcriptional networks. In the heart, the transcription factors GATA4 and NKX2-5 are required for cardiogenesis. GATA4 and NKX2-5 interact physically, and the activation of GATA4, in cooperation with NKX2-5, is essential for stretch-induced cardiomyocyte hypertrophy. Here, we report the identification of four small molecule families that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. A fragment-based screening, reporter gene assay, and pharmacophore search were utilized for the small molecule screening, identification, and optimization. The compounds modulated the hypertrophic agonist-induced cardiac gene expression. The most potent hit compound, N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide (3, IC50 = 3 mu M), exhibited no activity on the protein kinases involved in the regulation of GATA4 phosphorylation. The identified and chemically and biologically characterized active compound, and its derivatives may provide a novel class of small molecules for modulating heart regeneration.Peer reviewe

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4–NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4–NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II -mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4–NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.Peer reviewe

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4-NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II-mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro-and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4-NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries

Kuntoarvio ja parvekkeiden korjausvaihtoehtojen tarkastelu As Oy Kajaanin Tehdaskatu 6:een

Tämä insinöörityö on tehty 1960-luvulla rakennettuun kerrostaloon Kajaanissa. Työ käsittää kuntoarvion ja parvekkeiden korjausvaihtoehtojen tarkastelua. Kuntoarvio keskittyy rakennustekniseen arvioon. Uudistunut asunto-osakeyhtiölaki astui voimaan 1.7.2010. Laki velvoittaa asunto-osakeyhtiöitä esittämään kirjallisena seuraavan viiden vuoden korjaustarpeen. Lain tuoma velvoite on nostanut kysyntää kuntoarvioille. Kuntoarvio antaa pohjan tälle korjaus- ja kunnossapitosuunnitelmalle. Kuntoarvion tavoite on edistää kiinteistön kunnossapidon suunnitelmallista, turvallista, terveellistä ja kokonaistaloudellista kehitystä. Insinöörityössä perehdytään tutkimaan tarkemmin parvekkeiden korjaustarvetta ja verrataan eri korjausvaihtoehtoja. Parvekkeet ovat tuolle ajalle tyypillisiä avonaisia parvekkeita. Työssä selvitetään rakenteiden riskitekijät ja kartoittamaan vauriot.. Parvekkeiden eri korjausvaihtoehtojen vertailulla on tarkoitus löytää kiinteistön parvekkeille kannattava ja taloudellinen korjausratkaisu. Tämän ikäisellä kerrostalon monet rakennustekniset osat alkavat tulla elinkaarensa loppupäähän. Vaurioiden korjaamisella ja riskien vähentämisellä saadaan kiinteistölle lisää elinaikaa. Kuntoarviossa ehdotetaan korjaustoimenpiteitä, joilla on vaikutus rakenteellisesti, ulkonäöllisesti sekä laadullisesti kiinteistön elinkaareen. Korjaustoimenpiteet ovat myös energiataloudellisia. Esimerkiksi ikkunoiden ja parvekeovien uusimisella saadaan lämmityskustannuksiin säästöjä. Ajoissa suoritetut korjaustoimenpiteet pitävät yllä kiinteistön arvoa ja lisäävät asumismukavuutta.The target of this Bachelor’s thesis was a block of flats in Kajaani. The block of flats was built in the 1960s. The thesis includes a condition evaluation and comparison of balcony repair alternatives. The purpose the thesis is to promote real estate maintenance. The latest law on housing companies has been valid since July 2011. The law requires that housing companies make a five-year renovation plan. Because of the law condition evaluations have been in demand. Condition evaluation is based on the maintenance and repair plan. Condition evaluation is concentrated on the structural estimate. It was made in accordance with the construction information card. Condition evaluation included questions to occupants, visiting houses and a written report. In the thesis the condition of the balcony structures was studied in more detail and repair alternatives were searched for. Condition evaluation promotes the maintenance and repair plan. It has effects on the life cycle of the real estate considering structures, appearance and quality. The comparison of the different balcony repair alternatives was made to find a reasonable and economical alternative. Maintenance and repair actions implemented early enough keep the value of the real estate and increase comfort of living. Real estate maintenance is systematic, safe, healthy and economic development

Identification and characterization of small molecule modulators of cardiac hypertrophy

Abstract Heart failure (HF) is a clinical syndrome arising from diverse causes leading to abnormalities in cardiac structure and function and is characterized by typical symptoms, e.g., breathlessness, ankle swelling and fatigue. Pathophysiological stimuli (e.g., pressure overload, cardiac injury) activate several pathways and processes (e.g., neurohumoral mechanisms, inflammation, fibrosis, cell death) leading to the cardiac hypertrophy and remodeling, and finally cardiac dysfunction. Although improvements in treatments and their implementation have increased survival and reduced the hospitalization rate in patients with HF, cardiovascular disease is still the most common cause of death worldwide. Therefore, new approaches to improve outcomes in HF are needed. The aim of the present study was to identify novel small molecule modulators for the transcriptional synergy of cardiac hypertrophy associated transcription factors GATA4 and NKX2-5 by a phenotypic cell-based reporter assay. The most potent compound showed antihypertrophic actions in various in vitro assays and experimental models of pressure overload and myocardial ischemia in vivo. Micro- and nanoparticles (MNPs) are promising tools in different diagnostic and therapeutic applications in medicine. In the present study, the biosafety of the porous silicon MNPs for cardiac drug delivery was investigated and differences in biocompatibility between different types of MNPs were found. In summary, the results suggest that modulation of the key transcription factors involved in cardiac hypertrophy may provide the possibility for novel therapeutic interventions for HF. Moreover, porous silicon MNPs were shown to be applicable biomaterials for drug delivery into the heart.Tiivistelmä Sydämen vajaatoiminta on kliininen oireyhtymä, jonka tyypilliset oireet (mm. hengenahdistus, lihasväsymys ja nilkkaturvotus) ovat seurausta alentuneeseen sydämen minuuttitilavuuteen johtavista sydämen rakenteellisista ja/tai toiminnallisista muutoksista. Erilaiset patofysiologiset ärsykkeet (esim. paineylikuormitus ja sydänlihasvaurio) aktivoivat useita mekanismeja (esim. neurohumoraalinen aktivaatio, tulehdus, solukuolema, sidekudostuminen), jotka johtavat sydänlihaksen kasvuun ja uudelleen muovautumiseen, ja lopulta sydämen toimintahäiriöön. Vaikka sydämen vajaatoimintapotilaiden ennuste on parantunut ja sairaalahoitojaksojen määrä vähentynyt lääkehoitojen ansiosta, sydän- ja verenkiertoelinten sairaudet ovat edelleen yleisin kuolinsyy maailmanlaajuisesti. Siksi tarvitaan uusia lähestymistapoja sydämen vajaatoiminnan lääkehoidon tehostamiseksi. Tämän väitöstutkimuksen tavoitteena oli fenotyyppisen solupohjaisen reportterikokeen avulla tunnistaa uudenlaisia pienimolekyylisiä yhdisteitä muuntelemaan transkriptiotekijöiden GATA4 ja NKX2-5 sydänlihaksen kasvuun liittyvää transkriptionaalista yhteisvaikutusta. Voimakkaimmalla yhteisvaikutuksen estäjäyhdisteellä oli sydänlihaksen kasvua estäviä vaikutuksia useissa soluviljelymalleissa ja kokeellisissa paineylikuormituksen ja sydänlihaksen iskemian koe-eläinmalleissa. Mikro- ja nanokokoiset partikkelit ovat lupaavia erilaisissa lääketieteen diagnostisissa ja hoidollisissa sovelluksissa. Tässä väitöstutkimuksessa tutkittiin huokoisesta piistä valmistettujen mikro- ja nanopartikkelien bioturvallisuutta annosteltaessa yhdisteitä sydänkudokseen. Väitöstutkimuksen tulosten perusteella voidaan päätellä, että sydämen hypertrofiassa keskeisten transkriptiotekijöiden toiminnan muuntelu tarjoaa mahdollisuuden uudenlaisille sydämen vajaatoiminnan lääkehoidoille. Lisäksi havaittiin, että huokoinen pii soveltuu käytettäväksi yhdisteiden annosteluun sydänkudokseen

Nuclear receptor like structure of GATA4-NKX2-5 interaction.

<p>(A) DNA-binding domain of the estrogen receptor (Protein Data Bank: 1HCQ) showing R63, K66 and C67 mediating the interaction between the zinc fingers. (B) Homology model of the C-terminal zinc finger of GATA4 (green) and the homeodomain of NKX2-5 (blue) showing amino acids R190, K193 and C194 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144145#pone.0144145.s003" target="_blank">S3 File</a>). Structure similarity between GATA model and Estrogen receptor (1HCQ) is 1.81 RMSD (alpha-carbon). Due the sequence and structural fold differences between NKX2-5 and Estrogen receptor, RMSD values are not reported. Zinc atoms are represented as light blue spheres.</p

Homology models of transcription factor GATA4 bound to DNA.

<p>(A) Double GATA4 bound to a tandem GATA binding site. (B) C-terminal zinc finger of GATA4 bound to the major groove of DNA along with the N-terminal zinc finger bound to the minor groove. Amino acids N272 and R283 are highlighted as ball spheres and the location of amino acids H234, R264, M298, K299 and R319 are indicated. (C) Amino acid sequence of the mouse GATA4 zinc fingers. Darker shades indicate residues mutated in this study.</p

Effect of GATA4 mutations on ANP promoter activation in NIH3T3 cells.

<p>The -699bp ANP promoter was utilized to study the transcriptional activity of the GATA4 mutants on a validated GATA4 target gene. (A) Promoter activation by the indicated GATA4 proteins; activation by wtGATA4 is set at 1. (B) Effect of mutated GATA4 on synergy with NKX2-5 and (C) on synergy with KLF13. The data shown are from two independent experiments conducted in duplicate using five different DNA concentrations. Results (mean±SEM) are expressed as fold-changes compared to the control groups wtGATA4 in (A), wtGATA4-NKX2-5 in (B), and wtGATA4-KLF13 in (C). * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 vs. control group with Student's t-test.</p