Screening of novel therapeutic targets and risk alleles for cardiac disorders

Ischemic heart disease is the leading cause of death globally, even though the prognosis of the disease has improved with advancing treatments. One particular phenomenon which could be treated more effectively is ischemia-reperfusion injury. While reperfusion, the return of blood flow to the heart after infarction, is necessary for the functionality of the heart, it is a stress itself and contributes to the myocardial damage. Ischemia-reperfusion injury could be prevented pharmacologically, and novel targets for this are needed. Heart failure is a life-threatening disorder with high death rates within years from its diagnosis. In addition to acquired causes, there is a strong heritable component to its risk. Only a few of genetic variants attributing to the risk are well characterized. This thesis aimed to identify novel therapeutic targets for ischemic heart disease, especially ischemia-reperfusion injury, and genetic markers associated with risk of heart failure. Large-scale screenings on cell and tissue samples, as well as genome¬wide screening on human genetic data from the FINRISK study cohort, were carried out. First, the family of receptor tyrosine kinases (RTK), a family of cell surface receptors involved in cellular survival and proliferation, was assessed in the context of ischemia-reperfusion injury. A member of the family, ROR1, was detected as a potential candidate for therapeutic targeting. In the second study, an unbiased screen exploiting a combination of drug and shRNA libraries identified another member of RTK family, EGFR, as a putative therapeutic target for ischemia-reperfusion. Gefitinib, an EGFR tyrosine kinase inhibitor clinically used as a cancer drug, was identified as a candidate for drug repurposing. The genetic association study on the FINRISK population identified an E140K variant of the TRIM55 gene to be associated with heart failure. TRIM55 E140K was also shown to negatively affect cardiomyocyte-specific functions in vitro and in vivo. Altogether, the findings demonstrate that novel candidates for therapeutic targets and genetic markers for cardiac disorders can be effectively identified by preclinical and genetic screening methods. ROR1 and EGFR were identified as potential targets for treatment of ischemia-reperfusion injury, and TRIM55 E140K as a genetic marker potentially modifying the risk for heart failure.Sydänsairauksien uusien lääkevaikutuskohteiden ja riskialleelien seulonta Iskeeminen sydänsairaus on maailmanlaajuisesti yleisin kuolinsyy. Vaikka taudin hoidossa on edistytty, siihen liittyy yhä merkittävää sairastavuutta ja kuolleisuutta. Iskemia-reperfuusiovaurio on tautiin liittyvä alihoidettu ilmiö. Reperfuusio eli verenkierron palautuminen sydämeen infarktin jälkeen on tarpeellista sydämen toimintakyvyn palautumiselle, mutta se on itsessään stressitekijä, joka tekee lisänsä sydänlihasvaurioon. Vauriota voidaan estää lääkehoitojen avulla, ja uusien lääkevaikutuskohteiden tunnistaminen tähän tarkoitukseen on tarpeen. Sydämen vajaatoiminta on hengenvaarallinen sairaus, joka johtaa usein kuolemaan jo muutamassa vuodessa. Hankinnaisten syiden lisäksi perinnölliset tekijät vaikuttavat vahvasti sen riskiin. Kuitenkin vain kourallinen riskiä lisääviä geneettisiä variantteja on kuvattu tarkasti. Tämän väitöskirjan tavoitteena oli tunnistaa uusia lääkevaikutuskohteita iskeemisen sydänsairauden, etenkin iskemia-reperfuusiovaurion, hoitoon, ja sydämen vajaatoiminnan riskiin liittyviä geneettisiä tekijöitä. Työssä hyödynnettiin seulontoja solu-ja kudosnäytteillä ja toteutettiin genominlaajuinen seulonta FINRISKI-tutkimuksen geenidatasta. Seuloimme iskemia-reperfuusiovaurioon liittyen reseptorityrosiinikinaasien (RTK) perhettä. RTK-perhe koostuu solunpinnan reseptoreista, jotka säätelevät solujen selviytymistä ja jakautumista. Perheen jäsen ROR1 tunnistettiin lupaavana vaurion hoitokohteena. Toisessa osatyössä tunnistettiin lääkeaine-ja shRNA-kirjastoja hyödyntävän seulonnan avulla toinen RTK-perheen jäsen, EGFR, mahdollisena lääkehoitokohteena. EGFR:ää estävä syöpälääke, tyrosiinikinaasi-inhibiittori gefitinibi, tunnistettiin potentiaalisena lääkkeenä, joka voidaan uudelleenkohdistaa iskemia-reperfuusiovaurion hoitoon. FINRISKI-aineiston geneettisessä assosiaatiotutkimuksessa havaittiin TRIM55¬geenin E140K-variantin liittyvän sydämen vajaatoiminnan riskiin. Variantin osoitettiin vaikuttavan negatiivisesti sydänsolujen toimintaan in vivo ja in vitro. Uusia lääkehoitokohteita ja geenitekijöitä voidaan tehokkaasti löytää prekliinisten ja geneettisten seulontametodien avulla. ROR1 ja EGFR tunnistettiin lupaavina iskemia-reperfuusiovaurion lääkehoitokohteina, ja TRIM55 E140K sydämen vajaatoiminnan riskiin vaikuttavana geneettisenä tekijänä

Farmakogenetiikka saapuu klinikkaan

English summaryPeer reviewe

Combined genetic and chemical screens indicate protective potential for EGFR inhibition to cardiomyocytes under hypoxia

The return of blood flow to ischemic heart after myocardial infarction causes ischemia-reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia-reperfusion injury. Here we screened for targets for the treatment of ischemia-reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein-protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target

BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass,was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.Peer reviewe

Genetic and functional implications of an exonic TRIM55 variant in heart failure

Background: To tackle the missing heritability of sporadic heart failure, we screened for novel heart failure associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo. Methods and results: Heart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/- cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wild type genotype or overexpression of WT TRIM55, respectively. Conclusions: A previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.Peer reviewe

Genetic and functional implications of an exonic TRIM55 variant in heart failure

BackgroundTo tackle the missing heritability of sporadic heart failure, we screened for novel heart failure-associated genetic variants in the Finnish population and functionally characterized a novel variant in vitro and in vivo.Methods and resultsHeart failure-associated variants were screened in genotyping array data of the FINRISK study, consisting of 994 cases and 20,118 controls. Based on logistic regression analysis, a potentially damaging variant in TRIM55 (rs138811034), encoding an E140K variant, was selected for validations. In HL-1 cardiomyocytes, we used CRISPR/Cas9 technology to introduce the variant in the endogenous locus, and additionally TRIM55 wildtype or E140K was overexpressed from plasmid. Functional responses were profiled using whole-genome RNA sequencing, RT-PCR and Western analyses, cell viability and cell cycle assays and cell surface area measurements. In zebrafish embryos, cardiac contractility was measured using videomicroscopy after CRISPR-mediated knockout of trim55a or plasmid overexpression of TRIM55 WT or E140K. Genes related to muscle contraction and cardiac stress were highly regulated in Trim55 E140K/− cardiomyocytes. When compared to the WT/WT cells, the variant cells demonstrated reduced viability, significant hypertrophic response to isoproterenol, p21 protein overexpression and impaired cell cycle progression. In zebrafish embryos, the deletion of trim55a or overexpression of TRIM55 E140K reduced cardiac contractility as compared to embryos with wildtype genotype or overexpression of WT TRIM55, respectively.ConclusionsA previously uncharacterized TRIM55 E140K variant demonstrated a number of functional implications for cardiomyocyte functions in vitro and in vivo. These findings suggest a novel role for TRIM55 polymorphism in predisposing to heart failure.</div

Receptor tyrosine kinase profiling of ischemic heart identifies ROR1 as a potential therapeutic target

BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass,was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury

Farmakogenetiikka saapui klinikkaan - miten hyödyntää genomitietoa potilastyössä

Siirretty Doriast

Combined genetic and chemical screens indicate protective potential for EGFR inhibition to cardiomyocytes under hypoxia

AbstractThe return of blood flow to ischemic heart after myocardial infarction causes ischemia–reperfusion injury. There is a clinical need for novel therapeutic targets to treat myocardial ischemia–reperfusion injury. Here we screened for targets for the treatment of ischemia–reperfusion injury using a combination of shRNA and drug library analyses in HL-1 mouse cardiomyocytes subjected to hypoxia and reoxygenation. The shRNA library included lentiviral constructs targeting 4625 genes and the drug library 689 chemical compounds approved by the Food and Drug Administration (FDA). Data were analyzed using protein–protein interaction and pathway analyses. EGFR inhibition was identified as a cardioprotective mechanism in both approaches. Inhibition of EGFR kinase activity with gefitinib improved cardiomyocyte viability in vitro. In addition, gefitinib preserved cardiac contractility in zebrafish embryos exposed to hypoxia-reoxygenation in vivo. These findings indicate that the EGFR inhibitor gefitinib is a potential candidate for further studies of repurposing the drug for the treatment of myocardial infarction.</jats:p