Identification of novel sarcomeric modifiers of hypertrophy in hypertrophic cardiomyopathy using the yeast two-hybrid system

Todd, Carol (2013-03)

Thesis (MScMedSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) occurs when the cardiomyocytes in the left ventricle become enlarged by increasing in mass in response to haemodynamic pressure overload. This can either be attributed to a normal physiological response to exercise or can be the result of a maladaptive process or disease state, such as chronic hypertension. Hypertrophic cardiomyopathy (HCM) is the most common form of Mendelian-inherited cardiac disease. A defining characteristic thereof is primary LVH that occurs when there are no other hypertrophy-predisposing conditions present. Therefore, HCM provides a unique opportunity to study the molecular determinants of LVH in the context of a Mendelian disorder, instead of in more complex disorders such as hypertension. Over 1000 HCM-causing mutations in 19 genes have been identified thus far, most of them encoding sarcomeric proteins residing in the sarcomeric C-zone. However, for many HCM patients no disease-causing genes have been identified. Moreover, studies have shown phenotypic variation in presentation of disease in, as well as between, families in which the same HCM-causing mutation segregates. This has led many investigators to conclude that genetic modifiers of hypertrophy exist. The aim of the study was to identify novel plausible HCM-causing or modifier genes by searching for interactors of a known HCM-causing protein, namely titin. The hypothesis was that genes encoding proteins, which interact with proteins that are encoded by known HCM-causative genes, may also be considered HCM-causing or may modify the HCM phenotype. To this end, the aim was to identify novel interactors of the 11-domain super-repeat region of titin, which resides within the sarcomeric C-zone, using yeast two-hybrid analysis. Five putative interactors of the 11-domain super-repeat region of titin were identified in this study. These interactions were subsequently verified by colocalisation in H9C2 rat cardiomyocytes, providing further evidence for possible interactions between titin and these proteins. The putative interactor proteins of titin determined from the Y2H library screen were: filamin C (FLNC), phosphatidylethanolamine-binding protein 4 (PEBP4), heart-type fatty acid binding protein 3 (H-FABP3), myomesin 2 (MYOM2) and myomesin 1 (MYOM1). The FLNC gene could be a candidate for cardiac diseases, especially cardiomyopathies that are associated with hypertrophy or developmental defects. The putative interaction of titin and PEBP4 is speculated to be indicative of the formation of the interstitial fibrosis and myocyte disarray seen in HCM. Heart-type fatty acid-binding protein 3 has prognostic value to predict recurrent cardiac events. Its suggested interaction with titin is speculated to play a role in inhibiting its functional abilities. Myomesin 2 is jointly responsible, with MYOM1, for the formation of a head structure on one end of the titin string that connects the Z and M bands of the sarcomere. This is speculated to be linked to a developmental error with the result being a defect in sarcomeric structure formation, which could result in pathologies such as HCM. Therefore, these identified proteins could likely play a functional role in HCM due to their interactions with titin. This research could thus help with new insights into the further understanding of HCM patho-aetiology.

AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) ontstaan wanneer die kardiomyosiete in die linkerventrikel vergroot as gevolg van 'n verhoging in massa in reaksie op hemodinamiese drukoorlading. Dit kan toegeskryf word aan 'n normale fisiologiese respons op oefening of kan die gevolg wees van 'n wanaangepaste of siektetoestand, soos chroniese hipertensie. Hipertrofiese kardiomiopatie (HKM) is die mees algemene vorm van Mendeliese oorerflike hartsiekte. 'n Bepalende eienskap daarvan is primêre LVH, wat plaasvind wanneer daar geen ander hipertrofie-predisponerende voorwaardes teenwoordig is nie. Gevolglik bied HKM 'n unieke geleentheid om die molekulêre derterminante van LVH te bestudeer, in die konteks van 'n Mendeliese oorerflike siekte, in plaas van om dit in die meer komplekse siektes soos hoë bloeddruk te bestudeer. Meer as 1000 HKM-veroorsakende mutasies is tot dusver in 19 gene geïdentifiseer. Die meeste van hulle kodeer vir sarkomeriese proteïene wat in die C-sone voorkom. Egter, vir baie HKM-pasiënte is geen siekte-veroorsakende gene al geïdentifiseer nie. Daarbenewens het studies getoon dat variasie in fenotipiese aanbieding van die siekte in, sowel as tussen, families voorkom wat dieselfde HKM-veroorsakende mutasie het. Dit het daartoe gelei dat baie navorsers tot die gevolgtrekking gekom het dat genetiese wysigers van hipertrofie wel bestaan. Die doel van die studie was om nuwe moontlike HKM-veroorsakende of wysiger-gene te identifiseer deur te soek vir interaktors van 'n bekende HKM-veroorsakende proteïen, naamlik titin. Die hipotese was dat gene wat vir proteïene kodeer, wat in wisselwerking is met proteïene wat geïnkripteer word deur bekende HKM-veroorsakende gene, ook oorweeg kan word om HKM te veroorsaak. Dit kan ook die HKM fenotipe verander. Dus was die doel om nuwe interaktors van die 11-domein super-herhaalstreek van titin, soos gevind binne die sarkomeriese C-sone, te identifiseer deur middel van gis-twee-hibried-analise. Vyf vermeende interaktors van die 11-domein super-herhaalstreek van titin is in hierdie studie geïdentifiseer. Hierdie interaksies is later geverifieer met behulp van ko-lokalisering in H9C2-rotkardiomyosiete, wat verdere bewyse vir moontlike interaksies tussen titin en hierdie proteïene verskaf. Die vermeende interaktor-proteïene van titin wat bepaal is vanaf die gis-twee-hibried-biblioteeksifting was as volg: filamin C (FLNC), phosphatidylethanolamine-bindingsproteïen 4 (PEBP4), hart-tipe-vetsuur bindingsproteïen 3 (H-FABP3), myomesin 2 (MYOM2) en myomesin 1 (MYOM1). Die FLNC-geen kan 'n kandidaat vir kardiale siektes, veral kardiomiopatieë, wees wat geassosieer word met hipertrofie of ontwikkelingsafwykings. Die vermeende interaksie van titin en PEBP4 dui daarop om 'n aanduiding te wees vir die vorming van die interstisiële fibrose en miokardiale wanorde, soos gesien in HKM. Hart-tipe-vetsuur bindingsproteïen 3 het prognostiese waarde om herhalende kardiale gebeure te voorspel. Verder dui sy voorgestelde interaksie met titin moontlik daarop dat dit 'n rol kan speel in die inhibering van sy funksionele vermoëns. Myomesin 2 tesame met MYOM1 is verantwoordelik vir die vorming van 'n kopstruktuur aan die een kant van die titinstring wat dan die Z- en M-bande van die sarkomeer verbind. Daar word vermoed dat dit gekoppel is aan 'n ontwikkelingsfout, met die gevolg dat daar 'n defek is in sarkomeriese struktuurvorming, wat weer kan lei tot patologieë soos HKM.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79819
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