The identification of ligands of cardiac Connexin 45 (Cx45) and their possible association with the development of the cardiac conduction disorder, Progressive Familial Heart Block Type II (PFHBII)

Nxumalo, Nqobile (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Connexins are gap junction proteins which allow selective permeability of small metabolites and ions between cells. Three main cardiac isoforms (Cx40, Cx43 and Cx45) which have been extensively studied are unequally distributed throughout the heart, suggesting specific functional roles. Connexins 40, 43 and 45 null mice have been shown to develop cardiac abnormalities, including conduction disturbances similar to those of known human diseases. Interestingly, features of human myotonic dystrophy (DM) include cardiac conduction disturbances; the DM-causative gene encodes a protein kinase (DMPK) that is a ligand of the COOH-terminus of Cx43. This has lead to the suggestion that other unidentified Cx ligands may be involved in cardiac conduction, and, if defective, may cause conduction disease. It is proposed that such ligands may be involved in the pathophysiology of the conduction disease PFHB II. To date, most studies have focused on Cx43; hence the main aim of this study was to assess functional specificity of cardiac Cx45 to further understand its role in cardiac function and possibly in the development of cardiac conduction diseases. Yeast-2-hybrid technology was applied to identify putative Cx45 ligands; by constructing a bait clone encoding the Cx45 COOH-terminus domain and using it to screen a cardiac cDNA library in S. cerivisiae. Successive selection stages reduced the number of putative ligands from 371 to 25. Selected ligands were identified by sequence homology searches in Genbank databases and prioritised for further study based on likely biological relevance and subcellular localisation. The authenticity of putative protein interactions was further assessed by mammalian 2 hybrid analysis. The priortised ligands included three mitochondrial proteins (NADH dehydrogenase subunit IV, thioredoxin 2, and cytochrome C oxidase subunit I), and four cytoplasmic proteins (obscurin, myomegalin, CD63-antigen and SCF-apoptosis response protein 1) which bore biological relevance to cardiac function. In contrast, in another study (R. Keyser, 2007, MSc Thesis – University of Stellenbosch) most Cx40 ligands were cytoplasmic proteins. In addition to Y2H and M2H, whole exome sequencing (WES) was conducted to identify PFHBII-causing mutations. Numerous filtering and variant prioritization tools were used to identify plausible PFHBII-causing variants in the PFHBII patients based on in silico predictions of their potential to cause disease and the function of these genes its situated in. These included two variants associated with obscurin, a Cx 45 ligand. This variant was was also identified in three of the patients which could be predicted to cause disease. Based on chromosome mapping, the Cx 45 ligands identified in the current study could be excluded from involvement in PFHB II. Obsurin, in spite its chromosomal location, is an exception due to its clinical association with dilated cardiomyopathy, a clinical symptom of PFHBII. The link between obscurin and dilated cardiomyopathy in PFHBII patients needs to be investigated further.

AFRIKAANSE OPSOMMING: Connexins (Cx) is gaping verbindings proteïene wat die selektiewe deurlaatbaarheid van klein metaboliete en ione tussen selle toelaat. Drie goed-bestudeerde hart iso-forme (Cxs40, 43 en 45) is oneweredig deur die hart versprei, wat dui op hul spesifieke funksionele rolle. Daar is aangetoon dat Cx uit-klop muise hartkwale ontwikkel, onder meer geleidingsongeruimdhede soortgelyk aan sekere bekende menssiektes. Dit is interessant om op te merk dat menslike miotoniese distrofie (DM) kenmerke soos hartgeleidingsongeruimdhede insluit; die DM-veroorsakende geen kodeer vir ‘n proteïen-kinase (DMPK) wat ‘n ligand van die karboksielterminus van Cx43 is. Hierdie observasie het gelei tot die voorstel dat ander, ongeïdentifiseerde Cx ligande betrokke kan wees by hartgeleiding en, indien defektief, geleidingsiekte mag veroorsaak. Daar is voorgestel dat sulke ligande betrokke kan wees by die patofisiologie van die geleidingssiekte PFHBII. Tot op hede het meeste studies gefokus op Cx43; derhalwe was die hoofdoel van hierdie studie om die funksionele spesifisiteit van hart Cx45 te bepaal om sodoende ‘n beter begrip van Cx45 se rol in hartfunksie, of moontlik in die ontwikkeling van hartgeleidings siektes, te vorm. Gis-2-hibried-tegnologie is aangewend om moontlike Cx45 ligande te identifiseer; ‘n aas-kloon wat kodeer vir die COOH-terminale domein van Cx45 is gekonstrueer en is gebruik om ‘n hart kDNS-biblioteek te fynkam in S. cerevisiae. Opeenvolgende selekteerstadiums het die hoeveelheid moontlike ligande verminder van 371 na 25. Geselekteerde ligande is geïdentifiseer deur sekwensie-homologie soektogte in Genbank databasisse en is geprioritiseer vir verdere studie op grond van hul waarskynlike biologiese relevansie en subsellulêre lokalisering. Die egtheid van moontlike proteïen-interaksies is verder bepaal deur soogdier 2-hibried analise. Die geprioritiseerde ligande sluit in drie mitokondriale proteïene (NADH dehidrogenase sub-eenheid IV, tioredoksien 2 en sitokroom C oksidase sub-eenheid I) en vier sitoplasmiese proteïene (obskurien, miomegalien, CD63-antigeen en SCF-apoptosis respons proteïen 1) wat biologies relevant is tot hartfunksie. Hierteenoor het ‘n studie (R. Keyser, 2007, MSc Thesis – University of Stellenbosch),) bevind dat meeste Cx40 ligande sitoplasmiese proteïene is. Behalwe vir Y2H en M2H, is wieele exome sequencing (WES) uitgevoer om PFHBII-veroorsakende mutasies te identifiseer. Verskeie filter- en variantprioriteringsinstrumente is gebruik om waarskynlike PFHBII-veroorsakende variante in die PFHBII-pasiënte te identifiseer gebaseer op siliko voorspellings van hul potensiaal om siekte te veroorsaak en die funksie van hierdie gene is geleë in. Dit sluit twee variante in wat verband hou met obscurine, 'n Cx 45 ligand. Hierdie variant was ook geïdentifiseer in drie van die pasiënte wat voorspel kan word om siekte te veroorsaak. Gebaseer op chromosoom kartering, kan die Cx 45 ligande wat in die huidige studie geïdentifiseer is, uitgesluit word van betrokkenheid by PFHB II. Obsurien, ten spyte van sy chromosomale ligging, is 'n uitsondering as gevolg van sy kliniese assosiasie met verwydde kardiomyopatie, 'n kliniese simptoom van PFHBII. Die verband tussen obskurine en verwydde kardiomyopatie in PFHBII pasiënte moet verder ondersoek word.

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