Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / The results presented in this thesis show the existence of TREK-1(Twik-RElated K+ channel; KCNK2), variant A and C of TREK-2 (KCNK10) and TRAAK (Twik-Related Arachidonic Acid-stimulated K+ channel; KCNK4) in human heart; the localization of TREK-1 and variants of TREK-2 on the membrane and in cytoplasmic areas of human cardiomyocyte; the notably high-level expression of TREK-1 in diseased human heart; the reverse expression of variant A and C of TREK-2 in normal and diseased human heart. These observations strongly indicate TREK channels play important roles in arrhythmia genesis and TREK-sensitive cardiac remodelling within the development of cardiac hypertrophy, ischemia cardiomyopathy and idiopathic dilated cardiomyopathy. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1248412 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2006

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: In hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder, hypertrophy is variable within and between families carrying the same causal mutation, suggesting a role for modifier genes. Associations between left ventricular hypertrophy and left ventricular pressure overload suggested that sequence variants in genes involved in the Renin-Angiotensin Aldosterone System (RAAS) may act as hypertrophy modifiers in HCM, but some of these studies may have been confounded by, amongst other things, lack of adjustment for hypertrophy covariates. To investigate this hypothesis, twenty one polymorphic loci spread across six genes (ACE1, AGT, AGTR1, CYP11B2, CMA and ACE2) of the RAAS were genotyped in 353 subjects from 22 South African HCM-families, in which founder mutations segregate. Genotypes were compared to 17 echocardiographically-derived hypertrophic indices of left ventricular wall thickness at 16 segments covering three longitudinal levels. Family-based association was performed by quantitative transmission disequilibrium testing (QTDT), and mixed effects models to analyse the X-linked gene ACE2, with concurrent adjustment for hypertrophy covariates (age, sex, systolic blood pressure (BP), diastolic BP, body surface area, heart rate and mutation status). Strong evidence of linkage in the absence of association was detected between polymorphisms at ACE1 and posterior and anterior wall thickness (PW and AW, respectively) at the papillary muscle level (pap) and apex level (apx). In single-locus analysis, statistically significant associations were generated between the CYP11B2 rs3097 polymorphism and PW at the mitral valve level (mit) and both PWpap and inferior wall thickness (IW)pap. Statistically significant associations were generated at three AGTR1 polymorphisms, namely, between rs2640539 and AWmit, rs 3772627 and anterior interventricular septum thickness at pap and rs5182 and both IWpap and AWapx. Furthermore, mixed effects model detected statistically significant association between the ACE2 rs879922 polymorphism and both posterior interventricular septum thickness and lateral wall thickness at mit in females only. These data indicate a role for RAAS gene variants, independent of hypertrophy covariates, in modifying the phenotypic expression of hypertrophy in HCM-affected individuals. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HCM), ‘n autosomale dominante afwyking, toon hoogs variërende hipertrofie binne en tussen families wat dieselfde siekte-veroorsakende mutasie het, hierdie dui op die moontlike betrokkenheid van geassosieerde modifiserende gene. Assosiasies tussen linker ventrikulêre hipertrofie en linker ventrikulêre druk-oorlading stel voor dat volgorde variasies in gene betrokke in die Renin-Angiotensin Aldosteroon Sisteem (RAAS) mag optree as hipertrofie modifiseerders in HCM. Sommige van hierdie soort studies is egter beperk omdat hulle nie gekompenseer het vir kovariante van hipertrofie nie. Om hierdie hipotese te ondersoek, is die genotipe bepaal by een-en-twintig polimorfiese lokusse, verspreid regoor ses RAAS gene (ACE1, AGT, AGTR1, CYP11B2, CMA and ACE2), in 353 kandidate vanuit 22 Suid-Afrikaanse HCM-families in wie stigter mutasies segregeer. Genotipes was vergelyk met 17 eggokardiografies afgeleide hipertrofiese indekse van linker ventrikulêre wanddikte by 16 segmente wat oor drie longitudinale vlakke strek. Familie-gebaseerde assosiasies was bestudeer deur kwantitatiewe transmissie disequilibrium toetsing (QTDT) en gemengde effek modelle om die X-gekoppelde geen ACE2 te analiseer, met gelyktydige kompensasie vir hipertrofie kovariate (ouderdom, geslag, sistoliese bloed druk (BP), diastoliese BP, liggaamsoppervlak area, hartritme en mutasie-status). Sterk indikasies van koppeling in die afwesigheid van assosiasie is waargeneem tussen ACE1 lokusse en posterior wanddikte (PW) asook anterior wanddikte (AW) by die papillêre spier vlak (pap) en die apeks vlak (apx). In enkel-lokus analises is statisties-betekenisvolle assosiasies gevind tussen die CYP11B2 rs3097 polimorfisme en PW by die mitraalklep vlak (mit) en beide die PWpap en inferior wanddikte (IW)pap. Statisties-betekenisvolle assosiasies was verder gevind by drie AGTR1 polimorfismes, naamlik, tussen rs2640539 polimorfisme en AWmit, rs3772627 en die anterior interventrikulêre septumdikte (aIVS) by die pap en rs5182 by beide die IWpap en AWapx. Gemengde-effek modelle het verder assosiasies aangetoon tussen die ACE2 rs879922 polimorfisme en die posterior interventrikulêre septumdikte en die laterale wanddikte by die mit, slegs in vrouens. Hierdie data dui op ‘n kovariaat-onafhanklike rol vir RAAS genetiese variante in die modifisering van die fenotipiese uitdrukking van hipertrofie in HCM-geaffekteerde individue.

Thesis (PhD)--University of Stellenbosch, 2004. / 426 Leaves printed single pages, preliminary pages i-xxiv and i-xxvii and 399 numberd pages. Includes bibliography. List of figures, List of tables, List of abbreviations. / ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease. The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and an increased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring in thirteen genes, have been identified. As the vast majority of HCM-causing mutations occur in components of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere. Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed that HCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausing mutations in the gamma two subunit of adenosine monophosphate activated protein kinase highlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports a hypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion. Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second most prevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomeric thick filament, where it acts as a regulator of thick filament structure and cardiac contractility. Although cMyBPC has been studied extensively, the mechanisms through which it fulfill these functions have remained elusive, largely due to a lack of a comprehensive understanding of its interactions with other sarcomeric components and its quaternary structure. The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in a panel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC in which HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individuals was screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)- based single-strand conformation polymorphism method, as well as restriction enzyme digestion, DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands of domains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandand library-assays were performed. Three novel and two previously described putative HCM-causing mutations were identified in MYBPC3. Data generated in this and other studies, however, suggest that two of these “mutations” are likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausing mutations. Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This finding identified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specific C7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 and domain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no known sarcomeric ligands of the N-terminal region of cMyBPC. Identification of the ligands of specific domains of cMyBPC led to the development of detailed models of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fully phosphorylated. The integration of these models into an existing model of thick filament quaternary structure allows new insights into the functioning of cMyBPC as a regulator of both thick filament structure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HKM) is ‘n outsosomaal dominante primêre hartsiekte. Die primêre kenmerke van HKM is linker ventrikulêre hipertrofie, miokardiale wanorde, fibrose en ‘n verhoogde risiko van skielike dood. Tot dusver is 260 HKM-veroorsakende mutasies in 13 gene geïdentifiseer. Aangesien die oorgrote meerderheid van HKM-veroorsakende mutasies in komponente van die kardiale sarkomeer voorkom, is HKM as ‘n siekte van die kardiale sarkomeer beskryf. Funksionele analise van HKM-veroorsakende mutasies in sarkomeriese protein-koderende gene het aan die lig gebring dat hierdie mutasies ‘n wye spektrum van gevolge op kontraktiele funksie het. Die ontdekking van HKM-veroorsakende mutasies in die gamma-twee subeenheid van adenosien monofosfaat-geaktiveerde proteïen kinase het die feit dat mutasies nie-sarkomeriese proteïene ook HKM kan veroorsaak onderstreep en ondersteun ‘n hipotese dat HKM-veroorsakende mutasies energievermorsing en energie uitputting tot gevolg het. Mutasies in die kardiale miosien-bindingsproteïen C (kMiBPC) geen (MYBPC3) is die tweede mees algemene oorsaak van HKM. kMiBPC is ‘n modulêre protein wat ‘n integrale deel van die sarkomeriese dik filament vorm, waar dit die struktuur van die dik filament en kardiale kontraktiliteit reguleer. Nieteenstaande die feit dat kMiBPC intensief bestudeer is, word die meganismes hoe hierdie funksies vervul word swak verstaan, grotendeels weens die afwesigheid van ‘n in diepte begrip van sy interaksies met ander komponente van die sarkomeer asook sy kwaternêre struktuur. Die doelstellings van hierdie studie was, eerstens, om MYBPC3 vir HKM-veroorsakende mutasies in ‘n paneel van HKM-geaffekteerde individue te deursoek en tweedens, om die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is te identifiseer.‘n Paneel van deoksiribonukleïensuur (DNS) monsters verkry van onverwante HKM-geaffekteerde individue is deursoek vir HKM-veroorsakende mutasies in MYBPC3, deur middel van die polimerase ketting-reaksie (PKR)-gebasseerde enkelstrand konformasie polimorfisme metode, sowel as restriksie ensiem vertering, DNS volgordebepaling en terugtranskripsie PKR tegnieke. Die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is, is geïdentifiseer deur middel van gis twee-hibried (G2H) kandidaat-ligand en biblioteek-siftings eksperimente. Drie onbeskryfde en twee voorheen beskryfde vermeende HKM-veroorsakende mutasies in MYPBC3 is geïdentifiseer. Data gegenereer in hierdie en ander studies dui daarop dat twee van hierdie “mutasies” eerder polimorfismes, of siekte-modifiserende faktore, as hoof-lokus HKMveroorsakende mutasies is. Onlangse bevindings het ‘n spesifieke interaksie tussend die C5 en C8 domeine van kMiBPC getoon. Hierdie bevindings het óf domein C6, óf C10, as kandidaat-ligande van domein C7 geïdentifiseer. G2H eksperimente het ‘n spesifieke interaksie tussen domains C7 en C10 getoon. Addisionele G2H eksperimente het ook C-zone titin as ‘n ligand van domein C7 sowel as domein C10 as ‘n ligand van domein C3 geïdentifiseer. Verdere G2H eksperimente het egter geen sarkomeriese ligande van die N-terminale gedeelte van kMiBPC geïdentifiseer nie. Die identifikasie van ligande van spesifieke domeins van kMiBPC het gelei tot die ontwikkelling van ‘n gedetaileerde model van kMiBPC kwaternêre struktuur wanneer kMiBPC beide ongefosforileerd en ten volle gefosforileerd is. Die intergrasie van hierdie modelle in bestaande modelle van dik filament kwaternêre struktuur werp nuwe lig op die funksionering van kMiBPC as ‘n reguleerder van beide dik filament struktuur en kardiale kontraktiliteit, sowel as die patofisiologie van kMiBPCgeassosieerde HKM.

Thesis (PhD (Biomedical Sciences))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac muscle disorder worldwide. The disease is characterized by extreme variability in the amount of hypertrophy that develops in different patients in response to sarcomeric protein-encoding gene mutations. The underlying defect in HCM is altered contractility of the sarcomere, primarily due to a defective sarcomere. Although numerous disease-causing genes have been identified for HCM, the factors that modify the amount of hypertrophy that develops in a given person are still unknown, it can be hypothesized that molecules that affect contractility can act as modifiers of the hypertrophic signal, and therefore influence the development of hypertrophy. Cardiac contractility is regulated by dynamic phosphorylation of proteins within the sarcomere by kinases such as cAMP-activated protein kinase A (PKA). Because speed and energy efficiency of cardiac muscle contraction has to be regulated in order to match the body’s needs, PKA is anchored close to its targets by A-kinase anchoring proteins (AKAPs) to enable spatio-temporal control of phosphorylation. Cardiac myosin binding protein-C (cMyBPC) and cardiac troponin I (cTNI) are HCM-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation. In a previous study, our laboratory identified a phosphodiesterase 4D-interacting protein as ligand of the N-terminal of cMyBPC via a yeast-two-hybrid (Y2H) cardiac library screen. This protein is also known in the literature as myomegalin (MMGL) isoform 4. Because phosphodiesterases and PKA are sometimes anchored by the same anchoring protein (AKAP), we hypothesized that MMGL isoform 4 acts as an AKAP by anchoring PKA to the phosphorylatable N-terminal of cMyBPC, and tested this by direct protein-protein interaction analyses in a yeast-based system. The MMGL cDNA was cloned into a bait vector, which was directly assessed for interaction with two distinct PKA regulatory-subunit preys. We further investigated the function of MMGL itself by using the Y2H bait to screen a cardiac cDNA library for novel MMGL interactors. All the prey clones identified via these Y2H analyses were subsequently sequenced to determine their identity. Based on their identities and subcellular localization, all putative Y2H MMGL-prey interactions were further assessed by additional, separate biochemical techniques viz. in vivo co-immunoprecipitation and in vivo 3D co-localization. The interactions between MMGL and its known PKA-phosphorylatable sarcomeric ligands were also investigated under conditions of β-adrenergic stress, by quantitatively measuring levels of co-localization before and upon addition of the β-adrenergic agonist isoproterenol. Furthermore, in order to evaluate the role of MMGL in cMyBPC phosphorylation, we assessed the expression of the different phosphorylation isoforms of cMyBPC, with and without β-adrenergic stimulation, in the context of siRNA-mediated MMGL knockdown. We further hypothesized that MMGL and PKA may serve as modifiers of the hypertrophic phenotype. This was tested by conducting a single nucleotide polymorphism (SNP) genotyping study of the genes encoding MMGL and the regulatory subunits of PKA viz. PDE4DIP, PRKAR1A and PRKAR2A, respectively, and comparing genotypic data with clinical phenotypic traits in a family-based association study. A panel of 353 individuals, including genetically and clinically affected as well as unaffected HCM individuals, was identified. All these individuals were screened for the presence or absence of all three South African HCM founder mutations, and blood was collected and DNA extracted. Genotypes at multiple SNPs in each gene were determined by subjecting the DNA samples to TaqMan® allelic discrimination technology. Statistical analysis using quantitative transmission disequilibrium testing (QTDT) was done in order to establish whether the difference in genotype in these three genes might have an effect on HCM phenotype. Our results showed that MMGL interacted with both PKA regulatory subunits as well as with other cardiac proteins that are PKA targets, including the sarcomeric protein cTNI. It was confirmed that two regulatory subunits of PKA (PRKAR1A and PRKAR2A), cardiac ankyrin repeat protein (CARP), copper metabolism gene MURR1 domain 4 (COMMD4), α-enolase (ENO1), β-enolase (ENO3) and cTNI are novel interactors of MMGL. In order to classify a protein as an AKAP, interaction with one of PKA’s regulatory subunits are prerequisite; MMGL showed interaction with both, confirming our hypothesis of MMGL being an AKAP, moreover, classifying it as a novel dual-specific sarcomeric AKAP. The identities of the AKAPs involved in the phosphorylation of cMyBPC and cTNI had been unknown; our results indicate that MMGL is the AKAP involved in the phosphorylation of both these PKA targets. We also showed that quantitatively more interaction occurs between MMGL and its sarcomeric ligands cMyBPC and cTNI under β-adrenergic stress. This implicates that under elevated cAMP levels, PKA is dynamically recruited by MMGL to the PKA targets cMyBPC and cTNI, presumably to mediate cardiac stress responses and leading to increased cardiac contractility. Furthermore, siRNA-mediated knockdown of MMGL lead to a reduction of cMyBPC levels under conditions of β-adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage. The SNP modifier study indicated that one variant in PDE4DIP (rs1664005) showed strong association with numerous clinical hypertrophy traits, including maximal interventricular septum thickness, as well as a number of other composite score traits. Two variants in PRKAR1A (rs11651687 and rs3785906) also showed strong association with some of these clinical hypertrophy traits. These results therefore suggest that variants in these two genes may act as modifiers of the HCM phenotype. In conclusion, this study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP and appears to be involved in the phosphorylation of cMyBPC as well as cTNI; hence MMGL is likely to be an important component in the regulation of cardiac contractility, and by extension, in the development of hypertrophy. This has further implications for understanding the patho-aetiology of mutations in cMyBPC and cTNI, and raises the question of whether MMGL might itself be considered a candidate HCM-causing factor. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HKM) is die mees algemeenste oorerflike hartspier siekte wêreldwyd. Die siekte word gekenmerk deur die uiterste variasie in die hoeveelheid hipertrofie wat in verskillende pasiënte ontwikkel as gevolg van sarkomeriese proteïen-koderende mutasies. Die onderliggende gebrek in HKM is geaffekteerde kontraktiliteit van die sarkomeer, hoofsaaklik as gevolg van ‘n gebrekkige sarkomeer. Alhoewel daar verskeie siekte-veroorsakende gene vir HKM geïdentifiseer is, bly die faktore wat die hoeveelheid hipertrofie in ‘n gegewe persoon modifiseer, onbekend. Daar kan dus gehipotiseer word dat molekules wat kontraktiliteit beïnvloed as modifiseerders van die hipertrofiese sein kan optree, en dus die ontwikkeling van hipertrofie beïnvloed. Hartspier kontraktiliteit word gereguleer deur die dinamiese fosforilasie van proteïene binne die sarkomeer deur kinases soos bv. cAMP-geaktiveerde proteïen kinase A (PKA). Die spoed en energie doeltreffendheid van hartspier kontraksie moet gereguleer word om by die liggaam se behoeftes aan te pas; dus word PKA naby sy teikens deur A-kinase anker proteïene (AKAPs) geanker om sodoende die beheer van fosforilasie beide in die korrekte area sowel as tydsduur te reguleer. Kardiale miosien-bindingsproteïen C (cMyBPC), asook kardiale troponien I (cTNI), is beide HKM-veroorsakende sarkomeriese proteïene wat kontraktiliteit beheer deur middel van fosforilasie deur PKA. In ‘n vorige studie in ons laboratorium is ‘n fosfodiesterase 4D-interaksie proteïen as bindingsgenoot van die N-terminaal van cMyBPC geïdentifiseer deur middel van ‘n gis-twee-hibried (G2H) kardiale biblioteek sifting. In die literatuur staan dié proteïen ook bekend as miomegalin (MMGL) isovorm 4. Fosfodiesterases en PKA word soms deur dieselfde anker proteïen (AKAP) geanker, dus het ons hipotiseer dat MMGL isovorm 4 ook as AKAP kan optree deur PKA aan die fosforileerbare N-terminaal van cMyBPC te anker. Die hipotese is getoets deur middel van direkte proteïen-proteïen interaksie analises in ‘n gis-gebaseerde sisteem. Die MMGL cDNA was in ‘n jag-plasmied gekloneer, wat toe direk ge-evalueer is vir interaksie met twee verskillende PKA regulatoriese-subeenheid prooi-plasmiede. Die funksie van MMGL self is verder ondersoek deur die G2H jag-plasmied te gebruik om ‘n kardiale cDNA biblioteek te sif, sodoende om nuwe MMGL bindingsgenote te identifiseer. Alle prooi klone wat deur dié G2H analises geïdentifiseer is, was daarna onderworpe aan DNA-volgorde bepaling om hul identiteit vas te stel. Afhangende van hul identiteite en subsellulêre lokalisering, is alle moontlike G2H MMGL-prooi interaksies verder ge-evalueer deur bykomende, afsonderlike biochemiese tegnieke viz. in vivo ko-immunopresipitasie asook in vivo 3D ko-lokalisering. Die interaksie tussen MMGL en sy bekende PKA-gefosforileerde sarkomeriese bindingsgenote was ook ondersoek onder kondisies van β-adrenergiese stres, deur kwantitatief die vlakke van ko-lokalisering te meet voor en na byvoeging van die β-adrenergiese agonis isoproterenol. Om verder die rol van MMGL in cMyBPC fosforilasie te ondersoek, het ons die uitdrukking van die verskillende fosforilasie isovorms van cMyBPC, met en sonder β-adrenergiese stimulasie, in die konteks van siRNA-bemiddelde MMGL uitklop, bepaal. Ons het verder hipotiseer dat MMGL en PKA as modifiseerders van die hipertrofiese fenotipe mag dien. Dit is getoets deur ‘n enkel nukleotied polimorfisme (SNP) genotiperings studie van die gene wat kodeer vir MMGL en die regulatoriese subeenhede van PKA, viz. PDE4DIP, PRKAR1A en PRKAR2A, en daarna dié genotipiese data met kliniese fenotipiese data te vergelyk in ‘n familie-gebaseerde assosiasie studie. ‘n Paneel van 353 individue wat genetiese en klinies geaffekteerde, sowel as ongeaffekteerde HKM individue insluit, was geidentifiseerd. Alle individue was ondersoek vir die aanwesigheid of afwesigheid van al drie Suid-Afrikaanse HKM stigter mutasies; bloedmonsters is gekollekteer en DNA uitgetrek. Die genotipes van veelvoudige SNPs in elke geen was bepaal deur die DNA monsters aan TaqMan® alleliese diskriminasie tegnologie met behulp van die ABI TaqMan® Validated SNP Genotyping Assays sisteem te analiseer. Statistiese analises deur middel van kwantitatiewe transmissie disekwilibrium toetse (QTDT) was gedoen om te bepaal of die verskil in genotipe in hierdie drie gene ‘n effek op HKM fenotipe het. Ons resultate het gewys dat MMGL interaksie toon met beide PKA regulatoriese subeenhede, sowel as met ander kardiale proteïene wat ook PKA teikens is, insluitende die sarkomeriese proteïen cTNI. Dit is bevestig dat die twee regulatoriese subeenhede van PKA (PRKAR1A en PRKAR2A), kardiale ankyrin herhaal proteïen (CARP), koper metabolisme geen MURR1 domein 4 (COMMD4), α-enolase (ENO1), β-enolase (ENO3) en cTNI almal nuwe bindingsgenote van MMGL is. ‘n Proteïen moet interaksie met een van die regulatoriese subeenhede van PKA toon om as AKAP geklassifiseer te word; MMGL het interaksie met beide getoon, wat ons hipotese bevestig dat MMGL ‘n AKAP is, asook dat MMGL as ‘n nuwe dubbel-spesifieke sarkomeriese AKAP geklassifiseer kan word. Die identiteite van die AKAPs wat betrokke is in die fosforilasie van cMyBPC en cTNI was onbekend tot nou; ons resultate wys dat MMGL die AKAP is wat betrokke is in die fosforilasie van beide hierdie PKA teikens. Ons wys ook dat daar kwantitatief meer interaksie plaasvind tussen MMGL en sy sarkomeriese bindingsgenote cMyBPC en cTNI onder kondisies van β-adrenergiese stres. Dit impliseer dat PKA dinamies verwerf word deur MMGL, onder verhoogde vlakke van cAMP, tot by die PKA teikens cMyBPC en cTNI, moontlik om kardiale stres-response te bemiddel en dus te lei na verhoogde spierkontraksie. Verder het siRNA-bemiddelde uitklop van MMGL gelei na ‘n vermindering van cMyBPC vlakke onder kondisies van β-adrenergiese stres. Dit dui aan dat fosforilasie deur middel van MMGL-bystand ‘n voorvereiste is vir beskerming van cMyBPC teen proteolise. Die SNP modifiseerder studie het gewys dat een variant in PDE4DIP (rs1664005) sterk assosiasie toon met verskeie kliniese hipertrofie kenmerke, insluitende maksimale interventrikulêre septum diktheid, sowel as ander van die saamgestelde telling kenmerke. Twee variante in PRKAR1A (rs11651687 en rs3785906) het ook sterk assosiasie getoon met verskeie van die kliniese hipertropfie kenmerke. Hierdie resultate dui dus daarop dat variante in hierdie twee gene as modifiseerders van die HKM fenotipe mag optree. In samevatting skryf hierdie studie ‘n nuwe funksie aan MMGL isovorm 4 toe: dit voldoen aan alle vereistes om as AKAP geklassifiseer te word en dit blyk of dit betrokke is in die fosforilasie van cMyBPC en cTNI; dus is MMGL waarskynlik ‘n belangrike komponent in die regulasie van hartspier sametrekking, en dus met uitbreiding, in die ontwikkeling van hipertrofie. Dit hou verdere implikasies in om die siekte-oorsaak van mutasies in cMyBPC en cTNI te verstaan, en stel die vraag of MMGL self as ‘n kandidaat HKM-veroorsakende geen kan beskou word. / Medical Research Council / University of Stellenbosch / Prof Paul van Helden

The regulation of cardiac-specific genes such as GATA-4 and its co-factor FOG-2 is paramount for normal heart development and function. Indeed, those mechanisms that regulate GATA-4 and FOG-2 function, such as nuclear transport and the post-translational modification of SUMOylation, are of critical importance for cardiogenesis. Therefore the aims of this study were to: i) elucidate the nuclear transport mechanisms of GATA-4; ii) determine the function of SUMOylation on the biological activity of both GATA-4 and FOG-2; and iii) examine how these mechanisms impact on the role of GATA-4 and FOG-2 in cardiac hypertrophy. Firstly, we characterised a non-classical nuclear localisation signal that mediates active import of GATA-4 in both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial residues within this region that interacted with importin ?? to mediate GATA-4 import via the non-classical import pathway. In addition, a cardiac myocyte-specific CRM1-dependent nuclear export signal, which consists of three essential leucine residues, was identified. We also investigated the residues of GATA-4 that are responsible for its DNAbinding activity and therefore transcriptional control of cardiac-specific genes. Secondly, we demonstrated that SUMOylation of both GATA-4 and FOG-2 is exclusively carried out by SUMO-2/3. Moreover, SUMOylation is involved in the nuclear localisation of both GATA-4 and FOG-2 in cardiac myocytes as well as the transcriptional regulation of cardiac-specific genes, such as cardiac troponin I. Finally, and perhaps most biologically significant, we showed that nuclear transport as well as SUMOylation of GATA-4 is imperative for the ability of GATA-4 to induce cardiac hypertrophy. Moreover, it was determined that FOG-2 SUMOylation is involved in the ability of FOG-2 to protect against cardiac hypertrophy. In conclusion, the current study provides detailed information on the nuclear transport pathways of GATA-4 as well as the SUMOylation of both GATA-4 and FOG-2 and the role these two mechanisms play in gene transcription and cardiac hypertrophy.

The effects of an 8-week swim training program and a 3-week detraining period on the size and protein composition of the heart of female Wistar rats were examined. The animals were separated into three groups: trained, detrained, and sedentary control. The training program, 6 hours/day, 5 days/week, resulted in a significantly larger dry heart weight, a normal collagen concentration, and a nonsignificantly greater total protein content. The greatest changes occurred between 4 and 6 weeks of training. The mild physiologic hypertrophy was assumed to be typical, and therefore simply an enlargement of a normal heart with proportional increases in myocyte size, and connective and vascular tissue hyperplasia. The detraining period produced a return of the heart mass to normal by the end of 2 weeks of detraining. The total protein content and collagen concentration remained elevated, though nonsignificant. The results indicate the detrained heart is composed of more connective, vascular, and metabolically active tissue, all of which may be important in the detrained heart’s response to increased work.Ball State UniversityMuncie, IN 47306

The regulation of cardiac-specific genes such as GATA-4 and its co-factor FOG-2 is paramount for normal heart development and function. Indeed, those mechanisms that regulate GATA-4 and FOG-2 function, such as nuclear transport and the post-translational modification of SUMOylation, are of critical importance for cardiogenesis. Therefore the aims of this study were to: i) elucidate the nuclear transport mechanisms of GATA-4; ii) determine the function of SUMOylation on the biological activity of both GATA-4 and FOG-2; and iii) examine how these mechanisms impact on the role of GATA-4 and FOG-2 in cardiac hypertrophy. Firstly, we characterised a non-classical nuclear localisation signal that mediates active import of GATA-4 in both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial residues within this region that interacted with importin ?? to mediate GATA-4 import via the non-classical import pathway. In addition, a cardiac myocyte-specific CRM1-dependent nuclear export signal, which consists of three essential leucine residues, was identified. We also investigated the residues of GATA-4 that are responsible for its DNAbinding activity and therefore transcriptional control of cardiac-specific genes. Secondly, we demonstrated that SUMOylation of both GATA-4 and FOG-2 is exclusively carried out by SUMO-2/3. Moreover, SUMOylation is involved in the nuclear localisation of both GATA-4 and FOG-2 in cardiac myocytes as well as the transcriptional regulation of cardiac-specific genes, such as cardiac troponin I. Finally, and perhaps most biologically significant, we showed that nuclear transport as well as SUMOylation of GATA-4 is imperative for the ability of GATA-4 to induce cardiac hypertrophy. Moreover, it was determined that FOG-2 SUMOylation is involved in the ability of FOG-2 to protect against cardiac hypertrophy. In conclusion, the current study provides detailed information on the nuclear transport pathways of GATA-4 as well as the SUMOylation of both GATA-4 and FOG-2 and the role these two mechanisms play in gene transcription and cardiac hypertrophy.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 2001. / Typescript. Bibliography: leaves 95-112.

O Cor pulmonale induzido pela administração intraperitoneal de monocrotalina é um dos modelos mais utilizados para estudar os efeitos dessa síndrome sobre o sistema cardiovascular. Essa síndrome é caracterizada por um desequilíbrio no estado redox celular e uma consequente alteração no imunoconteúdo de proteínas sinalizadoras para a hipertrofia e insuficiência cardíaca. Normalmente, o peróxido de hidrogênio caracteriza-se como a espécie reativa do oxigênio mais estável, e por isso, a molécula mais envolvida com a modulação dessa sinalização. O exercício físico aeróbio tem sido extensamente estudado devido ao fato de ser uma prática que altera o estado redox celular e, consequentemente, a sinalização nos cardiomiócitos. Dessa forma, o objetivo deste estudo foi testar a hipótese de que o exercício físico poderia modular o estado redox no ventrículo direito em animais tratados com monocrotalina e, ao mesmo tempo, provocar alterações na sinalização celular, estrutura e função cardíaca. Ratos Wistar machos com aproximadamente 180 gramas de massa corporal foram treinados por quatro semanas após a injeção de monocrotalina ou solução salina. Os grupos experimentais (n=6-9 animais/grupo) foram: controle sedentário (CS) – ratos sedentários que receberam uma dose única de solução salina (i.p), monocrotalina sedentário (MS) – ratos sedentários que receberam uma dose única de monocrotalina (i.p), controle treinado (CT) - ratos treinados que receberam uma dose única de solução salina (i.p) e monocrotalina treinado (MT) - ratos treinados que receberam uma dose única de monocrotalina (i.p). As medidas hemodinâmicas foram realizadas após 24 horas da última sessão de exercício físico aeróbio. Os dados de pressão diastólica final do ventrículo direito (PDFVD), pressão sistólica do ventrículo direito (PSVD), derivada pressão/tempo máxima (dP/dtmax) e derivada pressão/tempo mínima (dP/dtmin) foram analisados. Após a análise hemodinâmica, os ratos foram mortos por deslocamento cervical e seus órgãos (coração, pulmão e fígado) foram coletados para análises morfométricas, bioquímicas e moleculares. As concentrações de peróxido de hidrogênio e a relação GSH/GSSG foram medidas obtidas com o objetivo de verificar o estado redox nos diferentes grupos experimentais. A massa dos órgãos foi utilizada para analisar a hipertrofia cardíaca, congestão pulmonar e hepática. A massa ventricular direita foi utlizada em cortes histológicos e para a análise de proteínas relacionadas com o remodelamento cardíaco pela técnica de Western Blot. A administração de monocrotalina provocou hipertrofia do ventrículo direito, congestão pulmonar, aumento da PDFVD, da PSVD, da dP/dtmax e da dP/dtmin nos animais MS e MT. Além disso, nos animais MS e CT, notamos uma redução nas concentrações de peróxido de hidrogênio, sugerindo uma modificação do sistema de defesa antioxidante provocada pela monocrotalina e pelo exercício físico. Já quanto ao volume de vasos, no grupo MS tivemos uma diminuição dessa variável em relação ao seu controle (CS), sendo que, o exercício físico preveniu este efeito nos animais MT em relação ao grupo MS. O exercício físico aeróbio promoveu uma redução no volume intersticial e na espessura da túnica média da artéria pulmonar nos animais MT. A monocrotalina provocou uma redução na relação p-GSK-3β/GSK-3β no grupo MT, sendo que que o exercício físico foi capaz de provocar um aumento nessa relação. Dessa forma, acreditamos que o exercício físico aeróbio possa alterar a função de proteínas redox-sensíveis e, dessa forma, modular a hipertrofia cardíaca nos animais que receberam a monocrotalina. / The Cor pulmonale induced by monocrotaline intraperitoneal administration is one of the most widel used models to study the effects of this syndrome on the cardiovascular system. This syndrome is characterized by an imbalance in cellular redox state and a consequent change in immunocontent of signaling proteins for hypertrophy and heart failure. Mostly, hydrogen peroxide is characterized as the most stable reactive oxygen species, and therefore the most involved molecule in the modulation of these signaling pathways. The aerobic exercise has been extensively studied due to the fact that it is a practice that alters the cellular redox state and thus signaling in cardiomyocytes. Thus, the aim of this study was to test the hypothesis that exercise could modulate the redox state of the right ventricle in animals treated with monocrotaline and, at the same time, cause changes in cell signaling, structure and cardiac function. Male Wistar rats approximately 180 grams of body mass were trained for four weeks after injection of monocrotaline or saline. The experimental groups (n = 6-9 animals / group) were: sedentary control (SC) - sedentary rats that received a single dose of saline (ip), sedentary monocrotaline (SM) - sedentary rats that received a single dose of monocrotaline (ip), trained control (TC) - trained rats that received a single dose of saline (ip) and trained monocrotaline (TM) - trained rats that received a single dose of monocrotaline (ip). Hemodynamic measurements were performed 24 hours after the last session of aerobic exercise. Data for end-diastolic pressure of the right ventricle (EDPRV), right ventricular systolic pressure (RVSP), maximum derived from pressure/time (dP/ dtmax) and minimum derived pressure/time (dP/dtmin) were analyzed. After hemodynamic analysis, mice were killed by cervical dislocation and their organs (heart, lung and liver) were colected. The hydrogen peroxide concentrations and GSH/GSSG are measurements with the aim of verifying the redox state in different experimental groups. The organ weights was used to analyze cardiac hypertrophy, pulmonary and hepatic congestion. A portion of the right ventricular mass was utilized for histological examination and other part fot analyze proteins related to cardiac remodeling, by Western blot. Morphometric analysis were performed after the removal of organs. Administration of monocrotaline caused right ventricular hypertrophy, pulmonary congestion, increased EDPRV, RVSP, dP/dtmax and dP/dtmin in SM and TM animals. Furthermore, in animals and SM and TC, we visualized a reduction in the concentrations of hydrogen peroxide, suggesting a change in the antioxidant defense system caused by monocrotaline and by exercise. Already for the volume of vessels in the SM group, we note a decrease of this variable in relation to its control (SC). At the same time, TM animals had an increase in the volume of vessels in relation to the SM group, showing that exercise promotes an increase in this item. The aerobic exercise promoted a decrease in interstitial volume and thickness of the muscle layer of the pulmonary artery in animals TM. The monocrotaline caused a decrease in the p-GSK-3β/GSK-3β in the TM group, showed that exercise was able to cause an increase in this ratio. Thus, we believe that aerobic exercise can alter the function of signaling proteins and thereby modulate cardiac hypertrophy in animals that received monocrotaline.

O Cor pulmonale induzido pela administração intraperitoneal de monocrotalina é um dos modelos mais utilizados para estudar os efeitos dessa síndrome sobre o sistema cardiovascular. Essa síndrome é caracterizada por um desequilíbrio no estado redox celular e uma consequente alteração no imunoconteúdo de proteínas sinalizadoras para a hipertrofia e insuficiência cardíaca. Normalmente, o peróxido de hidrogênio caracteriza-se como a espécie reativa do oxigênio mais estável, e por isso, a molécula mais envolvida com a modulação dessa sinalização. O exercício físico aeróbio tem sido extensamente estudado devido ao fato de ser uma prática que altera o estado redox celular e, consequentemente, a sinalização nos cardiomiócitos. Dessa forma, o objetivo deste estudo foi testar a hipótese de que o exercício físico poderia modular o estado redox no ventrículo direito em animais tratados com monocrotalina e, ao mesmo tempo, provocar alterações na sinalização celular, estrutura e função cardíaca. Ratos Wistar machos com aproximadamente 180 gramas de massa corporal foram treinados por quatro semanas após a injeção de monocrotalina ou solução salina. Os grupos experimentais (n=6-9 animais/grupo) foram: controle sedentário (CS) – ratos sedentários que receberam uma dose única de solução salina (i.p), monocrotalina sedentário (MS) – ratos sedentários que receberam uma dose única de monocrotalina (i.p), controle treinado (CT) - ratos treinados que receberam uma dose única de solução salina (i.p) e monocrotalina treinado (MT) - ratos treinados que receberam uma dose única de monocrotalina (i.p). As medidas hemodinâmicas foram realizadas após 24 horas da última sessão de exercício físico aeróbio. Os dados de pressão diastólica final do ventrículo direito (PDFVD), pressão sistólica do ventrículo direito (PSVD), derivada pressão/tempo máxima (dP/dtmax) e derivada pressão/tempo mínima (dP/dtmin) foram analisados. Após a análise hemodinâmica, os ratos foram mortos por deslocamento cervical e seus órgãos (coração, pulmão e fígado) foram coletados para análises morfométricas, bioquímicas e moleculares. As concentrações de peróxido de hidrogênio e a relação GSH/GSSG foram medidas obtidas com o objetivo de verificar o estado redox nos diferentes grupos experimentais. A massa dos órgãos foi utilizada para analisar a hipertrofia cardíaca, congestão pulmonar e hepática. A massa ventricular direita foi utlizada em cortes histológicos e para a análise de proteínas relacionadas com o remodelamento cardíaco pela técnica de Western Blot. A administração de monocrotalina provocou hipertrofia do ventrículo direito, congestão pulmonar, aumento da PDFVD, da PSVD, da dP/dtmax e da dP/dtmin nos animais MS e MT. Além disso, nos animais MS e CT, notamos uma redução nas concentrações de peróxido de hidrogênio, sugerindo uma modificação do sistema de defesa antioxidante provocada pela monocrotalina e pelo exercício físico. Já quanto ao volume de vasos, no grupo MS tivemos uma diminuição dessa variável em relação ao seu controle (CS), sendo que, o exercício físico preveniu este efeito nos animais MT em relação ao grupo MS. O exercício físico aeróbio promoveu uma redução no volume intersticial e na espessura da túnica média da artéria pulmonar nos animais MT. A monocrotalina provocou uma redução na relação p-GSK-3β/GSK-3β no grupo MT, sendo que que o exercício físico foi capaz de provocar um aumento nessa relação. Dessa forma, acreditamos que o exercício físico aeróbio possa alterar a função de proteínas redox-sensíveis e, dessa forma, modular a hipertrofia cardíaca nos animais que receberam a monocrotalina. / The Cor pulmonale induced by monocrotaline intraperitoneal administration is one of the most widel used models to study the effects of this syndrome on the cardiovascular system. This syndrome is characterized by an imbalance in cellular redox state and a consequent change in immunocontent of signaling proteins for hypertrophy and heart failure. Mostly, hydrogen peroxide is characterized as the most stable reactive oxygen species, and therefore the most involved molecule in the modulation of these signaling pathways. The aerobic exercise has been extensively studied due to the fact that it is a practice that alters the cellular redox state and thus signaling in cardiomyocytes. Thus, the aim of this study was to test the hypothesis that exercise could modulate the redox state of the right ventricle in animals treated with monocrotaline and, at the same time, cause changes in cell signaling, structure and cardiac function. Male Wistar rats approximately 180 grams of body mass were trained for four weeks after injection of monocrotaline or saline. The experimental groups (n = 6-9 animals / group) were: sedentary control (SC) - sedentary rats that received a single dose of saline (ip), sedentary monocrotaline (SM) - sedentary rats that received a single dose of monocrotaline (ip), trained control (TC) - trained rats that received a single dose of saline (ip) and trained monocrotaline (TM) - trained rats that received a single dose of monocrotaline (ip). Hemodynamic measurements were performed 24 hours after the last session of aerobic exercise. Data for end-diastolic pressure of the right ventricle (EDPRV), right ventricular systolic pressure (RVSP), maximum derived from pressure/time (dP/ dtmax) and minimum derived pressure/time (dP/dtmin) were analyzed. After hemodynamic analysis, mice were killed by cervical dislocation and their organs (heart, lung and liver) were colected. The hydrogen peroxide concentrations and GSH/GSSG are measurements with the aim of verifying the redox state in different experimental groups. The organ weights was used to analyze cardiac hypertrophy, pulmonary and hepatic congestion. A portion of the right ventricular mass was utilized for histological examination and other part fot analyze proteins related to cardiac remodeling, by Western blot. Morphometric analysis were performed after the removal of organs. Administration of monocrotaline caused right ventricular hypertrophy, pulmonary congestion, increased EDPRV, RVSP, dP/dtmax and dP/dtmin in SM and TM animals. Furthermore, in animals and SM and TC, we visualized a reduction in the concentrations of hydrogen peroxide, suggesting a change in the antioxidant defense system caused by monocrotaline and by exercise. Already for the volume of vessels in the SM group, we note a decrease of this variable in relation to its control (SC). At the same time, TM animals had an increase in the volume of vessels in relation to the SM group, showing that exercise promotes an increase in this item. The aerobic exercise promoted a decrease in interstitial volume and thickness of the muscle layer of the pulmonary artery in animals TM. The monocrotaline caused a decrease in the p-GSK-3β/GSK-3β in the TM group, showed that exercise was able to cause an increase in this ratio. Thus, we believe that aerobic exercise can alter the function of signaling proteins and thereby modulate cardiac hypertrophy in animals that received monocrotaline.