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Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The worldwide escalation in the incidence of obesity and its strong association with insulin resistance, type 2 diabetes and the cardiovascular complications that accompany these disease states have elicited interest in the underlying mechanisms of these pathologies. Preliminary data generated in our laboratory showed that obesity is associated with abnormalities in the insulin signalling pathway. Specifically, we found a down-regulation of protein kinase B (PKB/Akt), which is known to mediate the metabolic effects of insulin. One of the downstream targets of PKB/Akt is glycogen synthase kinase-3 (GSK-3), which is inhibited by this phosphorylation. Detrimental effects of unopposed activity of GSK-3 have recently been described. This may play a pivotal role in some of the adverse consequences of insulin resistance in the heart. Hypothesis: Chronic inhibition of GSK-3 will induce myocardial hypertrophy or exacerbate the development of existing hypertrophy in a pre-diabetic model of diet induced obesity and insulin resistance. Objectives: (1) Assess the extent of the development of myocardial hypertrophy in a rat model of diet induced obesity (DIO) and insulin resistance. (2) Assess the effect of inhibition of GSK-3 protein on the development of myocardial hypertrophy. Methods: Two groups of age-matched male Wistar rats were used. Control animals received standard rat chow, while obese animals received a high caloric diet for 20 weeks. After 12 weeks, half of the animals in both groups received GSK-3 inhibitor treatment (CHIR118637, 30mg/kg/day, Novartis). At the end of 20 weeks, three series of experiments were conducted. (i) The animals were subjected to echocardiography to determine in vivo myocardial function, and biometric, metabolic and biochemical parameters were evaluated. (ii) The ability of the cardiomyocytes to accumulate deoxy-glucose after stimulation with insulin was determined, and (iii) the localization of key proteins was monitored using fluorescence microscopy and cell size was determined using light microscopy and flow activated cell sorter analysis. Results and discussion: The high caloric diet increased body weight (p<0.005) and intraperitoneal fat mass (p<0.01) when compared to controls. Complications associated with obesity, such as impaired glucose tolerance (p<0.05), hyperinsulinemia (p<0.0005) and an increased HOMA-IR index (p<0.01) were observed. Additionally, cardiomyocytes from the DIO animals had a significantly impaired response to insulin, specifically when 10nM (p<0.05) and 100nM (p<0.05) of insulin were used as stimulus. We also found a dysregulation in PKB/Akt, indicated by a down-regulation of phosphorylated PKB/Akt (p<0.01). The diet promoted the development of myocardial hypertrophy, since the ventricular weight (p<0.05) and ventricular weight to tibia length ratio were increased (p<0.01). Echocardiography experiments showed an increase in end diastolic diameter in the DIO animals (p<0.05). Additionally, there was an increase in the cardiomyocyte cell width in the DIO rats (p<0.0001) and a tendency for peri-nuclear localization of NFATc3. GSK-3 inhibition promoted the development of insulin resistance in control animals, as indicated by an increase in the body weight (p<0.05), serum insulin levels (p<0.01) and HOMA-IR index (p<0.01). In the DIO animals, the GSK-3 inhibitor treatment improved insulin resistance, as a decrease in serum insulin concentration (p<0.05) was observed. The cardiomyocytes from the treated DIO animals also showed an increase in glucose uptake (p<0.05) when stimulated with 100nM of insulin. The GSK-3 inhibitor promoted the development of myocardial hypertrophy in the control animals, indicated by an increase in ventricular weight (p<0.05) and cardiomyocyte cell width (p<0.0001), but did not exacerbate hypertrophy in the DIO animals. Conclusion: Both the high caloric diet and the GSK-3 inhibitor promoted the development of insulin resistance and myocardial hypertrophy in the rats. In the DIO animals the GSK-3 inhibitor treatment ameliorated insulin resistance and did not promote the further development of myocardial hypertrophy. / AFRIKAANSE OPSOMMING: Inleiding: Die huidige styging in vetsugtigheid en die sterk assosiasie daarvan met insulien weerstandigheid, tipe 2 diabetes en kardiovaskulêre komplikasies soos hipertrofie, het ‘n belangstelling in die onderliggende meganismes van hierdie siektetoestande ontlok. Voorlopige data uit ons laboratorium het getoon dat vetsug geassosieerd is met abnormaliteite in die insulien seintransduksie-pad soos byvoorbeeld ‘n afregulering van miokardiale proteïen kinase B (PKB/Akt), wat bekend is om die metaboliese effekte van insulien te medieer. Een van die proteïene wat deur PKB/Akt gefosforileer en daardeur geïnhibeer word, is glikogeen sintase kinase-3 (GSK-3). Negatiewe effekte van onge-opponeerde aktiwiteit van GSK-3 is beskryf en dit mag ‘n sleutelrol speel in sommige van die nadelige gevolge van insulien weerstandigheid in die hart. Hipotese: Chroniese onderdrukking van GSK-3 sal miokardiale hipertrofie ontlok of die bestaande hipertrofie in ‘n pre-diabetiese model van dieet-geïnduseerde vetsug en insulien weerstandigheid vererger. Doelstellings: (1) Om die omvang van die ontwikkeling van miokardiale hipertrofie in ‘n rotmodel van dieet-geïnduseerde vetsug te ondersoek en (2) om die effek van inhibisie van GSK-3 op die ontwikkeling van hipertrofie te ondersoek. Metodes: Ouderdomsgepaarde manlike Wistarrotte is in hierdie studie gebruik. Die diere is vir ‘n periode van 20 weke aan verskillende diëte onderwerp, naamlik standaard kommersiële rotkos vir die kontrole diere en ‘n hoë kalorie dieet vir die eksperimenteel vet diere (DIO). Helfte van elke groep diere is vir 8 weke met ‘n GSK-3 inhibitor behandel (CHIR118637, 30mg/kg/day, Novartis). Na die 20 weke is 3 eksperimentele reekse uitgevoer: (i) Die diere is eggokardiografies ondersoek om in vivo miokardiale funksie te bepaal en biometriese, metaboliese en biochemiese parameters is evalueer. (ii) Die vermoë van kardiomiosiete om de-oksiglukose na insulien stimulasie te akkumuleer, is bepaal, en (iii) die lokalisering van sleutelproteïene is met behulp van fluoressensie mikroskopie en die selgrootte met behulp van ligmikroskopie bepaal. Resultate en bespreking: Die hoë kalorie dieet het gepaard gegaan met ‘n beduidende toename in liggaamsgewig (p<0.005) en intraperitoneale vetmassa (p<0.01) in vergelyking met diere op die kontrole dieet. Newe-effekte geassosieerd met vetsug nl. onderdrukte glucose toleransie (p<0.05), hiperinsulinemie (p<0.0005) en ‘n verhoogde HOMA-IR index (p<0.01) is ook waargeneem. Daar was ook ‘n beduidend ingekorte respons van glukose opname deur kardiomiosiete van die vet diere na stimulasie met 10nM (p<0.05) en 100nM (p<0.05) insulien. Disregulering van PKB/Akt is gevind in die vorm van ‘n afregulering van die fosforilering van die proteïen (p<0.01). Die dieet het ook gelei tot die ontwikkeling van miokardiale hipertrofie aangesien die ventrikulêre gewig (p<0.05) asook die verhouding van die ventrikulêre gewig teenoor tibia lengte beduidend toegeneem het (p<0.01). Eggokardiografie het ‘n toename in ventrikulêre end-diastoliese dimensie in die DIO diere aangetoon (p<0.05). Tesame hiermee het die breedte van kardiomiosiete van die DIO diere toegeneem (p<0.0001) en daar was ook ‘n peri-nukluêre lokalisering van NFATc3. Behandeling van kontrole diere met ‘n GSK-3 inhibitor het insulienweerstandigheid ontlok soos afgelei uit ‘n verhoging in liggaamsgewig (p<0.05), serum insulien-vlakke (p<0.01) en die HOMA-IR waarde (p<0.01). In teenstelling het behandeling van die DIO diere met die GSK-3 inhibitor tot ‘n verbetering van insulienweerstandigheid gelei aangesien ‘n verlaging in serum insulien konsentrasies gevind is (p<0.05). Kardiomiosiete vanaf die behandelde DIO diere het ook ‘n verhoogde insulien-gestimuleerde glukose opname met 100nM insulien getoon (p<0.05). Behandeling met die GSK-3 inhibitor het die ontwikkeling van miokardiale hipertrofie in die kontrole diere teweeggebring, soos aangetoon deur ‘n toename in die ventrikulêre gewig (p<0.05) en ‘n groter selwydte in kardiomiosiete terwyl dit geen invloed op die bestaande hipertrofie van die vet diere gehad het nie. Gevolgtrekking: Die huidige studie het getoon dat die betrokke dieet asook behandeling met ‘n GSK-3 inhibitor insulienweerstandigheid sowel as die ontwikkelling van miokardiale hipertrofie in rotte ontlok. In die DIO diere het die behandeling met die GSK-3 inhibitor bloedglukose en insulien-vlakke verlaag en het nie hipertrofie vererger nie.

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 (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Physiological cardiac hypertrophy is characterized by the heart’s ability to increase mass in a reversible fashion without leading to heart failure. In contrast, pathological cardiac hypertrophy leads to the onset of heart failure. For this study, we investigated a model of physiological hypobaric hypoxia-mediated right ventricular (RV) hypertrophy (RVH). Here our hypothesis was that the hypertrophic response and associated changes triggered in the RV in response to chronic hypobaric hypoxia (CHH) (increased RV mass, function and respiratory capacity) are reversible. To test our hypothesis we exposed male Wistar rats to 3 weeks of CHH and thereafter removed the hypoxic stimulus for 3 and 6 weeks, respectively. Adaptation to 3 weeks of CHH increased the RV to left ventricle (LV) plus interventricular septum ratio by increased (223.5 ± 7.03 vs. 397.4 ± 29.8, p<0.001 versus normoxic controls), indicative of RVH. Hematocrit levels, RV systolic pressure and RV developed pressure (RVDP) were increased in parallel. Mitochondrial respiratory capacity was not significantly altered when using both carbohydrate and fatty acid oxidative substrates. After the 3-week normoxia recovery period, the RV to LV ratio was increased but to a lesser extent compared to the 3-week hypoxic time-point, i.e. 244.7 ± 11.2 vs. 349.64 ± 3.8, p<0.001 versus normoxic controls. Moreover, hematocrit levels were completely normalized. However, the RV systolic pressure and the functional adaptations, i.e. increased RVDP induced by CHH exposure still persisted in the 3-week recovery (3HRe) group. Also, pyruvate utilization was increased versus matched controls (p<0.04 vs. matched controls). Interestingly, we found that at the 6-week recovery time point functional parameters were largely normalized. However, the RV to LV ratio was still increased by 269.3 ± 14.03 vs. 333.9 ± 11.7, p<0.0001 vs. matched controls. Furthermore, palmitoylcarnitine utilization was increased (p<0.03 vs. matched controls). In conclusion, we found that exposure to CHH resulted in various adaptive physiological changes, i.e. enhanced hematocrit levels, increased RV mass linked to greater RV contractility and respiratory function. It is important to note that all these changes only occurred in the RV and not in the LV. Furthermore, when a normoxic recovery period (3 and 6 weeks, respectively) were initiated, these physiological parameters largely normalized. Together, the findings of this thesis clearly show the establishment of a reversible model of RV physiological hypertrophy. Our future work will focus on disrupting signaling pathways underlying this process and to thereafter ascertain whether reversibility is abolished. Elucidation of such targets should provide a unique opportunity to develop novel therapeutic agents to treat patients and thereby reduce the burden of heart disease.

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

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality, and is a feature of common diseases, such as hypertension and diabetes. It is therefore vital to understand the underlying mechanisms influencing its development. However, investigating the mechanisms underlying LVH in such complex disorders can be challenging. For this reason, many researchers have focused their attention on the autosomal dominant cardiac muscle disorder, hypertrophic cardiomyopathy (HCM), since it is considered a model disease in which to study the causal molecular factors underlying isolated cardiac hypertrophy. HCM is a heterogeneous disease that manifests with various phenotypes and clinical symptoms, even in families with the same genetic defects, suggesting that additional factors contribute to the disease phenotype. Despite the identification of several HCM-causing genes, the genetic factors that modify the extent of hypertrophy in HCM patients remain relatively unknown. The gene encoding the sarcomeric protein, cardiac myosin binding protein C, cMyBPC (MyBPC3) is one of the most frequently implicated genes in HCM. Identification of proteins that interact with cMyBPC has led to improved insights into the function of this protein and its role in cardiac hypertrophy. However, very little is known about another member of the myosin binding protein family, myosin binding protein H (MyBPH). Given the sequence homology and similarity in structure between cMyBPC and MyBPH, we propose that MyBPH, like cMyBPC, may play a critical role in the structure and functionality of the cardiac sarcomere and could therefore be involved in HCM pathogenesis. The present study aimed to identify MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis and to verify these interactions using three-dimensional (3D) co-localisation and co-immunoprecipitation (Co-IP) analyses. We further hypothesized that both MyBPH and cMyBPC may be involved in autophagy. To test this hypothesis, both MyBPH and cMyBPC were analysed for co-localisation with a marker for autophagy, LC3b-II. The role of MyBPH and cMyBPC in cardiac cell contractility were analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to β-adrenergic stress after individual and concurrent siRNA-mediated knockdown of MyBPH and cMyBPC. In the present study we employed a family-based genetic association analysis approach to investigate the contribution of genes encoding the novel MyBPH-interacting proteins in modifying the hypertrophy phenotype. This study investigated the hypertrophy modifying effects of 38 SNPs and haplotypes in four candidate HCM modifier genes, in 388 individuals from 27 HCM families, in which three unique South African HCM-causing founder mutations segregate. Yeast two-hybrid analysis identified three putative MyBPH-interacting proteins namely, cardiac β-myosin heavy chain (MYH7), cardiac α-actin (ACTC1) and the SUMO-conjugating enzyme UBC9 (UBC9). These interactions were verified using both 3D co-localisation and Co-IP analyses. Furthermore, MyBPH and cMyBPC were implicated in autophagy, since both these proteins were being recruited to the membrane of autophagosomes. In addition, a cardiac contractility assay demonstrated that the concurrent siRNA-mediated knockdown of MyBPH and cMyBPC resulted in a significant reduction in cardiomyocyte contractility, compared to individual protein and control knockdowns under conditions of β-adrenergic stress. These results indicated that MyBPH could compensate for cMyBPC, and vice versa, further confirming that both these proteins are required for efficient sarcomere contraction. Results from genetic association analyses found a number of SNPs and haplotypes that had a significant effect on HCM hypertrophy. Single SNP and haplotype analyses identified SNPs and haplotypes within genes encoding MyBPH, MYH7, ACTC1 and UBC9, which contribute to the extent of hypertrophy in HCM. In addition, we found that several variants and haplotypes had markedly different statistical significant effects in the presence of each of the three HCM founder mutations. The results of this study ascribe novel functions to MyBPH. Cardiac MyBPC and MyBPH play a critical role in sarcomere contraction and have been implicated in autophagy. This has further implications for understanding the patho-etiology of HCM-causing mutations in the gene encoding MyBPH and its interacting proteins. This is to our knowledge the first genetic association analysis to investigate the modifying effect of interactors of MyBPH, as indication of the risk for developing LVH in the context of HCM. Our findings suggest that the hypertrophic phenotype of HCM is modulated by the compound effect of a number of variants and haplotypes in MyBPH, and genes encoding protein interactors of MyBPH. These results provide a basis for future studies to investigate the risk profile of hypertrophy development in the context of HCM, which could consequently lead to improved risk stratification and patient management. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is 'n primêre risikofaktor vir kardiovaskulêre morbiditeit en mortaliteit asook 'n kenmerk van algemene siektes soos hipertensie en diabetes. Daarom is dit van kardinale belang om te verstaan wat die onderliggende meganismes is wat die ontwikkeling van LVH beïnvloed. Die ondersoek na die onderliggende meganismes wat lei tot LVH in sulke komplekse siektes is ‟n uitdaging. Om hierdie rede fokus baie navorsers hul aandag op die autosomaal dominante hartspier siekte, hipertrofiese kardiomiopatie (HKM), wat beskou word as 'n model siekte om die molekulêre oorsake onderliggend tot geïsoleerde kardiovaskulêre hipertrofie te ondersoek. HKM is 'n heterogene siekte wat manifesteer met verskeie fenotipes en kliniese simptome, selfs in families met dieselfde genetiese defekte, wat impliseer dat addisionele faktore bydra tot die modifisering van die siekte fenotipe. Ten spyte van die identifisering van verskeie HKM-versoorsakende gene, bly die genetiese faktore wat die mate van hipertrofie in HKM pasiente modifiseer relatief onbekend. Die geen wat kodeer vir die sarkomeriese proteïen, kardiale miosien-bindingsproteïen C (kMyBPC) is die algemeenste betrokke in HKM. Die identifisering van proteïene wat bind met kMyBPC het gelei tot verbeterde insigte tot die funksie van hierdie proteïen en die rol wat hierdie proteïen in hipertrofie speel. Ten spyte hiervan, is daar baie min inligting beskikbaar oor 'n ander lid van die miosien-bindingsproteïen families, miosien-bindingsproteïen H (MyBPH). Gegewe die ooreenstemming tussen die DNA basispaar-volgorde en struktuur tussen hierdie twee proteïene, stel ons voor dat MyBPH, net soos kMyBPC, 'n kritiese rol in die struktuur en funksie van die kardiale sarkomeer speel en kan daarom betrokke wees in die patogenese van HKM. Die huidige studie het beoog om proteïene wat met MyBPH bind te identifiseer deur die gebruik van gis-twee-hibried (G2H) kardiale biblioteek sifting en om hierdie interaksies te verifieer met behulp van drie-dimensionele (3D) ko-lokalisering en ko-immunopresipitasie eksperimente. Ons het verder gehipotiseer dat beide MyBPH and kMyBPC betrokke kan wees in outofagie. Om hierdie hipotese te toets is beide MyBPH en kMyBPC geanaliseer vir ko-lokalisering met 'n merker vir outofagie, LC3b-II. Verder het ons beplan om die rol van MyBPH en kMyBPC in kardiale spiersel-sametrekking te ondersoek deur die oppervlak van gedifferensieerde H9c2 rot kardiomiosiete in reaksie op β-adrenergiese stres te meet, na individuele en gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC. In hierdie studie het ons 'n familie-gebaseerde genetiese assosiasie analise benadering gevolg om vas te stel of MyBPH en gene wat kodeer vir die geverifieerde bindingsgenote van MyBPH bydra tot die modifisering van die hipertrofiese fenotipe. Die doel van hierdie studie was om die hipertrofiese effek van 38 enkel nukleotied polimorfismes (SNPs) en haplotipes in vier kandidaat HKM modifiserende gene in 388 individue van 27 HCM families te toets, waarin drie unieke Suid-Afrikaanse HKM-stigters mutasies segregeer. G2H analise het drie verneemde MyBPH bindingsgenote geidentifiseer, naamlik miosien (MYH7), alfa kardiale aktien (ACTC1) en die SUMO-konjugerende ensiem UBC9 (UBC9). Hierdie interaksies is geverifieer deur middel van 3D ko-lokalisering en ko-immunopresipitasie analises. Verder is bewys dat MyBPH en kMyBPC betrokke is in outofagie, siende dat beide proteïene gewerf is tot die membraan van die outofagosoom. 'n Kardiale sametrekkings eksperiment het gevind dat die gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC 'n merkwaardige vermindering in die kardiomiosiet sametrekking veroorsaak het in reaksie op β-adrenergiese stres kondisies, in vergelyking met die individuele proteïen en kontrole uitkloppings eksperimente. Hierdie resultate bevestig dat MyBPH vir kMyBPC kan instaan en ook andersom, wat verder bevestig dat beide proteïene benodig word vir effektiewe sarkomeer sametrekking. Resultate van die genetiese assosiasie studie het gevind dat 'n aantal SNPs en haplotipes 'n beduidende effek of HKM hipertrofie het. Enkel SNP en haplotipe analises in gene wat kodeer vir MyBPH, MYH7, ACTC1 en UBC9 het SNPs en haplotipes geidentifiseer wat bydra tot die omvang van hipertrofie in HKM. Verder het ons gevind dat sekere SNPs en haplotipes kenmerkend verskillende statisties beduidende effekte in die teenwoordigheid van elk van die drie HKM-stigter mutasies gehad het. Die resultate van hierdie studie skryf twee nuwe funksies aan MyBPH toe. Kardiale MyBPC en MyBPH speel 'n kritiese rol in sarkomeer sametrekking en is betrokke in outofagie. Hierdie resultate het verdere implikasies vir die verstaan van die pato-etiologie van die HKM-veroorsakende mutasies in die MyBPH, MYH7, ACTC1 en UBC9 gene. So vêr dit ons kennis strek is dit die eerste genetiese assosiasie studie wat die modifiserende effek van bindingsgenote van MyBPH ondersoek as risiko aanduiding vir die ontwikkeling van LVH in die konteks van HKM. Ons bevindinge bewys dat die hipertrofiese fenotipe van HKM gemoduleer word deur die komplekse effekte van SNPs en haplotipes in die MyBPH geen en gene wat MyBPH proteïen-bindingsgenote enkodeer. Hierdie resultate verskaf dus 'n basis vir toekomstige studies om die risiko profiel van hipertrofie ontwikkeling met betrekking tot HKM te ondersoek, wat gevolglik kan bydra tot die verbeterde risiko stratifikasie en pasiënte bestuur.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

The primary focus of this study was to determine basal levels of myogenic (MRF4, myogenin, MyoD), proteolytic (FOXO3A, atrogin-1, MuRF-1), myostatin, and mitochondrial (PGC-1α & Tfam) mRNA in elderly women before and after aerobic training. This approach was taken to gain insight into the molecular adaptations associated with our observed increases in whole muscle cross sectional area (CSA) (11%, p<0.05), knee extensor muscle function (25%, p<0.05) and aerobic capacity (30%, p<0.05) with training. Nine elderly women (71±2y) underwent muscle biopsies obtained from the vastus lateralis before and after 12-weeks of aerobic training on a cycle ergometer. Post training biopsy samples were acquired 48 hours after the last exercise session. Aerobic training reduced (p < 0.05) resting levels of MRF4 by 25% while myogenin showed a trend to decrease (p = 0.09) after training. FOXO3A expression was 27% lower (p < 0.05) while atrogin-1 and MuRF-1 were unaltered after training. Additionally, myostatin gene expression was decreased (p < 0.05) by 57% after training. Lastly, aerobic training did not alter PGC-1α or Tfam mRNA. These findings suggest that aerobic training alters basal transcript levels of growth related genes in skeletal muscle of older women. Further, the reductions in FOXO3A and myostatin indicate the aerobic training induced muscle hypertrophy in older women may be due to alterations in proteolytic machinery. / School of Physical Education, Sport, and Exercise Science

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