Cardiovascular disease is the leading cause of death in the United States and the world. Progress in the development of new therapeutic strategies is hindered by shortcomings in our understanding of human myocardial pathophysiology and limitations in the ability of preclinical models to accurately predict successful clinical translation. The development of engineered models of the myocardium comprised of human cells derived from induced pluripotent stem cells has emerged as a promising strategy to overcome these problems. This dissertation builds on this work by developing a new engineered cardiac tissue platform and then utilizing it to investigate three distinct myocardial pathologies: (1) genetic restrictive cardiomyopathy, (2) autoimmune mediated myocardial injury, and (3) myocardial ischemia and reperfusion injury. Results from these studies provide new insights into therapeutic strategies for the first two conditions and describe substantial progress towards the creation of an innovative model of the third.

Submitted in fulfillment of the requirements for the Degree of Masters in Technology: Cardiology, Durban University of Technology, 2012. / Cardiomyopathies and their resultant heart failure (HF) remain a major cause of cardiovascular morbidity and mortality (Wood and Picard, 2004). Idiopathic dilated cardiomyopathy (IDCMO) is a primary myocardial disease of unknown cause, characterized by left ventricular (LV) or biventricular dilatation and impaired myocardial contractility. Dilated cardiomyopathy (DCMO), along with rheumatic heart disease and hypertension (HPT), is one of the leading causes of HF in Africa. In fact, in an epidemiology study of 884 patients in Soweto, IDCMO was the second major cause of HF. Thirty five percent of patients in the study, with HF, had IDCMO (Sliwa, Damasceno, Mayosi, 2005). Methodology: Patients referred to the cardiomyopathy (CMO) clinic at Chris Hani Baragwanath hospital, situated in the echocardiographic lab, were recruited, provided they satisfied the exclusion and inclusion criteria and were enrolled after obtaining voluntary informed consent. From May 2009 to September 2010, 70 patients with IDCMO were recruited for this trial. Patients with DCMO were identified by means of echocardiographic criteria which included a left ventricular ejection fraction (LVEF) of less than 45% and an end diastolic dimension (EDD) of greater than of 52 mm (2D in long parasternal axis). Results: In the present study the prevalence of left ventricular (LV) thrombus in patients with IDCMO was 18.6%. When using Univariate logistic regression, the only independent predictors of LV thrombus formation was LVEF and age. However, when multivariate logistic regression analysis was applied to the data, the only predictor with a significant association was age. The reason for this is not clear. It is postulated that perhaps younger patients have differences in the pathophysiology of their disease such as a greater smoldering inflammatory component which may therefore predispose them to thrombus formation. For example the presence of IL-6 may be important in the formation of LV clot in cases of LV dysfunction (Sosin, Bhatia, Davis, Lip, 2003). The association between LVEF and LV thrombus was borderline significant. Conclusion: The prevalence of LV thrombus formation in this cohort of patients with IDCMO was 18.6%. Echocardiographic parameters alone cannot predict which patients are more likely to develop thrombus formation. / National Research Foundation

Activation of A3 adenosine receptors has been shown to protect the myocardium from ischaemia reperfusion injury in a number of animal models. The PI3K - AKT and MEK1/2 - ERK1/2 cell survival pathways have been shown to play a critical role in regulating myocardial ischaemia reperfusion injury. In this study we investigated whether the A3 adenosine receptor agonist 2-CL-IB-MECA protects the myocardium from ischaemia reperfusion injury, when administered at reperfusion or post reperfusion and whether the protection involved the PI3K – AKT or MEK 1/2 – ERK1/2 cell survival pathways. In the Langendorff model of ischaemia reperfusion injury isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. Administration of 2-CL-IB-MECA (1nM) at reperfusion significantly decreased infarct size to risk ratio compared to non-treated ischeamic reperfused control hearts. This protection was abolished in the presence of the PI3K inhibitor Wortmannin or MEK1/2 inhibitor UO126. Western blot analysis determined that administration of 2-CL-IB-MECA (1 nM) upregulated ERK1/2 phosphorylation. In the adult rat cardiac myocyte model of hypoxia/reoxygenation cells underwent 6 hours of hypoxia and 18 hours of reoxygenation. Administration of 2-CL-IB-MECA (1 nM) at the onset of reoxygenation significantly decreased cellular apoptosis and necrosis. Administration of 2-CL-IB-MECA (1nM) in the presence of the Wortmannin or UO126 significantly reversed this anti-apoptotic effect and anti-necrotic effect. Our data further showed that 2-CL-IB-MECA protects myocytes subjected to hypoxia/reoxygenation injury via decreasing cleaved-caspase 3 activity that was abolished in presence of the PI3K inhibitor but not in the presence of the MEK1/2 inhibitor UO126. Administration of 2-CL-IB-MECA (100nM) at the onset of reperfusion also significantly decreased infarct size to risk ratio in the ischaemic reperfused rat heart compared to controls that was reversed in the presence of Wortmannin or Rapamycin. This protection was associated with an increase in PI3K-AKT / p70S6K / BAD phosphorylation. 2-CL-IB-MECA (100nM) administered at reoxygenation also significantly protected adult rat cardiac myocytes from hypoxia/reoxygenation injury 28 in an anti-apoptotic and anti-necrotic manner. This anti-apoptotic/necrotic effect of 2-CL-IB-MECA was abolished in the presence Wortmannin. Furthermore, that this protection afforded by 2-CL-IB-MECA (100nM) when administered at reoxygenation was associated with a decrease in cleaved caspase 3 activity that was abolished in the presence of the Wortmannin Interestingly, postponing the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion significantly protected the isolated perfused rat heart from ischaemia reperfusion injury in a Wortmannin and UO126 sensitive manner. This protection was associated with an increase in AKT and ERK1/2 phosphorylation. Administration of the A3 agonist 2-CL-IB-MECA 15 or 30 minutes after the onset of reoxygenation significantly protected isolated adult rat cardiac myocytes subjected to 6 hours of hypoxia and 18 hours of reoxygenation from injury in an anti-apoptotic/necrotic manner. This anti-apoptotic was abolished upon PI3K inhibition with Wortmannin or MEK1/2 inhibition with UO126. The anti-necrotic effect of 2-CL-IB-MECA when administered 15 or 30 minutes post-reperfusion was not abolished in the presence of the inhibitors. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after reoxygenation was associated with a decrease in cleaved-caspase 3 activity that was abolished in the presence of Wortmannin but not in the presence of the MEK 1/2inhibitor UO126. Collectively, we have demonstrated for the first time that administration of 2-CL-IB-MECA at the onset of reperfusion protects the ischaemic reperfused rat myocardium from lethal ischaemia reperfusion injury in a PI3K and MEK1/2 sensitive manner. Delaying the administration of 2-CL-IB-MECA to 15 or 30 minutes after the onset of reperfusion of reoxygenation also significantly protects the isolated perfused rat heart from ischaemia reperfusion injury and the adult rat cardiac myocyte from hypoxia/reoxygenation injury in an anti apoptotic / necrotic manner. Furthermore, that this protection is associated with recruitment of the PI3K-AKT and MEK1/2 – ERK1/2 cell survival pathways.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Protein kinases and phosphatases play important roles in the phosphorylation state of intracellular proteins under both physiologic and pathophysiologic conditions. Compared to the large number of studies investigating the significance of kinases, in particular the mitogen-activated protein kinases (MAPKs) in myocardial ischaemia/reperfusion and ischaemic preconditioning, relatively few studies have been done on the protein phosphatases in this scenario. Although several role players in the signal transduction cascade of ischaemia/reperfusion and ischaemic preconditioning have been identified thus far, the exact mechanism of cardioprotection still remains unclear. Previous studies from our laboratory have shown that the stress kinase, p38 MAPK, has a dual role in preconditioning: it acts as trigger of the process, while attenuation of its activation during sustained ischaemia and reperfusion is required for cardioprotection. Since the activation of p38 MAPK is dependent on both the upstream kinases for phosphorylation and phosphatases for dephosphorylation, we hypothesized that the balance between the activation state of the MAPKs and the induction of phosphatases may play a major role in determining the fate of cardiomyocytes exposed to ischaemic stress. The objectives of this study were: (i) to assess the activity of the myocardial protein phosphatases (PSPs and PP1) during sustained ischaemia and during reperfusion of non-preconditioned and ischaemic preconditioned hearts; (ii) to evaluate the significance of these phosphatases in ischaemia/reperfusion as well as in ischaemic preconditioning using available appropriate inhibitors; (iii) to give particular attention to the role of the phosphatase, mitogen-activated protein kinase phosphatase-1 (MKP-1), in ischaemia/reperfusion. MKP-1 is upregulated by stress conditions and selectively inactivates p38 MAPK by dephosphorylation of the regulatory Thr and Tyr residues. The glucocorticoid, dexamethasone which increases MKP-1 expression, was used as agonist to upregulate MKP-1 experimentally. The isolated perfused working rat heart was used as experimental model. After stabilization, hearts were subjected to either a one-cycle or multi-cycle ischaemic preconditioning protocol, followed by sustained global or regional ischaemia and reperfusion. Non-preconditioned hearts were subjected to ischaemia/reperfusion only. For Western blot analysis of MAPKs, PKB/Akt and MKP-1, hearts were freeze-clamped at different times during the perfusion protocol. Endpoints were infarct size, functional recovery and phosphorylation of the MAPKs (ERK and p38 MAPK) and PKB/Akt during reperfusion. Expression of MKP-1 was monitored. The results obtained showed that activation of PSPs and PP1 does not occur during sustained global ischaemia or reperfusion of non-preconditioned and preconditioned hearts. The role of the phosphatases was subsequently further investigated using two inhibitors namely cantharidin (5 μM, a concentration which inhibits both PP1 and PP2A) and okadaic acid (7.5 nM, a concentration which inhibits PP2A selectively). Administration of cantharidin or okadaic acid during the preconditioning phase, completely abolished preconditioning induced cardioprotection as indicated by mechanical failure during reperfusion and increased infarct size, associated with increased phosphorylation of p38 MAPK and PKB/Akt and dephosphorylation of ERK42/44. These results suggest a role for PP2A in the trigger phase of preconditioning. Administration of cantharidin or okadaic acid during early reperfusion of preconditioned hearts improved functional recovery. This was associated with increased phosphorylation of ERK42/44 and PKB, but not p38 MAPK. Dexamethasone, administered intraperitoneally to rats for 10 days (3mg/kg/day) or directly added to the perfusate (1 μM) resulted in significant cardioprotection of hearts subjected to 20 min sustained global ischaemia, followed by 30 min reperfusion. This is associated with a marked upregulation of MKP-1 and dephosphorylation of p38 MAPK during reperfusion. These studies suggest that the phosphatases are definitely involved in the phenomenon of ischaemia/reperfusion and ischaemic preconditioning. However, it also become clear that extensive further research is required to fully elucidate which phosphatases are involved and the mechanisms thereof. Due to the large size of the protein phosphatase family, this may prove to be a formidable task and far beyond the scope of this thesis. The results also suggested that pharmacological targetting of phosphatases involved in phosphorylation of the reperfusion injury salvage kinase (RISK) pathway (e.g. ERK42/44 and PKB/Akt) or dephosphorylation of pro-apoptotic kinases, such as p38 MAPK, may have significant clinical potential. / AFRIKAANSE OPSOMMING: Proteïenkinases en fosfatases speel 'n belangrike rol in die fosforileringstatus van intrasellulêre proteïene in beide fisiologiese en patofisiologiese toestande. In teenstelling met die groot aantal studies gedoen ten einde die rol van die kinases, veral die mitogeen-geaktiveerde proteïenkinases (MAPKs), in iskemie/herperfusie en iskemiese prekondisionering te ondersoek, is relatief min bekend aangaande die rol van die fosfatases in hierdie scenario. Hoewel verskeie rolspelers in die seintransduksieprosesse van iskemie/herperfusie en iskemiese prekondisionering reeds geïdentifiseer is, is die presiese meganisme van miokardiale beskerming steeds onbekend. Vroeëre studies vanuit ons laboratorium het getoon dat die streskinase, p38 MAPK, 'n tweeledige rol in prekondisionering speel: dit is 'n sneller ("trigger") van die proses, terwyl verlaagde aktivering tydens volgehoue iskemie en herperfusie, noodsaaklik vir beskerming is. Ons hipotese is dus dat die balans tussen die aktiveringstatus van die MAPKs en induksie van fosfatases die oorlewing van kardiomiosiete blootgestel aan iskemiese stres, bepaal. Die doelwitte van hierdie studie was: (1) bepaling van die aktiwiteit van miokardiale proteïen fosfatases (PSPs en PP1) tydens volgehoue iskemie en herperfusie van nie-geprekondisioneerde en iskemies-geprekondisioneerde harte; (ii) evaluering van die belang van fosfatases in iskemie/herperfusie beskadiging sowel as in iskemiese prekondisionering deur van geskikte inhibitore gebruik te maak; (iii) ondersoek na die rol van die fosfatase, mitogeen-geaktiveerde proteïen kinase fosfatase-1 (MPK-1) in iskemie/herperfusie beskadiging. Dit is bekend dat MKP-1 deur strestoestande opgereguleer word en p38 MAPK selektief deur defosforilasie van die regulatoriese Thr en Tyr residue inaktiveer word. Die glukokortikoïed, deksametasoon, wat MKP-1 uitdrukking stimuleer, is as agonis gebruik ten einde MKP-1 eksperimenteel op te reguleer. Die geïsoleerde, geperfuseerde werkende rothart is as eksperimentele model gebruik. Na stabilisasie, is die harte aan 'n enkel- of veelvuldige siklus iskemiese prekondisioneringsprotokol onderwerp, gevolg deur volgehoue globale of streeksiskemie. Nie-geprekondisioneerde harte is slegs aan iskemie/herperfusie onderwerp. Harte is op verskillende tye tydens die perfusieprotokol gevriesklamp vir Western blot analise van die MAPKs, PKB/Akt en MKP-1. Infarktgrootte en funksionele herstel tydens herperfusie is as indikators van iskemiese beskadiging gebruik. Fosforilasie van MAPKs en PKB/Akt sowel as uitdrukking van MKP-1 tydens vroeë herperfusie is gemonitor. Die resultate toon dat aktivering van PSP en PP1 tydens volgehoue iskemie en herperfusie nie plaasvind nie. Die rol van die fosfatases is verder ondersoek deur van twee inhibitore gebruik te maak, naamlik cantharidin (5 μM inhibeer beide PP1 en PP2A) en okadaic suur (7.5 nM inhibeer PP2A selektief). Toediening van of cantharidin of okadaic suur tydens die prekondisioneringsprotokol, hef prekondisionering-geïnduseerde beskerming totaal op, soos aangetoon deur hartversaking tydens herperfusie en 'n toename in infarktgrootte, tesame met 'n toename in die fosforilering van p38 MAPK en PKB/Akt en defosforilering van ERK42/44. Hierdie waarnemings dui op 'n rol vir PP2A as sneller in prekondisionering. Toediening van hierdie inhibitore tydens vroeë herperfusie het ook die miokardium beskerm, soos aangetoon deur 'n verbeterde meganiese herstel van geprekondisioneerde harte, tesame met ‘n verhoogde fosforilering van ERK42/44 en PKB (maar nie p38 MAPK nie). Deksametasoon, intraperitoneaal toegedien, vir 10 dae (3mg/kg/dag) of direk by die perfusaat gevoeg (1μM), het tot 'n hoogs beduidende beskerming teen iskemiese beskadiging gelei van harte blootgestel aan 20 min globale iskemie en 30 min herperfusie. Hierdie toename in funksionele herstel en afname in infarktgrootte het met 'n toename in MKP-1 uitdrukking en defosforilasie van p38 MAPK gepaard gegaan. Bogenoemde resultate dui op 'n definitiewe betrokkenheid van fosfatases in iskemie/herperfusie en iskemiese prekondisionering. Dit is egter ook duidelik dat intensiewe verdere navorsing benodig word om die presiese rol van die fosfatases te bepaal. Vanweë die grootte van die fosfatase familie, val dit egter buite die beskek van hierdie studie. Ten slotte, die resultate toon dat farmakologiese manipulasie van fosfatases betrokke by die fosforileringstatus van anti-apoptotiese kinases soos ERK42/44 en PKB/Akt en defosforilasie van pro-apoptotiese kinases, soos p38 MAPK, besondere kliniese toepassings mag hê.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2007. / Nitric oxide (NO) is a major signaling molecule in the heart with various biological effects. The putative role of NO as a cardioprotective agent against ischaemiareperfusion injury and in ischaemic preconditioning (IP) has made it one of the fastest growing fields in basic cardiovascular research. However, NO may also be associated with harmful effects, especially when released in excessive amounts. Little is known about the relative contributions to NO-production by the cardiac microvascular endothelial cells (CMECs) and the adjacent cardiomyocytes. Furthermore, the respective roles of endothelial NOS (eNOS) and inducible NOS (iNOS) are not well characterized in these cell types, particularly in hypoxia. In order to gain a better understanding of the role of NO in the hypoxic/ischaemic heart, the aims of this study were to: (1) develop an isolated cardiomyocyte model in which hypoxia and early IP can be induced and the role of NO assessed; (2) measure NOproduction in cardiomyocytes and CMECs under baseline and hypoxic conditions; and (3) evaluate the expression, regulation and activation of eNOS and iNOS in cardiomyocytes and CMECs (baseline and hypoxia) and establish the relationship with NO-production under these conditions. Cardiomyocytes isolated from adult rat hearts and commercially purchased rat CMECs were used as cell models.

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

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Chronic ventricular sympathectomy elicits changes in the coronary circulation, myocardial oxygen consumption and size of infarction resulting fromcoronary occlusion. These changes indicate a change occurring in the basic metabolism of the heart in response to the removal of its sympathetic nervous input. This hypothesis was tested using two groups of dogs, a shamoperated control and a ventricular sympathectomized group. The sympathectomy procedure was an intrapericardial surgical technique which selectively removes ventricular sympathetic input. Four weeks after surgery, left ventricular tissue samples were obtained and rapidly frozen to -80°C. Selected metabolic variables were then compared between the two groups.

Adaptation to chronic hypoxia increases myocardial resistance to acute ischemia/reperfusion (I/R) injury, similarly to application of exogenous erythropoietin (EPO). Nevertheless, it is not known if EPO induced by chronic hypoxia plays a role in its cardioprotective mechanism. The aim of this study was to find out if protective effect of exogenous EPO adds up to protection offered by chronic hypoxia. Adult male mice (ICR) were adapted to intermittent hypobaric hypoxia 8 hours per day, 5 days per week for 5 weeks. The degree of hypoxia corresponded to 7000 metres. Control animals were housed for the same time in normoxic environment. Resistance to I/R injury was assessed according to size of myocardial infarction induced by 45-min global ischemia and 1-h reperfusion of the heart in vitro. Animals were treated 24 h before the experiment with 200 or 5000 U/kg EPO. Treatment with 200 U/kg EPO was sufficient to significantly limit infarct size in normoxic animals (33,56 ± 2,93 % vs. 25,71 ± 2,29 %). Hypoxic adaptation decreased infarct area to 23,49 ± 2,30%, but additive effect of EPO in hypoxic group was not detected. The results indicate that exogenous EPO employs the same cardioprotective mechanisms as adaptation to chronic intermittent hypoxia. Preliminary results indicate that repeated application of EPO...

Diversos estudos indicam que a deficiência de estrógeno aumenta a incidência de doenças cardiovasculares em mulheres na pós-menopausa. Os efeitos decorrentes da deprivação de estrógeno no miocárdio ainda não estão totalmente esclarecidos. Estudos têm evidenciado efeitos benéficos da atividade física no sistema cardiovascular, em qualquer condição, em especial na menopausa. Utilizando ratos Wistar, como modelo experimental, os objetivos deste trabalho são: a) verificar se a ooforectomia e a atividade física produzem alterações no miocárdio do ventrículo esquerdo, b) verificar se a realização do exercício físico aeróbico tem efeito sobre essas alterações. Para a realização deste estudo foram utilizadas 15 ratas com 6 meses de idade, da linhagem Wistar, distribuídas em 3 grupos de 5 ratas cada: GC- Ratas com 6 meses de idade sem ooforectomia e sem atividade física; GS- Ratas com 6 meses de idade com ooforectomia bilateral sem atividade física; GA- Ratas com 6 meses de idade com ooforectomia bilateral que realizaram atividade física (corrida em esteira) durante 3 meses. Os animais dos 3 grupos foram eutanasiados com 9 meses de idade. A avaliação foi realizada através de microscopia de luz e estereologia. Os cortes histológicos foram corados por: Hematoxilina-Eosina e Picrosírius. Na parede do ventrículo esquerdo foram estimadas as densidades de volume dos cardiomiócitos, do tecido conjuntivo e a densidade de comprimento e densidade de volume dos vasos intramiocárdicos. Foi estimado o número total de núcleos de cardiomiócitos e sua densidade numérica foi estimada através do método disector. Os dados quantitativos dos três grupos foram estatisticamente comparados através do ANOVA e teste de Tukey. Os resultados mostraram que houve um aumento de peso nos animais do GS e GA em relação ao GC. Quanto ao treinamento, os animais do GA tiveram melhor rendimento nos TEMs. Quanto ao volume do VE, observamos uma diminuição, embora não significante, nos animais do GS e GA em relação ao GC. A densidade de volume dos vasos intramiocárdicos foi maior no GC que no GA. As diferenças não foram estatisticamente significantes em relação à ooforectomia e ao exercício físico para os parâmetros: densidade de volume dos cardiomiócitos e do tecido conjuntivo, densidade de comprimento dos vasos intramiocárdicos, densidade numérica dos núcleos dos cardiomiócitos, número total de núcleos de cardiomiócitos e densidade numérica do colágeno. Esses dados nos levam a concluir que a ooforectomia acarreta alterações no volume do ventrículo esquerdo e a atividade física diminui a densidade de volume dos vasos intramiocárdicos. Quanto aos outros itens, nem a ooforectomia, nem a atividade física, isoladamente ou associada, parecem influenciar de maneira significativa nestes dados. / Severals studies indicate that the estrogen deficiency increase the incidence of the cardiovascular diseases in women in the post-menopausal period. The effects of the estrogen deprivation in the myocardium still remain unclear. Studies have proved beneficial effects of the physical activity in the cardiovascular system, especially in the menopause. Using Wistar rats, as an experimental model, the aims of this work are: a-) to check if the ooforectomy and the physical activity produce alterations in the left ventricle of the myocardium; b-) to check if the aerobic exercise realization affects these alterations. To achieve these goals it was used 15 Wistar rats of 6 months of age, distributed in 3 groups of five rats each: Control group- Rats with six months of age without ooforectomy and physical activity; GS- Rats with six months of age with ofoorectomy bilateral and without physical activity and, GA- Rats with six months of age with ooforectomy bilateral and physical activity during 3 months. Al rats were euthanasied with 9 months of age. The results were obtained by using light microscopy were and stereology. The histological section was stained with Hematoxylin and Eosin, and Picrosirius stain. On the wall of the left ventricle were estimated the density of volume of the cardiomiocyt, of the connective tissue and the density of length and the density of volume of the intramyocardial vessels. It was estimated the total number of nuclei of cardiomiocyt and its numerical density was estimated a cross the disector method. The data of the three groups were statistically compared for the ANOVA and Tukey´s test. The results showed that there was an increase in the weight in the animals of GS and of GA. In relation to training, the animals of GA obtained the best income in the TEMs. In relation to the volume of the left ventricle, it was observed a decrease, although not significant, in the animals of GS and GA. The density of volume of the intramyocardial vessels was higher in the GC than GA. In relation to the ooforectomy and the physical exercise for the items: density of volume of the cardiomiocyt and of the connective tissue, density of length of the intramyocardial vessels, numerical density of nuclei of cardiomiocyt, total number of nuclei of cardiomiocyt and numerical density of collagen, the differences werent significant. We can conclude that the ooforectomy result in changes in the volume of the left ventricle and that the physical activity decrease the density of volume of the intramyocardial. In relation to the other items, neither the ooforectomy, neither the physical activity, alone or together, seem doesn\'t affect these data.