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))--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

Bibliography: leaves 94-106. / v, 106, [33] leaves, [2] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Assessment of myocardial metabolism with radiolabelled substrates and positron emission tomography provides a potentially sensitive technique to investigate physiological and pathological cardiac states "in vitro". Prior studies have indicated that overall metabolic activity cannot be estimated from rates of utilization of any one particular substrate. It was hypothesized that acetate labelled with carbon-11 would provide an index of oxidative metabolism, based on fundamental biochemical principles. The hypothesis is confirmed in studies using isolated perfused rabbit hearts and closed chest canine studies. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1995

Cholecystokinin, or "CCK" peptides, originally identified in the gastrointestinal tract, are now considered to be one of the most abundant peptide systems in the mammalian central nervous system. Prompted by recent findings that implicated the cholecystokinergic system in the pathophysiology of various illnesses, we developed a novel assay system to measure the various forms of cholecystokinin peptides in human plasma and cerebrospinal fluid. The system detects CCK-4, sulfated CCK-8 (CCK-8s) and nonsulfated CCK-8 (CCK-8ns) with equal affinity, with the lower detection limit of 2.7 fmol and an ED50 of 10.6 +/- 2.2 fmol. Using the assay system, we determined that mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was 12.9 +/- 2.1 pM CCK-4 equivalents. / After developing the cholecystokinin assay system, we were able to combine our unique methodology with other established techniques to investigate the role of CCK in illnesses such as premenstrual dysphoric disorder (PMDD), anxiety, bulimia nervosa, and cardiomyopathy. / Briefly, we observed no significant differences in plasma CCK levels between women with PMDD and healthy volunteers. However, we found that, independent of diagnosis, plasma cholecystokinin concentrations were higher in women during their first visit to the clinic to participate in the study, as compared to later visits. / In addition, application of our assay system allowed us to determine that oral ingestion of caffeine increased plasma CCK-LI levels 2--4 fold in humans. Moreover, we observed substantial variation in post-caffeine cholecystokinin levels among individuals. / In another study of cholecystokinin and anxiety, we used our CCK assay to determine the effects of ondansetron, a serotonin receptor antagonist, on cholecystokinin levels in plasma. We found that multiple oral doses of ondansetron influence the pharmacokinetic parameters of exogenous CCK. / We also used the three-step assay system to measure CCK-LI in patients with the eating disorder, bulimia nervosa. Baseline fasted cholecystokinin plasma levels were lower in bulimic women as compared to control subjects. However, at "satiety", or the post-binge stage, CCK levels in bulimic women were similar to those of control women. / Finally, our investigation into the role of cholecystokinin in cardiomyopathy revealed that neuronal cholecystokinin receptor density was altered in the cardiomyopathic hamster brain, as compared to age- and sex-matched control animals. (Abstract shortened by UMI.)

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 / M

Diabetes mellitus has been associated with both clinical and experimental cardiac dysfunction. Diabetic cardiomyopathy which is characterized by depressed cardiac contractility is accompanied by a variety of biochemical changes in Ca⁺⁺ metabolism. This cardiomyopathy may occur in the presence of normal coronary arteries and normal blood pressure. However, some studies have shown that hypertension is more prevalent among diabetics and can aggravate the cardiovascular abnormalities associated with diabetes. To understand the mechanisms of diabetic cardiomyopathy and consequences of combined hypertension and diabetes, experiments were designed to measure cardiac tissue responses to various inotropic agents in experimental diabetes. Six weeks following streptozotocin (STZ) administration, Wistar, spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats exhibited the 'classical signs' of diabetes which included: hyperglycemia, hypoinsulinemia, hyperlipidemia (except in WKY), and hypothyroidism. Decreased basal atrial rate and increased basal developed force (BDF) suggest a depressed SA node function and an alteration of Ca⁺⁺ utilization by diabetic ventricles. Decreased post quiescent potentiation (PQP) values (except in WKY) in ventricular tissues suggest a diminished amount of releasable Ca⁺⁺ from sarcoplasmic reticulum (SR). Decreased post stimulation potentiation (PSP) values in SHR papillary muscles (PM) are probably suggestive of a depressed sarcolemmal Na⁺-Ca⁺⁺ exchange function in this tissue. Diabetic rats show subsensitivity to β-adrenergic stimulation in ventricular tissues, supersensitivity and hyperresponsiveness to Ca⁺⁺ and α-adrenergic stimulation (except in WKY) in ventricular tissues and left atria (LA) and supersensitivity to BAY K 8644 in SHR LA and hyperresponsiveness to verapamil in ventricular strips. These alterations may be attributed to a change in receptor number and/or a post receptor alteration. Ryanodine decreased the PQP of Wistar and SHR PM and SHR LA in both controls and diabetics. It especially abolished PQP in SHR diabetic tissues, but had no effect on WKY tissues, which may suggest a difference in the SR function in these tissues. SR with impaired Ca⁺⁺ uptake may contribute to these phenomena in diabetic rats. Ryanodine also diminished (PQP + BDF) of SHR LA and (PQP/BDF) of Wistar and SHR PM, ˙but had no effects on control and other diabetic tissues. It appears that ryanodine has some influence on the Na⁺-Ca⁺⁺ exchange generated by sarcolemma (SL) of certain diabetic tissues. Further experiments are required to clarify this. SHR diabetic rats had greater changes in most of the measurements such as hyperlipidemia, depressed PQP and PSP values, and altered drug responses. This model exhibited very high mortality as compared to Wistar and WKY diabetic rats. As has been shown previously, the combination of hypertension and diabetes exerts a synergistic effect on the cardiac dysfunction in this model, and that altered lipid metabolism, SL and SR function are all involved in the development of cardiomyopathy. WKY diabetic rats, on the other hand, exhibited no significant changes in blood lipids, or in response to phenylephrine or to Ca⁺⁺ (LA) stimulation. Lack of change in these factors may explain the relatively normal cardiac function of this model as measured previously. / Pharmaceutical Sciences, Faculty of / Graduate

Heart failure resulting from different forms of cardiomyopathy is defined as the inability of the heart to pump sufficient blood to meet the body's metabolic demands. It is a major disease burden worldwide and the statistics show that 50% of the people who have the heart failure will eventually die from sudden cardiac death (SCD) associated with an arrhythmia. The central cause of disability and SCD is because of ventricular arrhythmias. Genetic mutations and acquired modifications to RyR2, the calcium release channel from sarcoplasmic reticulum, can increase the pathologic SR Ca2+ leak during diastole, which leads to defects in SR calcium handling and causes ventricular arrhythmias. The mechanism of RyR2 dysfunction includes abnormal phosphorylation, disrupted interaction with regulatory proteins and ions, or altered RyR2 domain interactions. Many pharmacological strategies have shown promising prospects to modulate the RyR2 as a therapy for treating cardiac arrhythmias. Here, we are trying to establish a novel approach to designing new drugs to treat heart failure and cardiac arrhythmias. Previously, we demonstrated that all pharmacological inhibitors of RyR channels are electron donors while all activators of RyR channels are electron acceptors. This was the first demonstration that an exchange of electrons was a common molecular mechanism involved in modifying the function of the RyR. Moreover, we found that there is a strong correlation between the strength of the electron donor/acceptor, and its potency as a channel inhibitor/activator, which could serve as a basis and direction for developing new drugs targeting the RyR. In this study, two new potent RyR inhibitors, 4-methoxy-3-methyl phenol (4-MmC) and the 1,3 dioxole derivative of K201, were synthesized which are derivatives of the known RyR modulators, 4-chloro-3-methyl phenol (4-CmC) and K201. The ability of K201, 1,3 dioxole derivative of K201 and 4-MmC to inhibit the cardiac calcium channel is examined and compared at the single channel level. All of these compounds inhibited the channel activity at low micromolar concentrations or sub-micromolar concentrations.

No description available.

Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a propancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg body weight for 2 consecutive days). Animals were divided into six experimental groups including control, treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, appropriate cells (1 X 106/mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 consecutive days. Animals were sacrificed and hearts were harvested at day 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess apoptosis and fibrosis. ERK1/2 phosphorylation was quantified using ELISAs. M-mode echocardiography fractional shortening was used to assess cardiac function. Results: Animals transplanted with ES cells, iPS cells, or both CMs showed a significant (p

Advances in genome editing, human induced pluripotent stem cells (iPSC), and cardiac tissue engineering have significantly improved the ability of in vitro models to model cardiac disease. The objective of this dissertation is to leverage cardiac tissue engineering to generate meaningful biological insights into human genetic cardiomyopathies. First, we studied a novel, de novo mutation in the filamin C (FLNC) gene which causes restrictive cardiomyopathy in a young patient. Using engineered cardiac tissues, we showed that this mutation causes a restrictive phenotype marked by increased passive tension and slowed contraction velocities. Complementing our engineered tissues, we used high-throughput calcium imaging to identify compounds which improved myocardial relaxation in mutant cardiomyocytes. These compounds improved function of mutant cardiac tissues, suggesting a potentially targetable pathway in the patient’s mutation. In another study, engineered cardiac tissues and stem cells were used to study BAG3, a dilated cardiomyopathy- related gene, in cardiac fibroblasts. BAG3-/- and wild-type iPSCs were differentiated to cardiac fibroblasts and cardiomyocytes. By generating fully isogenic cardiac tissues and altering cellular genotypes, we determined that the loss of BAG3 in cardiac fibroblasts was deleterious to cardiac tissue function despite genetically normal cardiomyocytes. Further work studying cardiac fibroblasts revealed a mechanistic function of BAG3 in regulating cardiac fibroblast extracellular matrix synthesis. Together, this work highlights the ability of cardiac tissues and stem cells to unravel the complexities of genetic heart disease.