Dissertations / Theses on the topic 'Cardiac hypertrophy'

Abstract :Cardiac hypertrophy has been identified as the most important independent risk factor for cardiovascular-related morbidity and mortality and is therefore regarded as a pathological condition. Despite this, beneficial physiological forms also appear to exist, such as in response to exercise, leading to maintained or improved cardiac function. The aim of this thesis was to examine two distinct rodent models, an endurance run-trained rat, and the DOCA-salt hypertensive rat, representing physiological and pathological hypertrophy, respectively, in order to develop a better understanding of the molecular changes associated with each condition. The thesis also examined the effect of dietary supplementation of L-arginine to the pathological model, a treatment that has been shown to ameliorate/prevent many of the cardiovascular impairments. Studies examined selected candidate genes (qRT-PCR), including conventional biomarkers of hypertrophy and exploratory analysis of adenosine-related genes (given adenosine’s established regulatory and protective role in the heart, yet minimally studied in cardiac hypertrophy), and explored global transcriptomic shifts via microarrays. The hypothesis of this work was that cardiac hypertrophy lies on a continuum, with similarities existing at the cardiac transcriptional level between early (adaptive) stages of pathological hypertrophy (DOCA-salt rat) and later stages of physiological hypertrophy (endurance run-trained rat). Examination of ten biomarkers of hypertrophy (ANF, BNP, -MHC, -MHC, cardiac -actin, skeletal -actin, SERCA2, PPAR, Coll I and III) revealed that the pathological model displayed alterations in the expression of many of these molecules in line with the literature. These changes were not observed in the physiological model. This therefore reinforces the value of conventional biomarkers in delineating pathological vs. physiological hypertrophies, and reveals fundamental differences in genesis of these two forms of hypertrophy. The adenosine system (receptors and purine handling molecules) was altered in the pathological hypertrophy model as evidenced by the modulation of genes corresponding to A3AR, Ada, and Adk, with a potential shift from purine salvage towards degradation of adenosine to inosine. Furthermore, this study represents the first report of altered regulation of the nucleoside transporter ENT3 in a pathological condition. None of these changes were seen in the physiological model with only modulation of the A2aAR evident. Examination of the transcriptional response to physiological hypertrophy revealed that short (6 week) and long (12 week) training programmes resulted in different profiles, likely reflecting progression of the hypertrophy process. The short programme stimulated genes associated with the mitochondria, oxidoreductase, receptor binding and coenzymemetabolismand repressed the expression of transcripts associated with phosphorylation, catalytic activity, defence/immunity and energy pathways. Thus, initial changes observed are primarily of a metabolic and signalling nature. In contrast, the longer programme resulted in shifts in protein handling and synthesis, and genes involved in structural molecule activity, nucleotide binding and cellular homeostasis. These patterns support a progression with time from initial metabolic adaptations to longer term shifts in protein phenotype and structural adaptations, consistent with longer term changes in heart structure. Similarly, the pathologicalmodel displayed different time-dependent gene expression profiles. Overall, the pattern of changewith early (2week) treatment is suggestive of changes in intracellular signalling and increasing transcriptional capacity with the later changes (at 4 weeks) indicative of structural adaptations (intra- and extracellularly) togetherwith an inflammation response. Genes coding for calciumhandling, ion channels, and gap junctions were altered throughout themodel andmay contribute to electrical conduction defects and cardiac dysfunction. The adrenergic signalling pathway was modulated as associated signalling molecules were down-regulated. The study revealed many expected and novel changes, of which further study should focus on: calcium regulation, metabolic regulation, gap junctions, and (as might be exii pected) signalling via the adrenergic pathway, insulin-like growth factor, PI3K, and Jak/STAT. L-Arginine modulated biomarker expression in pathological hypertrophy, with stimulation of PPAR and SERCA2 with little or no effect on the adenosine-related genes. L-Arginine affected the overall transcriptional response to DOCA-salt treatment, stimulating genes involved in cell growth andmaintenance, nerve transmission, heparin and glycosaminoglycan binding, peptide binding and protein targeting, as well as the repression of genes related to apoptosis (favouring a pro-apoptotic state), intracellular organisation and biogenesis, and enzyme inhibitor activity. The beneficial effects of L-arginine in the setting of pathological hypertrophy may be due to modulation of metabolism, improving calcium handling and overall enhancing cellular functioning. This work demonstrates that cardiac hypertrophy is clearly different at the transcriptional level depending upon the aetiology. This repudiated the hypothesis of the thesis that cardiac hypertrophy lies on a continuum with similarities existing at the cardiac transcriptional level between early (adaptive) stages of pathological hypertrophy and later stages of physiological hypertrophy. Whilst some of the data was in accordance with current knowledge of these states, novel changes were also discovered, contributing to our understanding of the molecular aspects of cardiac hypertrophy.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Griffith University. School of Medical Science.
Griffith Health
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Heart failure (HF) is a syndrome resulting from a complex genetic predisposition and multiple environmental factors: it is a leading cause of morbidity and mortality. Specific gene expression patterns are activated in the hypertrophic and failing heart and are thought to contribute to the development of HF. Many regulatory molecules are involved in the control of gene expression: among these, long non-coding RNA (lncRNA) is gaining importance for several cellular process and diseases. However, little is still known about its involvement in HF. Many functions have been attributed to lncRNAs, such as cell proliferation, apoptosis and cell invasion, indicating that they may represent a major regulatory component of the eukaryotic genome. Not surprisingly, lncRNAs have been found implicated in several aspects of cancer, and in many neuronal diseases. Despite this, and the known role of other ncRNAs, such as miRNA, in HF, the function of lncRNAs in this pathologic state has been not studied. Thus, the general hypothesis behind this project is that lncRNAs have an important role in defining gene expression re-programming in HF. Consequently, the overall scientific objective of this proposal is to study the role of lncRNAs in gene transcription regulation accompanying heart failure. To this end, we propose to use high-throughput RNA sequencing (RNA-seq) to identify lncRNAs that are modulated in cardiomyocytes during HF. In order do to this, we performed RNA-seq on cardiomyocytes isolated from mice after 1, 2, 4 and 7 days of transverse aortic constriction (TAC) and from sham-operated mice. The importance of this study lies not only in the furthering of our understanding of the pathological mechanisms leading HF, but aims to generate – in the light of recent progress in RNA-based therapeutic strategies – data that may be instrumental to the development of improved therapeutic strategies for this increasingly frequent pathology.

Abstract Cardiac hypertrophy is an adaptive response to increased cardiac workload. It is initially beneficial, since it helps to maintain cardiac output, but ultimately it is considered as an independent predictor for heart failure and sudden cardiac death. The cardiac hypertrophic response is triggered by mechanical and neurohumoral stimuli and is associated with the activation of complex changes in gene programming and intracellular signaling pathways. The purpose of this study was to investigate the expression of some novel load-induced factors i.e. melusin, thrombospondin (TSP)-1 and -4 and LIM and cysteine-rich domains protein 1 (LMCD1)/dyxin during the hypertrophic response. Melusin was expressed in cardiac tissue both in the atria and ventricles, furthermore its expression was very rapidly activated in response to multiple hypertrophic stimuli predominantly in the left atria. Melusin gene expression was activated when cultured cardiac myocytes were subjected to mechanical stretch or hypertrophic agonists such as endothelin-1 or angiotensin (Ang II). TSP-1 and TSP-4 gene expressions were rapidly activated at an early stage of pressure overload. Myocardial infarction (MI) induced the expression of both TSP-4 and TSP-1 mRNA in the heart. TSP-4 may also be an endothelial cell-specific marker of pressure overload since its expression was limited to endothelial cells in the adult heart. The expression of LMCD1/dyxin was found to be induced during the cardiac hypertrophic response and after MI. By itself, mechanical load was a critical regulator of LMCD1/dyxin gene expression. LMCD1/dyxin is a putative novel p38 mitogen-activated protein kinase (MAPK) target since adenovirus-mediated overexpression of p38 MAPK upregulated LMCD1/dyxin expression. In addition, during the Ang II-induced pressure overload p38 MAPK phosphorylation levels correlated with the early induction of LMCD1/dyxin expression. In conclusion, this study provides new information on the expression of melusin, TSP-1 and -4 and LMCD1/dyxin in the cardiac hypertrophic response. Early induction of their gene expression may represent an initial step in the adaptive and protective remodeling processes following increased workload in the heart
Tiivistelmä Sydänlihas mukautuu lisääntyneeseen kuormitukseen lihassolujen koon kasvun eli hypertrofian avulla. Pitkittyessään hypertrofinen kasvu on kuitenkin tärkeä sydämen vajaatoimintaa ja äkkikuolemaa ennakoiva riskitekijä. Hypertrofisessa vasteessa mekaaninen venytys sekä neurohumoraaliset tekijät saavat aikaan solunsisäisten signaalinvälitysreittien aktivoitumisen, mikä johtaa lisääntyneeseen geenien luentaan ja proteiinituotantoon. Väitöskirjassa tutkittiin uusien kuormitusaktivoituvien tekijöiden, melusiinin, trombospondiini (TSP) -1:n ja -4:n sekä dyksiinin ilmentymistä hypertrofisen vasteen aikana. Melusiinia ilmentyy sydämessä sekä kammioissa että eteisissä, mutta painekuormituksen myötä se aktivoituu nopeasti pääasiassa vasemmassa eteisessä. Sydänlihassolujen soluviljelymallissa melusiinin luenta lisääntyy suoraan mekaanisen venytyksen ja hypertrofisten agonistien vaikutuksesta. Painekuormitus aktivoi nopeasti myös TSP-1:n ja -4:n luentaa sydämessä. TSP-1:n ja -4:n geeniluenta lisääntyy myös kokeellisessa sydäninfarktimallissa. Lisäksi sydämessä TSP-4:ää havaittiin olevan ensisijaisesti endoteelisoluissa. Dyksiinin ilmentyminen lisääntyi sekä painekuormituksen että sydäninfarktin aiheuttaman sydänlihaksen uudelleenmuovautumisen aikana. Mekaaninen kuormitus riitti jo yksinään aktivoimaan dyksiinin geeniluentaa sydämessä. Lisäksi mitogeeni-aktivoituvan p38-proteiinikinaasin havaittiin säätelevän dyksiinin ilmentämistä. Väitöskirjatyössä saatiin uutta tietoa sydänlihaksen kuormituksen aikaisista muutoksista geenien luennassa sydänlihaksessa. Työssä osoitettiin, että painekuormitus aktivoi sydämessä aiemmin vähän tutkittujen geenien, melusiinin, TSP-1:n ja -4:n sekä dyksiinin, ilmentymistä. Näiden tekijöiden aktivoituminen hypertrofisen vasteen alkuvaiheessa antaa viitettä siitä, että tekijät osallistuvat kuormittuneen sydänlihaskudoksen uudelleenmuovautumiseen. Melusiini voi toimia erityisesti eteiskudosta kuormitukselta suojaavissa mekanismeissa, kun taas TSP-4 osoittautui aktivoituvan painekuormituksessa nimenomaan endoteelisoluissa

Cardiac hypertrophy often develops to compensate for hemodynamic overload and is associated with heart failure. Recent studies have revealed that overexpression and PKA-mediated phosphorylation of heat shock protein 20 (HSP20) at Ser16 can attenuate hypertrophic growth of cardiomyocytes and trigger cardioprotective functions following sustained β-adrenergic stimulation (Fan et al., 2004, 2005, 2006). However, the molecular mechanism of HSP20 induced cardioprotection remains to be fully elucidated. In order to gain insight into the protective mode of action of HSP20, I attempted to (1) investigate the spatiotemporal control of PKA-mediated phosphorylation of HSP20, as well as (2) identifying novel protein binding partners for HSP20 utilising cutting edge ProtoArray technology. Initially, I set up an in vitro hypertrophy model using sustained isoprenaline (ISO)-stimulated neonatal rat cardiomyocytes. Cell size, protein synthesis and fetal gene expression were assessed as parameters of hypertrophic growth. In the first section of my studies, members of the cAMP-specific PDE4 family were shown to form signalling complexes with HSP20, and that the PKA-mediated phosphorylation of HSP20 could be modulated by PDE4. Based on peptide array data, a cell-permeable peptide ‘bs906’ was developed to inhibit the interaction of PDE4 with HSP20. Interestingly, the disruption of the PDE4-HSP20 complex was shown to induce PKA-mediated phosphorylation of HSP20 and trigger cardioprotection against the hypertrophic response measured in neonatal cardiomyocytes upon chronic β-adrenergic stimulation. In the second part of my studies, protein kinase D1 (PKD1) was identified as one interacting partner that robustly associated with HSP20. This interaction was confirmed by biochemical and immunocytochemical means. Using similar approaches to those used for the PDE4-HSP20 interaction, a cell-permeable peptide ‘HJL09’ was generated to promote disruption of the PKD1-HSP20 complex. Experimentation using the peptide concluded that the disruption of the PKD1-HSP20 complex reduced HSP20 phosphorylation and attenuated the hypertrophic response in cultured cardiomyocytes as shown by reduced increases in cell size, protein content and actin reorganisation. In undertaking this work, I also defined a novel PKD phosphorylation site (Ser16) on HSP20 that conforms to the PKD phosphorylation motif of RxxS (also a PKA site). My biochemical data suggested that PKD1 may regulate the cardioprotective function of HSP20 via phosphorylation at Ser16. In situ proximity ligation assay (PLA) further revealed a role of HSP20 as ‘molecular escort’ in targeting the nuclear translocation of PKD1. This function, in part, may be responsible for the induction of fetal gene reexpression as selective disruption of PKD1-HSP20 complex using ‘HJL09’ hindered the nuclear influx of the complex, thereby attenuating hypertrophic signalling. In summary, these studies describe some exciting findings which provide further insight into novel signalling mechanism of cardiac hypertrophy in neonatal rat cardiomyocytes. I have shown that PKA and PKD1 exhibiting opposite functions despite sharing the phosphorylation site on HSP20. In this regard, HSP20 functions as a molecular nexus for the opposing actions of the PKA and PKD1 signalling pathways in hypertrophy, suggesting that crosstalk may occur between anti-hypertrophic and pro-hypertrophic pathways. The identification and characterisation of these complexes should help to build a better understanding of the hypertrophic signalling pathway, and may provide novel therapeutic strategies for the treatment of cardiac hypertrophy.

Left ventricular hypertrophy (LVH) is a significant risk factor for the development of heart failure (HF), the incidence of which is increased by obesity. Diets high in fat and sugar have been linked with the development of the metabolic syndrome and obesity, and may expose the heart to a unique environment via the differential actions of dietary macronutrients. The main objectives of this study were to determine the effect of differing dietary regimens upon (i) the progression of LVH and whole organism morphology (ii) function and metabolism in the hypertrophied heart, and (iii) cardiac ceramide content. Cardiac hypertrophy was surgically induced in male Sprague-Dawley rats via abdominal aortic constriction (AC). Animals were assigned to either a diet containing 5% sucrose/7% fat (standard diet, SD), 9 % sucrose/45 % fat (high-fat diet, HFD), or 14% sucrose/44% fat (western diet, WD) for 9 weeks. LVH was observed in all AC groups but was greatest in those fed a SD or WD. Both HFD and WD resulted in a significant increase in abdominal fat mass, which was positively associated with serum concentrations of leptin. In vitro cardiac function was unaltered by any dietary regimen alone, but was significantly enhanced in hypertrophied hearts from HFD and WD-fed animals, consistent with a compensated phase of hypertrophic remodelling. This was accompanied by a small reduction in palmitate oxidation and increased reliance upon lactate, an effect which was exacerbated in hearts from WD-fed animals. In WD-fed animals, there was a substantial increase in cardiac triglyceride (TG), which was not affected by AC. PPARα protein was reduced following AC in the hearts of animals fed a SD or WD, whereas the HFD prevented this decline. CD36 protein expression was not different between control and AC animals, but was highest in those fed a WD. In addition to elevated TG, WD hearts also exhibited a significant accumulation of long-chain ceramide species (C16-C24) compared with other dietary groups; consistent with metabolic remodelling. This effect was observed independent of AC. In order to simulate a model of HF, WD animals were treated with adriamycin (ADR), and cardiac ceramide content was further increased with the specific accumulation of C16 and C18 ceramide. These findings suggest that dietary macronutrient composition can have a profound effect upon the progression of LVH. Furthermore, the enhanced ceramide content in WD hearts indicates that the macronutrient composition of this dietary profile is most deleterious to the hypertrophied heart. Prolonged exposure of the hypertrophied heart to the WD may lead to increased apoptosis and accelerate the transition to HF.

Cardiac hypertrophy defines an adaptive process brought about in response to sustained increases in haemodynamic work. Cardiomyocytes undergo an initial compensatory phase in which enlargement and contractility alterations normalise wall stress and maintain adequate perfusion of organs. In pathological hypertrophy, this deteriorates to a decompensated state characterised by ventricular dysfunction and predisposition to heart failure. In contrast, physiological hypertrophy and associated enhanced cardiac functioning arising from chronic exercise training does not progress to heart failure. Determination of the molecular pathways underlying myocardial hypertrophy remains a challenge for cardiovascular research. The objective of the work presented in this thesis was to identify genes differentially expressed during pathological and physiological hypertrophy in order to enhance our knowledge of the mechanistic processes involved. A reverse Northern hybridisation method was applied to profile the expression of specifically selected genes in the hypertrophic models examined. Functional categories represented in the gene panel assembled included cardiac contractile and cytoskeletal markers, matrix metalloproteinases, vasoactive pathway factors, calcium handling genes, ion channels, cardiac regulatory factors, signalling pathway intermediates, apoptotic factors and histone deacetylases. In order to investigate pathological hypertrophy, a deoxycorticosterone acetate-salt (DOCA-salt) rat model was utilised. DOCA-salt treated rats used in this study demonstrated a 1.4-fold increase in heart weight to body weight ratio compared to controls. Impaired cardiac function indicative of a decompensated pathological phenotype in the DOCA-salt treated group was demonstrated by way of decreased chamber size, impaired myocardial compliance and significantly reduced cardiac output. Reverse Northern hybridisation analysis of 95 selected genes identified a number of candidates with differential expression in hearts of DOCA-salt treated rats. Increased gene expression was demonstrated for the collagenase MMP1 and stress-activated signal transduction factor Sin1. In contrast, the sarcoplasmic reticulum calcium ATPase SERCA-2 and anti-apoptotic factor BCL2l-10 genes exhibited decreased expression. To investigate changes in gene expression associated with physiological hypertrophy, use was made of an endurance run-trained rat model. The run-trained rats used in this study demonstrated a 24.1% increase in heart weight to body weight ratio and improvements in performance consistent with physiological cardiac adaptation. These performance indicators included improvements in systolic volume, cardiac output, myocardial compliance and bio-energetic function. Reverse Northern hybridisation expression analysis of 56 genes identified a number of differentially expressed mRNA transcripts in run-trained hypertrophied hearts. Four genes shown to demonstrate reduced expression in the run-trained rat model were interleukin-1 receptor associated kinase (IRAK1) and the developmentally expressed transcription factors Nkx-2.3, dHAND, and IRX-2. Based upon the reverse Northern hybridisation results, four genes were selected for Western blotting analysis of rat cardiac tissue. Of these, MMP1 and a putative isoform of Sin1 exhibited increased levels in DOCA-salt treated hypertrophic left ventricular tissue, results that correlate with the findings of increased mRNA expression for these two genes. Therefore, this study identified MMP1 and Sin1 as candidates involved in pathological but not physiological hypertrophy. This finding is in accord with other recent investigations demonstrating that pathological hypertrophy and physiological hypertrophy are associated with distinct molecular phenotypes. An aside to the major objective of identifying genes differentially regulated in left ventricular hypertrophy involved the application of the P19CL6 cell in vitro model of cardiomyogenesis to compare protein expression during hypertrophy and development. The Sin1 isoform, found to be up-regulated during DOCA-salt induced hypertrophy, was also shown to be more abundant in differentiating, than non-differentiating, P19CL6 cells. This result is consistent with the developing paradigm that implicates 'fetal' genes in the hypertrophic remodelling process.

Cardiac hypertrophy defines an adaptive process brought about in response to sustained increases in haemodynamic work. Cardiomyocytes undergo an initial compensatory phase in which enlargement and contractility alterations normalise wall stress and maintain adequate perfusion of organs. In pathological hypertrophy, this deteriorates to a decompensated state characterised by ventricular dysfunction and predisposition to heart failure. In contrast, physiological hypertrophy and associated enhanced cardiac functioning arising from chronic exercise training does not progress to heart failure. Determination of the molecular pathways underlying myocardial hypertrophy remains a challenge for cardiovascular research. The objective of the work presented in this thesis was to identify genes differentially expressed during pathological and physiological hypertrophy in order to enhance our knowledge of the mechanistic processes involved. A reverse Northern hybridisation method was applied to profile the expression of specifically selected genes in the hypertrophic models examined. Functional categories represented in the gene panel assembled included cardiac contractile and cytoskeletal markers, matrix metalloproteinases, vasoactive pathway factors, calcium handling genes, ion channels, cardiac regulatory factors, signalling pathway intermediates, apoptotic factors and histone deacetylases. In order to investigate pathological hypertrophy, a deoxycorticosterone acetate-salt (DOCA-salt) rat model was utilised. DOCA-salt treated rats used in this study demonstrated a 1.4-fold increase in heart weight to body weight ratio compared to controls. Impaired cardiac function indicative of a decompensated pathological phenotype in the DOCA-salt treated group was demonstrated by way of decreased chamber size, impaired myocardial compliance and significantly reduced cardiac output. Reverse Northern hybridisation analysis of 95 selected genes identified a number of candidates with differential expression in hearts of DOCA-salt treated rats. Increased gene expression was demonstrated for the collagenase MMP1 and stress-activated signal transduction factor Sin1. In contrast, the sarcoplasmic reticulum calcium ATPase SERCA-2 and anti-apoptotic factor BCL2l-10 genes exhibited decreased expression. To investigate changes in gene expression associated with physiological hypertrophy, use was made of an endurance run-trained rat model. The run-trained rats used in this study demonstrated a 24.1% increase in heart weight to body weight ratio and improvements in performance consistent with physiological cardiac adaptation. These performance indicators included improvements in systolic volume, cardiac output, myocardial compliance and bio-energetic function. Reverse Northern hybridisation expression analysis of 56 genes identified a number of differentially expressed mRNA transcripts in run-trained hypertrophied hearts. Four genes shown to demonstrate reduced expression in the run-trained rat model were interleukin-1 receptor associated kinase (IRAK1) and the developmentally expressed transcription factors Nkx-2.3, dHAND, and IRX-2. Based upon the reverse Northern hybridisation results, four genes were selected for Western blotting analysis of rat cardiac tissue. Of these, MMP1 and a putative isoform of Sin1 exhibited increased levels in DOCA-salt treated hypertrophic left ventricular tissue, results that correlate with the findings of increased mRNA expression for these two genes. Therefore, this study identified MMP1 and Sin1 as candidates involved in pathological but not physiological hypertrophy. This finding is in accord with other recent investigations demonstrating that pathological hypertrophy and physiological hypertrophy are associated with distinct molecular phenotypes. An aside to the major objective of identifying genes differentially regulated in left ventricular hypertrophy involved the application of the P19CL6 cell in vitro model of cardiomyogenesis to compare protein expression during hypertrophy and development. The Sin1 isoform, found to be up-regulated during DOCA-salt induced hypertrophy, was also shown to be more abundant in differentiating, than non-differentiating, P19CL6 cells. This result is consistent with the developing paradigm that implicates 'fetal' genes in the hypertrophic remodelling process.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Background: The aim of this thesis was to study transcription patterns and extracellular signalling of the hypertrophic heart to better understand the mechanisms initiating, controlling and maintaining cardiac hypertrophy. Cardiac hypertrophy is a risk factor for cardiovascular morbidity and mortality. Hypertrophy of the myocardium is a state, independent of underlying disease, where the myocardium strives to compensate for an increased workload. This remodelling of the heart includes physiological changes induced by a changed gene expression, alteration of the extracellular matrix and diverse cell-to-cell signalling. Shedding microvesicles and exosomes are membrane released vesicles derived from the plasma membrane, which can mediate messages between cells and induce various cell-related processes in target cells. Methods and materials: Two different microarray studies on different materials were performed. In the first study, cardiac myectomies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM) and 5 controls without cardiac disease were used. In the second study, myocardial tissue from 6 aorta ligated and 6 sham operated (controls) rats at three different time points (1, 6 and 42 days post-surgically) were analysed. To reveal differences in gene expression the materials were analyzed with Illumina whole genome microarray and multivariate data analysis (PCA and OPLS-DA). Cultured cardiomyocytes (HL-1) were incubated with and without growth factors (TGF-β2 or PDGF BB). Microvesicles and exosomes were collected and isolated after differential centrifugations and ultracentrifugations of the cell culture medium. The microvesicles and exosomes were characterized with dynamic light scattering (DLS), flow cytometry, western blot, electron microscopy and Illumina whole genome microarray. Results: The two different microarray studies revealed differentially expressed gene transcripts and groups of transcripts. When comparing HOCM patients to controls significant down-regulation of the MYH6 gene transcript and two immediate early genes (IEGs, EGR1 and FOS), as well as significant up-regulation of the ACE2, JAK2 and HDAC5 gene transcripts were found. In the rat model, 5 gene groups showed interesting clustering after multivariate data analysis (OPLS-DA) associated with the hypertrophic development: “Atherosclerosis”, “ECM and adhesion molecules”, “Fatty acid metabolism”, “Glucose metabolism” and “Mitochondria”. The shedding microvesicles were rounded vesicles, 40-300 nm in size and surrounded by a bilayered membrane. Chromosomal DNA sequences were identified in the microvesicles. The microvesicles could be taken up by fibroblasts resulting in an altered gene expression in the fibroblasts. The exosomes from cultured cardiomyocytes (incubated with TGF-β2 or PDGF BB) had an average diameter of 50-80 nm, similar to the unstimulated control exosomes. A large, for all cardiomyocyte derived exosomes, common pool of mRNA seems stable and a smaller pool varied in mRNA content according to treatment of the cardiomyocyte. Of the common mRNA about 14% were ribosomal, 14% were of unknown locus and 5% connected to the function of the mitochondria. Conclusions: The microarray studies showed that transcriptional regulation at a stable stage of the hypertrophic development is a balance of pro and anti hypertrophic mechanisms and that diverse gene groups are differently regulated at different time points in the hypertrophic progression. OPLS-DA is a very useful and powerful tool when analyzing gene expression data, especially in finding clusters of gene groups not seen with traditional statistics. The extracellular vesicle studies suggests that microvesicles and exosomes released from cardiomyocytes contain DNA and can be involved in events in target cells by facilitating an array of processes including gene expression changes. Different treatment of the cardiomyocyte influence the content of the exosome produced, indicating that the signal function of the exosome might vary according to the state of the cardiomyocyte.
Bakgrund: Syftet med den här avhandlingen var att studera transkriptions-mönster och extracellulär signalering vid hjärthypertrofi för att bättre förstå de mekanismer som startar, styr och underhåller tillväxten. Hjärthypertrofi, onormal tillväxt av hjärtmuskeln, är en riskfaktor för andra hjärt-kärlsjukdomar och dödlighet. Hypertrofi av hjärtmuskeln är ett tillstånd, oberoende av bakomliggande sjukdom, där hjärtmuskeln strävar efter att kompensera för ökad arbetsbelastning. Denna omställning av hjärtat innefattar fysiologiska förändringar orsakade av ett förändrat genuttryck, modifiering av miljön utanför cellen och ändrad cell-till-cell signalering. Mikrovesiklar och exosomer är små membranomslutna bubblor som frisätts från cellmembranet, ut i cellens omgivning. De kan förmedla budskap mellan celler och påverka olika processer i målceller. Metoder och material: Avhandlingen innefattar två olika microarraystudier på olika material. I den första studien användes hjärtbiopsier från 8 patienter med hypertrofisk obstruktiv kardiomyopati (HOCM) och 5 kontroller utan hjärtsjukdom. I det andra projektet användes hjärtvävnad från 6 aortaligerade och 6 skenopererade (kontroller) råttor vid tre olika tidpunkter (1, 6 och 42 dagar efter kirurgiskt ingrepp). För att påvisa skillnader i genuttryck analyserades proverna med Illumina helgenom microarray och multivariat dataanalys. Avhandlingens andra del innehåller två studier om mikrovesiklar och exosomer. Odlade hjärtmuskelceller (HL-1) stimulerades med tillväxt-faktorer (TGF-β2 eller PDGF BB) och ostimulerade celler användes som kontroll. Mikrovesiklar och exosomer renades fram med centrifugeringar och ultracentrifugering av cellodlingsmediet för att sedan karakteriseras med olika metoder för att studera storlek, ytmarkörer och innehåll. Illumina helgenom microarray användes för att studera microvesiklarnas och exosomernas mRNA innehåll. Resultat: I de två olika microarraystudierna hittades gentranskript och grupper av gentranskript som skiljde sig mellan kontroller och den hypertrofa hjärtvävnaden. När HOCM patientproverna jämfördes med kontroller hittades nedreglering av MYH6, EGR1 och FOS samt uppreglering av ACE2, JAK2 och HDAC5. Efter multivariat dataanalys av materialet från råtta, hittades 5 grupper av gentranskript med intressanta mönster som kunde kopplas till den hypertrofiska utvecklingen av hjärtmuskeln: "Ateroskleros", "ECM och adhesionsmolekyler", "Fettsyrametabolism", "Glukosmetabolis-men" och "Mitokondrien". Mikrovesiklarna hade en diameter på 40-300 nm och innehöll kromosomala DNA-sekvenser. När mikrovesiklarna överfördes till en annan celltyp (fibroblaster) resulterade det i ett förändrat genuttryck i fibroblasterna. Exosomer från hjärtmuskelcellerna som odlats med eller utan tillväxtfaktor hade en diameter på 50-80 nm. En stor pool av olika gentranskript var gemensam för alla exosomer oavsett stimulering eller ej. En mindre pool av gentranskript varierade i innehåll mellan de stimulerade och ostimulerade hjärtmuskelcellerna. I den gemensamma gentranskript poolen var ca 14 % ribosomala, ca 14 % var okända och ca 5 % var associerade till mitokondrien och dess funktion. Slutsats: Microarraystudierna visade att transkriptionsreglering i ett stabilt skede av hypertrofiutvecklingen är en balans mellan pro- och anti-hypertrofiska mekanismer och att olika gengrupper var olika reglerade vid olika tidpunkter i hjärtmuskeltillväxten. OPLS-DA är ett mycket användbart och kraftfullt verktyg när man analyserar genexpressionsdata, särskilt för att hitta grupper av gen-transkript som är svåra att upptäcka med traditionell statistik. Microvesikel- och exosomstudierna visade att mikrovesiklar och exosomer som frisätts från hjärtmuskelceller innehåller både DNA och RNA och kan vara inblandade i händelserna i målceller genom att underlätta en rad processer, inklusive ändringar av genuttryck. Olika stimulering av hjärtmuskelcellen kan påverka innehållet i exosomernas som produceras, vilket indikerar att exosomernas signalfunktion kan variera beroende på hjärtmuskelcellens tillstånd.

The adult heart is capable of remodelling in response to different pathological stimuli; in most cases, a phase of compensated hypertrophy evolves into frank dysfunction and heart failure. To identify microRNAs able to prevent cardiac hypertrophy and preserve cardiac function, we performed a high-content microscopy, high-throughput functional screening for human microRNAs able to reduce neonatal cardiomyocyte (CM) cell size using a whole-genome microRNA library. The most effective anti-hypertrophic microRNA was hsa-miR-665. In a model of transverse abdominal aortic constriction (TAC) in 8 weeks old CD1 mice (n=14 per group), AAV9-mediated delivery of miR-665 showed remarkable capacity to protect against pathological cardiac hypertrophy and preserve function over time. This effect was observed when the vectors were delivered either before (LVEF at 60 day after TAC: 51.3% ±5.8% in treated vs 34.82% ±0.77% in controls; P < 0.005) or after hypertrophy onset (LVEF at 60 days after TAC: 57.5%±5.60% in treated vs 28.4%±15% in controls; P < 0.001). Global mRNA changes in hearts treated with miR-665 were evaluated by mRNA deep sequencing. All the 43 genes, for which siRNA were available, out of the 67 genes that were found to be significantly expressed ≤2 fold over control were individually down-regulated by specific siRNAs and tested for being direct miR-665 targets. This approach identified three sarcomeric proteins as direct mediators of miR-665 activity, namely Enah, Fhl1 and Xirp2, which are known to be involved in sarcomeric mechanotransduction and myofibrillar remodelling. In conclusion, miR-665 represents an important tool to decipher the molecular mechanisms of hypertrophy and offers a potential lead for the development of new biotherapeutics.

Cardiac hypertrophy may be associated with an enhanced susceptibility to ischaemic/reperfusion injury but the mechanisms remain unresolved. There is evidence for an increased dependence on glucose metabolism in cardiac hypertrophy, which may be beneficial in normoxia but detrimental in ischaemia. The role of glycogen, the major endogenous substrate during ischaemia, to the enhanced susceptibility of the hypertrophied heart to ischaemic/reperfusion injury is unclear. Work in this thesis investigates the role of glycogenolysis to the severity of ischaemia, and assesses oxidative substrate utilisation following reperfusion, in the hypertrophied heart. Pressure overload cardiac hypertrophy was induced surgically in male Sprague-Dawley rats by intra-renal constriction. A moderate hypertrophy was observed nine weeks post surgery as evidenced by between a 4 and 25 % increase in heart weight: tibia length ratio. Hearts were perfused in an isovolumic mode, and function was recorded. ¹³C-NMR spectroscopy was performed on extracts from hypertrophied and control hearts reperfused with ¹³C labelled substrates to determine the profile of substrate use. Glycogen content was unchanged in hypertrophied hearts compared to control hearts and there was no evidence for glycogen loading in the presence of physiological substrates and insulin. In addition, no further glycogen loading occurred when insulin concentrations were increased to pharmacological levels. Provision of other carbohydrate substrates, such as lactate, did result in a further increase in myocardial glycogen content. Hypertrophied hearts experienced the same extent of ischaemia as controls with no evidence of increased ischaemic injury, implying that a compensated model of hypertrophy was generated in this study. Myocardial function decreased during low flow ischaemia and stopped during global ischaemia, but contracture was not observed. The severity of ischaemia was the determining factor in the degree of glycogen degradation. Increased glycogen degradation during ischaemia did not correlate with increased ischaemic injury, suggesting that the availability of glycogen for energy provision limited ischaemic injury. Recovery on reperfusion was markedly improved in the presence of insulin. This improvement appeared to be mediated by the inotropic actions of insulin rather than by alterations in substrate provision. The profile of substrate use in hypertrophied hearts during reperfusion was found to be the same as that in controls. No metabolic alterations were observed in the hypertrophied heart that enhanced susceptibility to ischaemic/reperfusion injury, implying that compensated hypertrophy is a beneficial response of the heart.

Rationale: Cardiac hypertrophy is the enlargement of the heart and can be induced by pathological and non-pathological events. The Fragile X family of RNA-binding proteins have been shown to be involved in cardiac structure and development (Mientjes et al, 2004) (Padje et al, 2009). Potential links between FXR1 and hypertrophy have not been significantly studied. Objective: To study the effect that varying expression of FXR1 has upon hypertrophy, and the molecular role FXR1 plays in hypertrophy. Method and Results: Following a voluntary running protocol, FXR1 wild-type mice had significant cardiac hypertrophy while FXR1 heterozygous mice (reduced FXR1 expression) had a blunted response with no significant hypertrophy. Cardiomyocyte size analysis showed that FXR1 overexpression (~9-fold increase) caused significant size reduction and FXR1 knockdown caused significant size increase. RNA immunoprecipitation showed multiple components of the PI3K/AKT/mTOR pathway associating with FXR1 in a protein/RNA complex. Western blotting showed that exercised FXR1 heterozygous mice had an increased expression of phosphorylated AKT versus non-exercised wild-type mice. Varying FXR1 expression in cells did not affect p-AKT expression. Conclusions: The amount of FXR1 in mice and cells appears to alter the severity of cardiac hypertrophy. FXR1 may regulate cardiac hypertrophy, but how this occurs is still unclear.

Abstract Cardiac loading induces left ventricular hypertrophy and cardiac remodeling which when prolonged, leads to heart failure, a complex syndrome affecting approximately 1-2% of the adult population of the Western world with a prevalence increasing with age. Pathological remodeling involves functional and structural changes that are associated with fetal gene expression, sarcomeric re-organization, hypertrophy of cardiomyocytes, fibrosis, inflammation, oxidative stress and impairment of metabolism. The aim of this study was to investigate the role of three novel factors during the cardiac remodeling process with different experimental models of cardiac overload. Phosphatase and actin regulator 1 (Phacr1) expression was rapidly downregulated due to myocardial infarction (MI). Adenovirus-mediated Phactr1 overexpression changed the skeletal α-actin to cardiac α-actin ratio in both healthy and infarcted rat hearts and cultured cardiomyocytes. Phactr1 could regulate the actin isoform switch via the serum response factor (SRF). The expression of transforming growth factor (TGF)- β-stimulated clone 22 (TSC-22) was rapidly induced by multiple hypertrophic stimuli and was also evident post-MI. In addition, TSC-22 could regulate collagen 3a1 expression in the heart. The expression of retinal degeneration 3-like (Rd3l) was downregulated in response to pressure overload and also downregulated post-MI. Rd3l knockout mice expressed increased myocyte hypertrophy and cardiac dysfunction in response to a transverse aortic constriction (TAC) induced pressure overload. This thesis provides novel information about Phactr1, TSC-22 and Rd3l in load-induced cardiac hypertrophy and remodeling. Collectively these studies increase our understanding of the regulatory mechanisms underlying the progression of heart failure
Tiivistelmä Sydämen kuormitus saa aikaan vasemman kammion liikakasvun eli hypertrofian ja sydämen uudelleenmuovautumisen, mikä pitkittyessään johtaa sydämen vajaatoimintaan. Sydämen vajaatoiminta on monimutkainen oireyhtymä, josta länsimaissa kärsii noin 1-2 % aikuisväestöstä, ja esiintyvyys nousee iän myötä. Patologisessa uudelleenmuovautumisessa tapahtuu toiminnallisia ja rakenteellisia muutoksia, joihin liittyy muutoksia geenien ilmentymisessä, sarkomeerin uudelleen järjestäytymistä, sydänlihassolujen koon kasvua, fibroosia, tulehdusta, oksidatiivista stressiä ja aineenvaihdunnan huonontumista. Tämän työn tarkoituksena oli tutkia kolmen uuden tekijän roolia sydämen uudelleenmuovautumisessa erilaisissa kokeellisissa sydämen kuormituksen malleissa. Fosfataasin ja aktiinin säätelijä 1:n (Phactr1) ilmentyminen väheni nopeasti infarktin seurauksena. Adenovirusvälitteinen Phactr1:n ylituotanto muutti luusto- ja sydänlihasaktiinien isomuotojen suhdetta sekä terveessä että infarktisydämessä, samoin viljellyissä sydänlihassoluissa. Phactr1 saattaa säädellä isomuotojen suhdetta seerumiresponsiivisen tekijän (SRF) avulla. Transformoituvan kasvutekijä β1:n stimuloima proteiini 22:n (TSC-22) ilmentyminen nousi nopeasti usean hypertrofisen stimuluksen seurauksena sekä infarktin jälkeen. Lisäksi TSC-22 voisi säädellä kollageeni 3a1:n ilmentymistä sydämessä. Retinan degeneroituvan proteiinin 3 kaltaisen tekijän (Rd3l) ilmentyminen väheni sekä painekuormituksen että infarktin seurauksena. Rd3l-poistogeenisillä hiirillä aortan ahtauman aiheuttama painekuormitus sai aikaan lisääntynyttä sydänlihassolujen hypertrofiaa ja sydämen toimintahäiriöitä. Tämä väitöskirjatutkimus tuo uutta tietoa Phactr1-, TSC-22- ja Rd3l-geeneistä kuormituksen aiheuttamassa sydämen hypertrofiassa ja uudelleenmuovautumisessa. Nämä tulokset auttavat osaltaan ymmärtämään monimutkaisia molekyylitason mekanismeja, jotka johtavat sydämen vaajatoiminnan kehittymiseen

Myocardial transcriptional response to stress is critically dependent on availability of the histone acetyltransferase p300. p300 is a transcription coactivator which is rapidly induced by hemodynamic stress in the myocardium. This induction is both necessary and sufficient to drive myocardial hypertrophy in vivo. Although short-term elevation of p300 may be adaptive, sustained elevation of p300 increases the risk of heart failure. The mechanism of p300 induction during stress is unknown. The downstream effectors of p300 have been only incompletely elucidated. In this context, the role of microRNAs in regulating p300-driven cardiac hypertrophy can be of potential interest. MicroRNAs are short ~18-25 nucleotide non-coding RNAs that inhibit gene expression by inducing messenger RNA cleavage and blocking translation. Engagement of microRNAs by various transcription factors/coactivators is an important regulatory mechanism in angiogenesis and hypertrophy. However, the extent to which microRNAs act as downstream effectors and/or modulators of the p300-driven hypertrophic response is unknown. Preliminary data showed that miR20a, miR142 and miR374-5p were regulated during p300-driven cardiac hypertrophy and in acute ischemia. In addition, these microRNAs may target p300 as part of a feedback loop as p300 is one of the predicted targets. The goal of this project was to dissect the role of a subset of microRNAs including miR-20a, miR142 and miR374 during p300 regulated hypertophic growth in vitro and in vivo and to verify its targets. This study is important to delineate the role of microRNAs in the p300-dependent signaling network in the myocardium and may result in therapeutic benefit.

The heart has the remarkable ability to adjust in response to varying stress stimuli and myocardium enlargement, referred to as cardiac hypertrophy, is a common form of stress adaptation. Divergent forms of hypertrophy can occur depending on the type and duration of the insult. The beneficial physiological form of hypertrophy is reversible and leads to improved cardiac function, while the pathological form is a maladaptive process that often transitions to heart failure. As a result of the prominence of cardiac disease, investigations into methods of reducing this detrimental form of cardiac remodeling are sought. Interestingly, pathological cardiac hypertrophy shares common features with the regulated form of cell death referred to as apoptosis. Here, we describe an essential role for apoptotic caspase-dependent signaling in the induction of pathological cardiac hypertrophy. Initially, we discovered that primary cardiomyocytes treated with hypertrophy agonists display transient activation of intrinsic-mediated apoptotic-signaling, including caspase 9 and caspase 3 activity. The necessity of functional caspase activation in hypertrophic signaling was shown by both in vitro and in vivo methods. We further investigated caspase cleavage targets histone deacetylase 3 (HDAC3) and gelsolin (GSN). HDAC3 cleavage was observed during early stages of hypertrophy and reduced in the presence of a caspase inhibitor. Caspase-mediated GSN cleavage occurred at latter stages, coincident with the cytoskeletal alterations that occur during this process. We demonstrated the requirement of GSN and its caspase-mediated processing by use of GSN expressing adenoviruses (AdVs). Use of a non-cleavable GSN-AdV provided evidence for not only the requirement of GSN in the hypertrophic response, but also for caspase mediated GSN cleavage. This body of work implicates caspase pathways and their targets as inductive signaling cues for pathological cardiac hypertrophy. These observations suggest that inhibitors that mute or suppress caspase activity and/or activity of its cognate substrates may offer novel therapeutic targets to limit the development of pathological hypertrophy.

Cardiac hypertrophy is an adaptive response in which the heart grows to normalize output during times of increased demand. This increase in size originates from the growth of cardiomyocytes rather than cellular division. Many cellular modifications observed during hypertrophy are reminiscent of apoptosis; caspase proteases, traditionally known for their role in apoptosis, have recently been implicated in non-apoptotic settings including cardiac differentiation. Studies have reported caspase-3 inhibition limits the heart`s ability to undergo pathological hypertrophy in vivo. Data presented here indicate that inhibition of caspase-3 and caspase-8 minimizes hypertrophic growth in primary cardiomyocytes. Phenylephrine induced an increase in cell size, which was attenuated upon addition of caspase inhibitors. These data suggest these proteins may be involved in hypertrophic growth of cardiomyocytes. Furthermore, results suggest that increased caspase activity may not be directly responsible for this effect. Rather, subcellular localization of caspase proteases may contribute to the effects seen during hypertrophy.

p38-mitogen activated protein kinases (p38-MAPKs) are stress activated serine/threonine kinases that are activated during several different cardiac pathologies. Classically, studies have focused solely on p38α signaling in the heart. However, there is also high cardiac expression of the p38γ isoform but little is known about its cardiac function. The aim of this study was to elucidate the signaling pathway of p38γ, with a particular focus on its role in the progression of pathological cardiac hypertrophy. Comparisons of cardiac function and structure of wild type (WT) and p38γ knock out (KO) mice, in response to abdominal aortic banding, found that KO mice developed less ventricular hypertrophy than their corresponding WT controls, and have preserved cardiac function. Basal p38γ myocardial staining was primarily localised at the membranes and throughout the cytoplasm. Following aortic constriction, nuclear staining of p38γ increased, but no accumulation of p38α was observed. This suggests that the two isoforms play distinct roles in the heart. To elucidate its signaling pathway, we generated an analogue sensitive p38γ, which is mutated at a gatekeeper residue, to specifically track and identify its endogenous substrates in the myocardium. The mutation allows only the mutant kinase, but not WT kinases, to utilise analogues of ATP that are expanded at the N6 position and contain a detectable tag on the γ-phosphate. Transfer of this tag to substrates allows subsequent isolation and identification. Furthermore, unlike other p38-MAPKs, p38γ contains a C-terminal PDZ domain interacting motif. We have utilised this motif in co pull-down assays to identify interacting proteins of p38γ in the heart. Using these techniques we have identified, amongst other substrates, LDB3 and calpastatin as novel substrates of p38γ and we have determined the residues that are targeted for phosphorylation. Lastly we have shown that phosphorylation of calpastatin reduces its efficiency as a calpain inhibitor in vitro, hence proposing a mechanism by which p38γ may mediate its pro-hypertrophic role.

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed July 28, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Doctor of Philosophy
The overall aim of this thesis was to determine the role of telomeres in the development of cardiac hypertrophy. It was hypothesised that changes in cardiomyocyte telomere length and/or maintenance cause cardiac hypertrophy.

Das Ziel der vorliegenden Arbeit war die Identifizierung von Geschlechterunterschieden (GU) in der Expression von miRNAs im späten Stadium der Myokardhypertrophie, sowie der möglichen Rolle von ERbeta bei der Regulierung dieser GU. Unsere früheren Studien identifizierten ERβ als determinierenden Faktor für die beobachteten GU bei Druckbelastung. Unter anderem führte eine Deletion des ERbeta zur Aufhebung der zuvor beobachteten GU auf physiologischer und fibrotischer Ebene, sowie in der Genexpression. In dieser Studie wurden insgesamt 30 miRNAs mit Geschlechter- und/oder Geschlecht*Operation-Interaktionseffekten 9 Wochen nach TAC in WT Mäusen identifiziert. Die gleichen Effekte waren in ERbeta-/- Tieren nicht zu beobachten, teilweise aufgrund einer höheren Expression dieser miRNAs in ERbeta-/- Weibchen als bei den Männchen. Die vorliegende Studie zeigt eine Hemmung vieler miRNAs durch Östrogen (E2) und seine Rezeptoren in weiblichen Kardiomyozyten, welches somit die in vivo-Ergebnisse bestätigt und die protektive Rolle von E2 und ERβ im weiblichen Herzen unterstreicht. Sechs der miRNAs mit GU in WT-, aber nicht in ERbeta-/- Hypertrophie-Modellen wurden als mögliche Fibroseregulatoren identifiziert, da ihnen gemeinsame Inhibitoren des ERK-MAPK-Signalwegs als Zielgene vorhergesagt wurden. Die Expression dieser miRNAs, miR-106a, miR-106b, miR-21, miR-24, miR-27a und miR-27b, war in kardialen Fibroblasten durch E2 geschlechterabhängig reguliert. Zusammengefasst bestätigt diese Arbeit die schützende Rolle von E2 und ERbeta im weiblichen Herzen. E2 und seine Rezeptoren hemmen die Expression vieler miRNAs in weiblichen Kardiomyozyten und kardialen Fibroblasten, sowie in vivo. In männlichen Herzen und kardialen Fibroblasten scheint ERalpha der Hauptakteur zu sein, welcher insbesondere mögliche Fibrose-bezogene miRNAs reguliert. Die verschiedenen Rollen der ERs in weiblichen und männlichen Herzen sind ein bestimmender Faktor der beobachteten GU bei Myokardhypertrophie.
The present study aimed to identify sex-differently expressed miRNAs in a late stage of hypertrophy (9 weeks) and the possible role of ERs in the regulation of these differences. Our previous studies identified ERbeta as an important determinant factor of the observed sex differences in pressure overload, playing different roles in males and females. This report identified a total of 30 miRNAs with sex and/or sex*surgery interaction effect 9 weeks after TAC in WT mice. The same effects were not observed in ERbeta-/- animals partially due to the higher expression of these miRNAs in ERbeta-/- females than in their WT counterparts. This study reveals a repression of a number of miRNAs by estradiol and its receptors alpha and beta in female cardiomyocytes, confirming the in vivo results and accentuating the important protective role of oestrogen and ERbeta in the female heart. Six of the miRNAs with sex differences in WT but not in ERbeta-/- hypertrophy models were found to be possible fibrosis regulators by putatively targeting common ERK/MAPK pathway inhibitors. MiR-106a, miR-106b, miR-21, miR-24, miR-27a and miR-27b were subjected to a different regulation by estradiol in cardiac fibroblasts in a sex-dependent manner. In conclusion, this study reinforces the oestrogen and ERbeta protective roles in the female hearts. Estradiol and ERs repress many miRNAs’ expression in both female cardiomyocytes and cardiac fibroblasts, as well as in vivo. In male hearts and cardiac fibroblasts, ERalpha is apparently the major player, regulating in particular potential fibrosis –related miRNAs. The different roles of ERs in male and female hearts are a determinant factor of the observed sex differences in cardiac hypertrophy.

Homer proteins are a family of scaffolding proteins involved in many intracellular signaling pathways, in both excitable and non-excitable cells. These proteins participate in the assembly and regulation of functional signaling complexes, facilitating the cross-talk between surface membrane receptors and channels in the membranes of intracellular compartments (Worley PF. et al., 2007). Homer proteins are constitutively expressed in the brain, where their scaffolding function is important for a variety of neuronal processes, such as intracellular Ca2+ homeostasis, synaptic plasticity associated with learning and memory in the mature brain, and neuronal development of the embryonic brain (Xiao B. et al., 1998; Worley PF. et al., 2007; Foa L. et al., 2009). Among the Homer splice variants, Homer 1a isoform acts as a natural dominant-negative by disassembling signalling complexes mediated by other Homer isoforms. The Homer 1a gene is transcribed as an immediate early gene (IEG), in neuronal cells its expression is low under normal conditions and increases rapidly after neuronal activation (Brakeman PR. et al., 1997). Homers proteins are also expressed in cardiac muscle, but their regulation and function remain still poorly understood. Despite their important role as regulators of multimeric signalling complex in nervous system, few reports have focused on the role of Homers in the heart. It has been reported that mRNA coding for Homer 1a rapidly and transiently increases in neonatal cardiomyocytes upon stimulation with either endothelin-1 (ET1) or other hypertrophic agonists (Kawamoto T. et al., 2006). The Homer 1a protein levels are also up-regulated following AngII-induced hypertrophy in neonatal cardiomyocytes (Guo WG. et al., 2010). Recently, it has been demonstrated that the variant Homer 1b/c positively regulates α1-adrenergic dependent hypertrophy, whereas Homer 1a is able to antagonize such effect (Grubb DR. et al., 2011). This study investigated the role of Homer 1a in the cardiac hypertrophic program. Our working hypothesis is that Homer 1a may be one of the molecular modulators of cardiac hypertrophy. For this purpose, we studied the presence, sub-cellular distribution and function of Homer1a in cardiac muscle. Under resting conditions we found that Homer 1a is constitutively expressed in cardiac muscle of both mouse and rat and in HL-1 cells (a specific cardiac cell line). In addition, using immunofluorescence confocal microscopy of adult rat heart sections, we showed that Homer 1a displays a peculiar localization: it is sarcomeric and peri-nuclear. We also analyzed Homer 1a expression under hypertrophic conditions. For this purpose, we used rat neonatal cardiomyocytes stimulated with the adrenergic agonist norepinephrine (NE). A significant increase in both Homer1a mRNA and protein was found after NE stimulation, whereas Homer 1b/c (a different Homer 1 isoform) expression remained unchanged. In this hypertrophic cellular model, we studied the adrenergic pathways involved in NE-inducted Homer 1a up-regulation by using specific α1- and β- adrenergic receptor blockers (prazosin and propranolol, respectively). The results showed that prazosin - but not propranolol - drastically reduced NE-induced up-regulation of Homer 1a mRNA, demonstrating that the α1-adrenergic pathway is involved. The effect of hypertrophic stimulation on Homer 1a expression was also confirmed in NE-stimulated HL-1 cardiomyocytes. In this cell line we found that 1 hour after NE stimulation Homer 1a content increased by a factor of 2.5. Overall, these results confirm our working hypothesis and demonstrate the involvement of Homer 1a in the α1-adrenergic pathway leading to cardiac hypertrophy. In the second part of the study we analyzed the effects of Homer 1a over-expression monitoring different hypertrophic markers, such as MAPK/ERK1/2 phosphorylation, NFAT nuclear translocation, ANF-promoter activity and increase in cell size. The results showed that during NE stimulation Homer 1a modulated many of them (except for NFAT nuclear translocation that did not appear to be affected by Homer 1a over-expression), whereas under resting conditions Homer 1a over-expression per sè was ineffective. In particular, we found that, in NE-stimulated HL-1 cells, over-expressed Homer 1a significantly reduced phosphorylation levels of ERK1/2 by about 40%, negatively modulating MAPK pathway. As regards the ANF promoter activity, this activity was significantly reduced by about 20% in NE-stimulated Homer 1a over-expressing cells. In order to verify the specificity of the Homer 1a effect on ANF, we performed the same experiment over-expressing Homer 1c and we found that, unlike Homer 1a, Homer 1c did not modulate the activity of ANF promoter in NE-stimulated HL-1 cells. Subsequently, we assessed the effect of Homer 1a over-expression on increase in cell size. The results obtained showed that Homer 1a counteracted the increase in NE-stimulated cell size. Finally, a preliminary analysis, in vivo, of Homer 1a expression was performed in three hypertrophic models, i.e. mice with chronic transverse aortic constriction, transgenic mice over-expressing Gαq and rats treated with monocrotaline. At variance with results observed in cellular models in vitro, in these models Homer 1a expression did not result affected by hypertrophic conditions, at least in the time span under investigation. However, for this approach in vivo, a broad time-course is needed and, therefore, further analyses are required. In summary, our data on Homer 1a presence and sub-cellular localization in cardiac tissue demonstrate that Homer 1a is constitutively expressed and displays a sarcomeric and peri-nuclear distribution. In our cellular models in vitro, Homer 1a up-regulation is an early event of the NE-induced hypertrophy and, as inferred from gain-of function studies, Homer 1a isoform antagonizes initiation and development of NE-induced events leading to α1-adrenergic-dependent hypertrophy. In conclusion, our results in vitro indicate that Homer 1a is inserted into a negative feedback mechanism in which acts as negative molecular modulator, counteracting early steps of hypertrophy. However, further studies are needed to elucidate the mechanisms underlying this process.
Le proteine Homer sono una famiglia di proteine coinvolte in molte vie di trasduzione del segnale intracellulare, in cellule eccitabili e non eccitabili. Queste proteine partecipano nell’assemblaggio e nella regolazione di complessi funzionali di ‘signalling’, facilitando il ‘cross-talk’ tra recettori della membrana plasmatica e canali posti sulle membrane dei compartimenti intracellulari (Worley PF. et al., 2007). Le proteine Homer sono costitutivamente espresse nel cervello, dove svolgono la funzione di ‘scaffold’ in molti processi neuronali, quali ad esempio l’omeostasi del calcio intracellulare, la plasticità sinaptica associata all’apprendimento ed alla memoria nel cervello maturo, lo sviluppo embrionale del cervello (Xiao B. et al., 1998; Worley PF. et al., 2007; Foa L. et al., 2009). Tra le diverse varianti di splicing alternativo, l’isoforma Homer 1a agisce da dominante negativo disassemblando i complessi di ‘signalling’ formati dalle altre isoforme Homer. Il gene Homer 1a è trascritto come gene immediato precoce, la sua espressione nelle cellule neuronali è bassa in condizioni basali ed aumenta rapidamente in seguito ad attivazione neuronale (Brakeman PR. et al., 1997). Le proteine Homer sono espresse anche nel muscolo cardiaco, ma la loro regolazione e la loro funzione è ancora poco conosciuta. Nonostante l’importanza degli Homer come proteine regolatrici di complessi coinvolti nelle vie di trasduzione del segnale, pochi studi si sono focalizzati sul loro ruolo nel cuore. A tal riguardo, è stato riportato che l’mRNA codificante per Homer 1a aumenta rapidamente e transientemente in colture di cardiomiociti neonatali in seguito a stimolazione con endotelina-1 ed con altri agonisti ipertrofici (Kawamoto T. et al., 2006). Un successivo lavoro ha evidenziato che, in condizioni di ipertrofia indotta da angiotensina II, anche i livelli di espressione della proteina Homer 1a risultano up-regolati in colture di cardiomiociti neonatali (Guo WG. et al., 2010). Un recente studio ha, invece, dimostrato che l’isoforma Homer 1b/c regola positivamente l’ipertrofia dovuta a stimolazione α-adrenergica, mentre l’isoforma Homer 1a antagonizza tale effetto (Grubb DR. et al., 2011). In questo studio abbiamo esaminato il ruolo della proteina Homer 1a nell’ipertrofia cardiaca. La nostra ipotesi di lavoro è che la proteina Homer 1a sia un modulatore molecolare dell’ipertrofia. A tal fine, abbiamo studiato la presenza, la localizzazione sub-cellulare e la funzione di Homer 1a nel muscolo cardiaco. Analizzando l’espressione di Homer1a in condizioni normali è emerso che la proteina Homer 1a è espressa costitutivamente nel muscolo cardiaco di topo e ratto e nelle cellule HL-1 (una specifica linea cellulare cardiaca). Mediante immunofluorescenze su sezioni di cuore di ratto adulto (analizzate utilizzando il microscopio confocale) abbiamo esaminato la localizzazione sub-cellulare di Homer 1a che risulta essere sarcomerica e perinucleare. Successivamente, abbiamo analizzato l’espressione di Homer 1a in condizioni ipertrofiche; per questa analisi sono stati utilizzati cardiomiociti neonatali di ratto stimolati con l’agonista adrenergico norepinefrina (NE). In questo sistema sperimentale, abbiamo riscontrato un aumento significativo sia dell’mRNA che della proteina Homer 1a in seguito alla stimolazione con NE, mentre non abbiamo rilevato nessuna variazione sull’espressione della proteina Homer 1b/c (una diversa isoforma degli Homer). In cardiomiociti in coltura stimolati con NE, sono state, inoltre, analizzate le vie di trasduzione del segnale adrenergico coinvolte nell’up-regolazione di Homer 1a indotta da NE, usando specifici inibitori dei recettori α1- and β- adrenergici (prazosin e propanololo, rispettivamente). I risultati ottenuti hanno evidenziato che il prazosin, ma non il propranololo, drasticamente riduce l’up-regolazione dell’mRNA di Homer 1a indotta da NE, dimostrando che la via di trasduzione del segnale α1-adrenergico è coinvolta. L’effetto della stimolazione ipertrofica sull’espressione di Homer 1a è stato confermato anche su cellule HL-1 stimolata con NE. In questa linea cellulare abbiamo osservato che un’ora dopo la stimolazione con NE la proteina Homer 1a aumenta di un fattore 2,5. Complessivamente, questi risultati confermano la nostra ipotesi di lavoro e dimostrano il coinvolgimento della proteina Homer 1a nella trasduzione del segnale α1-adrenergico che induce ipertrofia cardiaca. Nella seconda parte di questo studio abbiamo esaminato gli effetti dell’over-espressione di Homer 1a monitorando diversi markers ipertrofici, quali la fosforilazione delle proteine MAPK/ERK1/2, la traslocazione nucleare di NFAT, l’attivazione del promotore di ANF e l’aumento delle dimensioni cellulari. I risultati hanno dimostrato che durante la stimolazione con NE Homer 1a modula la maggior parte di questi (eccezion fatta per la traslocazione nucleare di NFAT che non risulta essere variata dall’over-espressione di Homer 1a), al contrario in condizioni basali (senza stimolazione con NE) l’over-espressione di Homer 1a di per sé non ha alcun effetto. Nello specifico, i risultati ottenuti hanno rilevato che in cellule HL-1 stimolate con NE la proteina Homer 1a over-espressa significativamente riduce i livelli di fosforilazione delle proteine ERK1/2 di circa il 40%, modulando negativamente la via di trasduzione del segnale MAPK/ERK1/2. Per quanto concerne l’attività promotoriale di ANF, questa attività è significativamente ridotta di circa il 20% nelle cellule HL-1 over-esprimenti Homer 1a e stimolate con NE. Al fine di verificare la specificità di questo effetto sul promotore ANF, abbiamo condotto lo stesso esperimento over-esprimendo l’isoforma Homer 1c ed abbiamo riscontrato che, diversamente da Homer 1a, la proteina Homer 1c non ha alcun effetto sull’attività del promotore ANF in cellule HL-1 stimolate con NE. Successivamente, abbiamo analizzato l’effetto dell’over-espressione di Homer 1a sull’aumento delle dimensioni cellulari durante stimolazione con NE. I risultati ottenuti hanno dimostrato che la proteina Homer 1a è in grado di bloccare significativamente l’aumento delle dimensioni delle cellule HL-1 stimolate con NE. Nell’ultima parte di questo lavoro, abbiamo condotto un’analisi preliminare, in vivo, dell’espressione della proteina Homer 1a in tre modelli di ipertrofia, quali topi con costrizione trasversale dell’aorta, topi transgenici over-esprimenti Gαq e ratti trattati con monocrotalina. Diversamente da quanto ottenuto nel modello cellulare in vitro, in questi modelli l’espressione della proteina Homer 1a non risulta alterata dalle condizioni ipertrofiche, almeno nell’intervallo di tempo considerato. Tuttavia, per quanto riguarda questo approccio in vivo, sarà necessario analizzare l’espressione della proteina Homer 1a in un intervallo di tempo più ampio e, di conseguenza, ulteriori analisi sono richieste. In sintesi, dai nostri risultati relativi alla presenza ed alla localizzazione sub-cellulare di Homer 1a nel tessuto cardiaco è emerso che la proteina Homer 1a è costitutivamente espressa e mostra una localizzazione sarcomerica e peri-nucleare. Nei nostri modelli cellulari in vitro, l’up-regolazione di Homer 1a è un evento precoce dell’ipertrofia indotta da NE e, come dimostrato dagli studi di gain-of fuction, la proteina Homer 1a è in grado di antagonizzare l’avvio e lo sviluppo degli eventi che portano all’ipertrofia α1- adrenergica dipendente. Concludendo, i nostri dati in vitro indicano che Homer 1a è inserito in un meccanismo di feedback negativo in cui agisce come modulatore negativo, bloccando gli steps precoci dell’ipertrofia cardiaca. Tuttavia, ulteriori studi sono necessari per definire il meccanismo alla base di questo processo.

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

Biomedical Sciences
Ph.D.
Increases in cardiac afterload caused by disease conditions results in remodeling of heart structure by hypertrophy and alterations in the molecular regulation of contractile performance. These adaptations can be regulated by various Ca2+-dependent signaling processes. STIM1 is an important regulator of Ca2+ signaling in different cell types by sensing endoplasmic reticular Ca2+ levels and coupling to plasma membrane Orai channels. The role of STIM1 in heart is not well understood, given the robust Ca2+ regulatory machinery present within cardiac myocytes. Previous reports indicate that STIM1 may play a role in regulation of cardiac hypertrophy. The goal of this work is to understand how STIM1 can affect contractile Ca2+ regulation in normal and diseased myocytes. We induced cardiac hypertrophy by slow progressive pressure overload in adult cats. Isolated adult feline ventricular myocytes (AFMs) exhibited increased STIM1 expression and activity, which correlated with altered Ca2+ handling. Use of BTP2 to block Orai channels resulted in a reduction of action potential (AP) duration and diastolic spark rate of hypertrophied myocytes, without affecting myocytes from sham-operated animals. Overexpressed STIM1 in cultured AFMs caused persistent Ca2+ influx that resulted in increased diastolic spark rates and prolonged APs, similar to myocytes from banded animals. STIM1 mediated Ca2+ influx could load the sarcoplasmic reticulum and activated CaMKII, which increased spark rates and lead to spontaneous APs. Importantly, STIM1 operated by associating with Orai channels because these effects could be blocked with either BTP2 or with a dominant negative Orai construct. Prolonged Ca2+ entry through this pathway eventually causes cell death. In conclusion, the work presented in this thesis establishes a role for STIM1-Orai in contractile Ca2+ regulation.
Temple University--Theses

Various pathological conditions that exert stress on cardiac muscle severely compromise the supply of oxygenated blood to peripheral tissues. To maintain steady cardiac output during adverse times, the myocardium is forced to undergo tissue remodeling in a process known as cardiac hypertrophy. The loss of cardiac myocytes during the transition to heart failure has been a subject of intense investigation, and this loss is hypothesized to originate from apoptosis/programmed cell death. In particular, caspase proteases have been implicated as primary components in this process. However, caspase activity has been linked to many cellular remodeling events independent of inducing cell death. Therefore, the possibility that alterations in caspase activity precipitate the cellular alterations that give rise to pathological cardiac hypertrophy was investigated in the current study: Using cultures of primary neonatal cardiomyocytes and a combination of pharmacological and biological inhibitors it is demonstrated that: (a) Physical growth of a myocyte is not dependant on activity of the apoptotic caspases; (b) Caspase activity is not necessary for the transcriptional control of natriuretic peptide production (ANF and BNP); (c) Transcriptional activity of a general stress-responsive transcription factor Nf-kB is dependent on caspase enzymatic activity in the neonatal cardiomyocyte.

This thesis aims to investigate differential changes in Ca2+ and Na+ regulation during the development from cardiac hypertrophy to heart failure (HF) between sexes. Clinical evidences show females are more resistant to the development of cardiac hypertrophy and have better survival in HF than males. Oestrogen is postulated to provide cardioprotection although this is still under debate. This work used guinea pigs (GPs), a species with electrophysiology akin to human, that were subjected to aortic constriction (AC) to study the progression from pressure-overload cardiac hypertrophy to HF between sexes. Selected female animals underwent ovariectomy (OVx), mimicking postmenopausal status, to examine the effects of long-term deprivation of ovarian hormones. The effect of oestradiol supplementation was also investigated. Ventricular myocytes isolated from hearts at cardiac hypertrophy had prolonged action potential duration (APD), increased Ca2+ transient amplitudes and SR Ca2+ content, reduced Na+/K+ ATPase (NKA) function and increased late sodium current (INa,L). Fractional shortening (FS) remained unchanged in these hearts. Compromised FS with detrimental Ca2+ handling, more reduced NKA function and enhanced INa,L were noted at HF. Males showed earlier declined NKA function, more compromised FS and more detrimental Ca2+ handling than females at HF. Ventricular myocytes from OVx animals showed increased L-type Ca2+ channel current with gating shifts and larger window current, larger Ca2+ transient amplitudes, greater SR Ca2+ content, and increased Ca2+ sparks and waves. OVx myocytes showed more early and delayed afterdepolarisations (EADs and DADs) with DAD-induced extrasystoles following β-adrenergic stimulation. AC with OVx GPs showed more reduced FS, more dysregulated Ca2+ handling, more reduced NKA function and larger INa,L than AC females. In conclusion, females were more resistant to pressure-overload. Long-term deprivation of ovarian hormones abolishes the slower onset of HF in females, and provides pro-arrhythmic substrates to females. Oestradiol supplementation offered protective effects on OVx GPs.

Myocardial protective strategies during cardiac surgery continue to improve yet they remain imperfect. Patients with left ventricular hypertrophy (LVH) are considered to be at greater risk of myocardial injury post cardiac surgery. Perhexiline is an anti-anginal agent known to modulate myocardial metabolism towards a more efficient glucose metabolic pathway. This metabolic modulation may improve myocardial protection. In this thesis I present a multi-centre double-blind randomised placebo controlled trial evaluating the role of perhexiline as an adjunct to standard myocardial protection in patients with LVH secondary to aortic stenosis undergoing an aortic valve replacement. Perhexiline does not augment myocardial protection. Magnetic Resonance Spectroscopy based energetic studies, echocardiographic and functional assessments in a homogenous patient cohort show no added benefit with perhexiline therapy in LVH. Therefore perhexiline should be limited to those patients refractory to maximum medical therapy. Metabolomic assessment of LVH has shown no change in the metabolomic profile within the myocardium. However any changes that do exist may be subtle. In LVH there is an increased activity of some innate cardioprotective mechanistic pathways in patients that do not sustain a low cardiac output episode post cardiac surgery. Further examination of these cardioprotective regulators is warranted.

The a of TNFa in title is the Greek alpha.
Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Background: Cardiac hypertrophy is an adaptive process occurring in response to mechanical overload or tissue injury. The stimuli for cardiac hypertrophy are diverse and vary from increased afterload on the heart to cardiac remodeling in response to cytokines. Amongst others, obesity is characterized by excessive body weight resulting in metabolic disorders. This excess body weight necessitates an increased blood and oxygen delivery to the peripheral tissues, which is achieved by an elevated cardiac output. Total blood volume is also increased in the obese due to the increased tissue volume and vascularity. With time, the obesity induced increase in cardiac preload results in left ventricular hypertrophy and dilatation. Obesity is also associated with complications such as hypertension, insulin resistance and impaired glucose metabolism. In addition, adipose tissue has been implicated to contribute to elevated circulating TNFa levels in obesity and may contribute to the pathophysiology of the heart in obese individuals. The heart is a major cytokine-producing organ that generates amongst others tumor necrosis factor a (TNFa). TNFa is a proinflammatory cytokine, which acts to increase its own production, has cytotoxic and cytostatic effects on certain tumor cells and influences growth and differentiation in virtually all cell types including cardiomyocytes. Elevated levels of TNFa are detected peripherally in almost all forms of cardiac injury and in hypertrophic cardiomyopathy. These elevations are proposed to be deleterious to the heart, although an adaptive role for low levels of TNFa has been proposed. Aim: The aim of the study was to determine whether there is a correlation between obesity and serum, myocardial, and adipose tissue TNFa levels and cardiac hypertrophy. We also wished to determine whether the hearts from the obese animals functioned normally under normoxic conditions and whether they responded differently to ischaemia/reperfusion when compared with their concurrent controls. Materials and Methods: Male Sprague-Dawley rats (n=100) were fed a high caloric diet (HCD) containing 33% rat chow, 33% condensed milk, 7% sucrose and 27% water, or standard laboratory rat chow for 6-12 weeks. Food consumption, body weight gain, heart weight and tibia length were measured. Serum glucose, insulin and lipid levels were also determined. Hearts were excised and perfused on the isolated Working Heart perfusion apparatus and cardiac function was monitored and documented. Hearts were then subjected to 15 minutes of total global ischaemia at 370C, and reperfused for 30 minutes. Cardiac function was again documented. A separate series of hearts were freeze-clamped at different time points during the experimental protocol and stored in liquid nitrogen for the determination of myocardial TNFa and cGMP levels. Serum TNFa levels were determined after 12 weeks on the high caloric or normal/control diet. After 12 weeks on the diet myocardial TNFa levels of the HCD fed animals and their concurrent controls were determined before and during ischaemia. Adipose tissue and myocardial tissue TNFa levels were also determined after 6, 9 and 12 weeks on the respective diets. Myocardial cGMP levels were measured in the HCD fed rats and the control rats after 6, 9, and 12 weeks. These data were used as an indirect index to determine whether the myocardial NOcGMP pathway was activated in the normoxic hearts on the respective diets. Results: The body weight of the HCO fed animals was significantly higher compared with their respective controls after 12 weeks on the diet (459.9 ± 173.8 g and 271.5 ± 102.6 g respectively (p<0.05». The HCO fed animals also had heart weight to body weight ratios that were significantly greater compared with the controls (4.2 ± 0.1 mglg and 3.7 ± 0.1 mglg respectively (p<0.05». The plasma glucose levels of the HCO fed animals were higher than their respective controls (9.2 ± 0.3 mmoiII and 7.8 ± 0.3 mmoiII respectively (p<0.05)), but their insulin levels were similar (12.87 ± 1.02 IlIUlml and 12.42 ± 5.06 IlIU/ml). Plasma lipid profiles (plasma cholesterol, high density lipoprotein (HOL) cholesterol and plasma triacylglyceride (TAG)) were abnormal in the HCO fed animals compared with the control rats. Plasma TAG levels in the HCO fed animals were significantly higher compared with the control rats (0.664 ± 0.062 mmoiII and 0.503 ± 0.043 (p<0.05», while plasma cholesterol levels (1.794 ± 0.058 mmoIII and 2.082 ± 0.062 mmoiII (p<0.05» and HOL cholesterol levels were significantly lower (1.207 ± 0.031 mmoiII and 1.451 ± 0.050 mmoiII (p<0.05». Cardiac mechanical function was similar for both groups before ischaemia, but the percentage aortic output recovery was lower for the hearts from the HCO fed animals when compared with their controls (47.86 ± 7.87% and 66.67 ± 3.76 % respectively (p<0.05». Serum TNFa levels of the HCO fed animals were higher compared with the control animals (51.04 ± 5.14 AU and 31.46 ± 3.72 AU respectively (p<0.05», but myocardial TNFa levels remained lower in these animals (312.0 ± 44.7 pglgram ww and 571.4 ± 132.9 pg/gram ww respectively (p<0.05)). During ischaemia these myocardial TNFa levels increased above those of the controls (442.9 ± 12.4 pg/gram ww and 410.0 ± 12.5 pg/gram ww respectively (p<0.05)). The adipose tissue TNFa levels were significantly increased after 12 weeks on the high caloric diet compared with the control animals (4.4 ± 0.4 pg/gram ww and 2.5 ± 0.3 pg/gram ww respectively (p<0.05)). There was no significant difference in the myocardial cGMP levels of the HCD rats compared with the conrol rats after 6, 9 and 12 weeks. Conclusion: 1) The high caloric diet induced obesity, which lead to cardiac hypertrophy in this study. 2) There was a strong correlation between elevated adipose tissue and serum TNFa levels, and cardiac hypertrophy. 3) Elevated serum TNFa levels did not lead to activation of the myocardial NO-cGMP pathway in the normoxic hearts in this model. 4) The hypertrophied hearts from the HCD fed animals had poorer post-ischaemie myocardial functions than their concurrent controls.
AFRIKAANSE OPSOMMING: Agtergrond: Miokardiale hipertrofie is In aanpassing wat gebeur as In gevolg van meganiese oorbelading of weefsel beskadiging. Verskillende stimuli kan tot miokardiale hipertrofie aanleiding gee soos byvoorbeeld In verhoging in nalading, of miokardiale hermodellering in respons op sitokiene. Verhoging van voorbelading in vetsug mag ook tot hipertrofie aanleiding gee. Vetsug word gekenmerk deur In oormatige liggaamsmassa wat tot metaboliese versteurings lei. Die oormatige liggaamsmassa vereis In verhoging in bloed- en suurstofverskaffing aan die perifere weefsel wat deur In verhoging in die kardiale uitset vermag kan word. Die bloed volume van In vetsugtige individu word ook verhoog as gevolg van In verhoging in weefselvolume en vaskulariteit en met verloop van tyd induseer die verhoogde kardiale voorbelading linker ventrikulêre hipertrofie en dilatasie. Vetsug word ook met verskeie ander siekte toestande soos hipertensie, insulien weerstandigheid en versteurde glukose metabolisme, geassosieer. Vetweefsel dra ook by tot verhoging van tumor nekrose faktor alfa (TNFa) vlakke in die bloed, wat op sy beurt tot miokardiale hipertrofie mag bydra. TNFa is In proinflammatoriese sitokien wat sy eie produksie kan stimuleer. Dit het ook sitotoksiese en sitostatiese effekte op sekere tumor selle en kan groei en differensiasie in bykans alle seltipes, insluitende kardiomiosiete, stimuleer. Die hart kan ook TNFa produseer en verhoogde TNFa vlakke word feitlik in alle vorms van miokardiale besering en hipertrofiese kardiomiopatie waargeneem. Daar word voorgestel dat verhoogde TNFa vlakke vir die hart nadelig is, ten spyte van die vermoeding dat die sitokien In potensiële aanpassings rol by laer vlakke het. Doelstelling: Die doel van hierdie studie was om vas te stelof daar 'n verband tussen vetsug en serum, miokardiale en vetweefsel TNFa vlakke en miokardiale hipertrofie, bestaan. Ons het ook gepoog om te bepaal of harte van vetsugtige diere normaal funksioneer en of die response van sulke harte op isgemie-herperfusie van die van ooreenstemmende kontroles verskil. Materiaal en tegnieke: Manlike Sprague-Dawley rotte (n=100) is vir 6-12 weke op 'n hoë kalorie dieët (HKD) geplaas. Die HKD het uit 33% rotkos, 33% gekondenseerde melk, 7% sukrose en 27% water bestaan. Kontrole diere het standaard laboratorium rotkos ontvang. Voedselinname, liggaamsmassa toename, serum insulien, glukose en lipied vlakke is ook bepaal. Harte is geïsoleer en geperfuseer volgens die Werk Hart perfusie metode en hart funksie is gemonitor en gedokumenteer. Harte is vervolgens aan 15 minute globale isgemie by 3rC blootgestel en daarna weer vir 30 minute geherperfuseer waartydens hartfunksie weer gedokumenteer is. 'n Aparte groep harte is op spesifieke tydsintervalle gedurende die eksperimentele protokol gevriesklamp en in vloeibare stikstof gestoor vir die bepaling van miokardiale TNFa en sGMP vlakke. Serum TNFa vlakke is bepaal na 12 weke op die dieët. Na die diere 12 weke op die HKD was, is hierdie diere en hulooreenstemmende kontroles se miokardiale TNFa vlakke voor en na isgemie bepaal. Vetweefsel en miokardiale TNFa vlakke is ook onderskeidelik na 6, 9 en 12 weke bepaal. Miokardiale sGMP vlakke is in die HKD diere en in die kontrole diere na 6, 9 en 12 weke bepaal. sGMP vlakke is gebruik as 'n indirekte indeks van aktivering van die miokardiale NO-sGMP boodskapper pad. Resultate: Na 12 weke op die dieët was die liggaamsmassa van die HKD diere beduidend hoër in vergeleke met hulooreenstemmende kontroles (459.9 ± 173.8 g en 271.5 ± 102.6 g (p<0.05)). Die HKD diere se hart massa tot liggaam massa verhouding was ook beduidend hoër in vergelyking met die van kontroles (4.2 ± 0.1 mglg en 3.7 ± 0.1 mglg (p<0.05)). Alhoewel insulien vlakke dieselfde was (12.42 ± 5.06 j.lIU/ml en 12.87 ± 1.02 j.lIU/ml), was serum glukose vlakke van die HKD diere hoër as die van die ooreenstemmende kontroles (9.2 ± 0.3 mmoiii en 7.8 ± 0.3 mmoiii (p<0.05)). Plasma lipied profiele (HOL cholesterol, plasma cholesterol en trigliseriede) was abnormaal in die HKD diere. Plasma TAG vlakke in die HKD diere was beduidend hoër as die van die kontroles (0.664 ± 0.062 mmoiii en 0.503 ± 0.043 (p<0.05)), terwyl plasma cholesterol vlakke (1.794 ± 0.058 mmoiii en 2.082 ± 0.062 mmoiii (p<0.05)) en HOL cholesterol vlakke beduidend laer was (1.207 ± 0.031 mmoiii en 1.451 ± 0.050 mmoiii (p<0.05)). Miokardiale meganiese funksie was dieselfde vir beide groepe voor isgemie, maar die persentasie aorta omset herstel tydens herperfusie was laer in die HKD diere in vergelyking met die van kontrole diere (47.86 ±. 7.87% en 66.67 ± 3.76% (p<0.05)). Serum TNFa vlakke van die HKD diere was beduidend hoër as die van kontrole diere (51.04 ± 5.14 AU en 31.46 ± 3.72 AU (p<0.05)), maar miokardiale TNFa vlakke was laer (312.0 ± 44.7 pglgram nat gewig en 571.4 ± 132.9 pglgram nat gewig (p<0.05)). Die vetweefsel TNFa vlakke was ook beduidend verhoog na 12 weke op "n hoë kalorie dieët wanneer dit vergelyk word met die van kontrole diere (4.4 ± 0.4 pglgram nat gewig en 2.5 ± 0.3 pglgram nat gewig respektiewelik (p<0.05)). Daar was geenbeduidende verskille in die miocardiale vlakke van sGMP in die HKD diere in vergelyking met die kontroles na 6, 9 en 12 weke. Gevolgtrekkings: 1) "n Hoë kalorie dieët het in dié studie vetsug geïnduseer en tot miokardiale hipertrofie gelei. 2) Daar was "n positiewe korrelasie tussen verhoogde vetweefsel en serum TNFa vlakke, en miokardiale hipertrofie. 3) Verhoogde serum TNFa vlakke het nie tot die aktivering van die miokardiale NO-sGMP pad in hierdie model gelei nie. 4) Die hipertrofiese harte het tydens herperfusie ná isgemie swakker as hulooreenstemmende kontroles gefunksioneer.

Biomedical Sciences
Ph.D.
Pathological cardiac hypertrophy (PCH) occurs in response to pathological stimuli affecting the heart such as coronary artery disease, myocardial infarction, or hypertension. PCH is also be independent risk factor for cardiac events and/or sudden death. Despite therapeutic advancements in the treatment of cardiovascular diseases (CVD) and heart failure, deaths due to CVD remain the leading cause of mortality worldwide. Furthermore, treatment of these cardiovascular diseases slows their progression, but individuals eventually progress to heart failure, which has a 5-year survival rate of approximately 50 percent. There is a clear need for development of new therapies that can reverse PCH and the associated damage to the heart. As healthcare improves, populations are living longer, and illness due to age increases. One issue that occurs with aging is loss of normal cardiac function leading to heart failure. This functional decline is accompanied by morphological changes in the heart, including hypertrophy. Although it is well documented that myocardial remodeling occurs with aging, the mechanisms underlying these changes are poorly understood. Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor β (TGF-β) superfamily of proteins, which regulate a number of cellular processes. Shared circulation of a young mouse with an old mouse or a single daily intraperitoneal (IP) injection of GDF11 for 30 days was shown to reverse aging-induced pathological cardiac hypertrophy. This molecule is highly homologous with another TGF-β family member, myostatin, which is a known negative growth regulator of skeletal muscle. We began by attempting to validate published data claiming that a single daily intraperitoneal (IP) injection of 0.1 mg/kg/day of GDF11 could reverse aging induced cardiac hypertrophy. We performed a blinded study during which treated 24-month-old C57BL/6 male mice with a single IP injection of 0.1 mg/kg/day of GDF11for 28 days and monitored changes in cardiac function and structure using echocardiography (ECHO). We also looked for differences in fibrosis, myocyte size, markers of pathological hypertrophy and heart weight. We were unable to find any differences between vehicle treated age mice and GDF11 treated aged mice in any of the measured parameters. While we did find an increase in heart weight between 8-week-old mice and the 24-month-old mice, there was no difference in the heart weight to body weight ratios of these groups of animals. From these data we concluded that our aged- mice did not have pathological hypertrophy and the dose of GDF11 used in this study did not have any effect on cardiac structure or function. Hypertensive heart disease results in changes in cardiac structure and function including left ventricular hypertrophy, systolic and/or diastolic dysfunction. It is also a leading cause of heart failure. Members of the TGF-β superfamily of proteins have been shown to be involved in many of the processes that occur in the heart in response to hypertension, such as the fibrotic response. Although it was previously shown that treatment with 0.1 mg/kg of GDF11 did not prevent pressure overload induced cardiac hypertrophy, we found this dose was too low to alter cardiac structure in our aging study. In addition, a single GDF11 dose is insufficient to fully address this issue. We therefore performed a blinded dose-ranging study to investigate the effects of GDF11 on pressure overload induced cardiac hypertrophy using transverse aortic constriction (TAC) which mimics the effects of chronic hypertension on the heart. In this study, animals received TAC surgery and were assigned to treatment groups so that there were no differences in wall thickness, cardiac function, or pressure gradients across the aortic constriction at the start of the treatments 1 week after TAC. Mice were given 0.5 mg/kg/day of GDF11, 1.0 mg/kg/day GDF11, 5.0 mg/kg/day of GDF11, or vehicle via a single daily IP injection for 14 days. Using these higher doses, we found that GDF11 had dose dependent effects on both cardiac structure and function following TAC. Myocyte cross sectional area was dose-dependently decreased compared to vehicle treated mice in both sham and TAC conditions. Cardiac function was preserved in the 1.0 and 5.0 mg/kg groups treatment groups after TAC. Left ventricular internal chamber dimensions were preserved with the 1.0 mg/kg treatment group. Treatment with GDF11 caused a dose dependent decrease on both body weight and heart weight in both normal and TAC mice, but with an effect on heart weight in the TAC mice that was independent of body weight. However, the 5.0 mg/kg dose caused large reductions in body weight (cachexia) and death. Our results show that GDF11 can reduce pathological hypertrophy and cardiac remodeling after pressure overload, but there is a narrow therapeutic range.
Temple University--Theses

Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways.

Introduction: Cardiac hypertrophy is an adaptive response to increased myocardial workload that becomes maladaptive when hypertrophied hearts are exposed to an acute metabolic stress, such as ischemia/reperfusion. Acceleration of glycolysis occurs as part of the hypertrophic response and may be maladaptive because it enhances glycolytic metabolite accumulation and proton production. Activation of AMP-activated protein kinase (AMPK), a kinase involved in the regulation of energy metabolism, is proposed as a mechanism for the acceleration of glycolysis in hypertrophied hearts. However, this concept has not yet been proven conclusively. Additionally, several studies suggest that AMPK is involved in hypertrophic remodeling of the heart by influencing cardiac myocyte growth, a suggestion that remains controversial. Hypothesis: AMPK mediates hypertrophic remodeling in response to pressure overload. Specifically, AMPK activation is a cellular signal responsible for accelerated rates of glycolysis in hypertrophied hearts. Additionally, AMPK influences myocardial structural remodeling and gene expression by limiting hypertrophic growth. Experimental Approach: To test this hypothesis, H9c2 cells, derived from embryonic rat hearts, were treated with (1 µM) arginine vasopressin (AVP) to induce hypertrophy. Substrate utilization was measured and the effects of AMPK inhibition by either Compound C or by adenovirus-mediated transfer of dominant negative AMPK were determined. Subsequently, adenovirus-mediated transfer of constitutively active form of AMPK (CA-AMPK) was expressed in H9c2 to specifically increase AMPK activity and, thereby, further characterize the role of AMPK in hypertrophic remodeling. Results: AVP induced a metabolic profile in hypertrophied H9c2 cells similar to that in intact hypertrophied hearts. Glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation. These changes were associated with AMPK activation, and inhibition of AMPK ameliorated but did not normalize the hypertrophy-associated increase in glycolysis. CA-AMPK stimulated both glycolysis and fatty acid oxidation, and also increased protein synthesis and content. Howver, CA-AMPK did not induce a pathological hypertrophic phenotype as assessed by atrial natriuretic peptide expression. Conclusion: Acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK. AMPK is a positive regulator of cell growth in these cells, but does not induce pathological hypertrophy when acting alone.