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

<p>Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.</p><p> In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.</p><p>In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.</p><p>In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.</p><p>In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.</p><p>Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.</p> / Dissertation

Durant le développement myocardique, les cardiomyocytes s'allongent et se connectent entre eux grâce à une structure spécialisée, le disque intercalaire. Cette organisation des cardiomyocytes est essentielle pour le couplage mécanique et la conduction électrique. Un des éléments responsables de l'insuffisance cardiaque est la perturbation de ces sites de contact intercellulaire. Actuellement, aucun facteur n'est connu pour coordonner l'organisation polarisée des cardiomyocytes. Ici, nous présentons une augmentation importante de Pdzrn3 dans des cardiomyopathies hypertrophiques humaines et dans des myocardes murins, corrélée à une perte de l'élongation polarisée des cardiomyocytes. De plus la délétion spécifique intramyocardique de l'expression de Pdzrn3, dans un modèle murin, protège de la survenue d'une insuffisance cardiaque secondaire à une cardiomyopathie hypertrophique. Nos résultats révèlent une nouvelle voie de signalisation qui contrôle un programme génétique essentiel pour le développement myocardique, le maintien de la géométrie et de la fonction contractile des cardiomyocytes. Cette voie de signalisation implique PDZRN3 et cette molécule constitue une cible thérapeutique potentielle pour la protection de l’insuffisance cardiaque chez l’homme. / During heart maturation, individual cardiomyocytes stretch out and connect some with the others via their extremities by intercalated disk protein complexes. This planar and directionnel organization of the myocyte sis crucial for the machanical coupling and the anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure concerns alterations in the contact sites between cardiomyocytes. Yet no factors on its own is known to coordinate cardiomyocyte polarized organization. Here we reported enhanced levels of Pdzrn3 in the diseased hypertrophic human and mouse myocardium, correlated with the loss of cardiomyocyte polarized elongation. Furthermore, mouse cardiac Pdzrn3 deficiency protected against heart failure in a mouse model of hypertrophic cardiomyopathy. Our results reveal a novel signaling that controls a genetic program essential for heart maturation and for maintain of cardiomyocyte overall geometry and contractile function and implicates PDZRN3 as a potential therapeutic target for human heart failure protection.

A cardiomiopatia hipertrófica (CMH) é a principal cardiopatia dos felinos, caracterizada por hipertrofia ventricular esquerda, sem dilatação. A prevalência em humanos é de um a cada 500 indivíduos e, em pelo menos 60% dos casos, a doença é de origem familial. Há mais de 1400 mutações em mais de 11 genes que codificam proteínas do sarcômero relacionadas à CMH. Em algumas famílias de gatos, a CMH é transmitida de forma autossômica dominante sendo muito similar à humana. No Maine Coon, redução na miomesina e mutação no gene que codifica a proteína C miosina ligante (MYBPC3) são alterações encontradas nos acometidos pela CMH. No Ragdoll, a CMH está relacionada com mutação no mesmo gene, porém em um códon diferente e altamente conservado na espécie. Em outras raças como Persa, British Shorthair, Norwegian Forest também há evidências da CMH familial, porém não há comprovação do tipo de herança envolvida. No presente estudo, uma população de 100 gatos da raça Persa foi avaliada por meio de exames ecocardiográfico, eletrocardiográfico, laboratoriais, mesuração da pressão arterial e pesquisa da mutação relacionada à doença renal policística (PKD), prevalente em gatis de Persas. Os animais foram classificados quanto à presença ou não da CMH e, após seleção dos grupos experimentais (20 gatos sem CMH e 22 gatos com CMH), amostras de sangue foram submetidas à extração do DNA, genotipagem pela técnica de PCR e sequenciamento dos genes da alfa-actina cardíaca (exon 5 do gene ACTC1) e da proteína C miosina ligante (exon 27 do gene MYBPC3), com posterior correlação das mutações com a presença da afecção. À avaliação da população total, a CMH foi mais prevalente em gatos machos e de maior faixa etária; ocorreu em 22 animais; e a forma assimétrica com hipertrofia miocárdica em região septal basal foi a mais comum na raça. A presença de mutação relacionda à PKD foi mais comum nos gatos com hipertrofia ventricular, apesar dos mesmos apresentarem pressão arterial e função renal normais. Foi identificado um polimorfismo de nucleotídeo único (SNP) na posição 890 do exon 5 do gene ACTC1 e três SNP no intron 5-6 do mesmo gene. Nenhum polimorfismo, adição ou deleção foi observado em outras regiões do gene ACTC1 ou no gene MYBPC3. Apesar dos SNP observados no estudo, os mesmos não se enquadram nos critérios de mutação causal da CMH porque não provocam mudança em aminoácidos e não ocorreram exclusivamente em animais com CMH. Desta forma, a mutação causal da CMH em gatos da raça Persa não foi elucidada e mutações nestes dois exons de genes cardíacos não parecem ser a causa da cardiomiopatia na referida raça. Avaliações de genes cardíacos adicionais são necessárias para a identificação da causa molecular desta cardiopatia no Persa. Em relação aos resultados encontrados nos gatos PKD positivos, há necessidade de mais estudos para avaliar a relação causal (PKD e hipertrofia) ou associação genética entre ambas. Faz-se necessária a avaliação cardiológica de gatos PKD positivos, bem como é necessário incluir a PDK como diagnóstico diferencial da CMH no Persa. / Hypertrophic cardiomyopathy (HCM) is the most important feline heart disease and it is characterized by ventricular hypertrophy in absence of dilated left ventricle. In humans, the prevalence is 1 to 500 individuals and the familial HCM occurs in at least 60% of cases. There are more than 1400 mutations in more than 11 sarcomeres genes related to HCM. In some families of cats, HCM is an autosomal dominant genetic disease very similar to the human HCM. Reduction in miomesine and a mutation in myosin binding protein C gene (MYBPC3) are observed in Maine Coon cats with HCM. In Ragdoll cats, HCM is associated with a mutation in the same gene, but in a different codon highly conserved in feline species. In other breeds such as Persian, British Shorthair and Norwegian Forest there is also evidence of familial HCM, but the type of genetic inheritance is unknown. In this study, a population of 100 Persian cats was assessed by: echocardiography, electrocardiography, laboratorial tests, blood pressure determination and genetic test for the presence of the polycystic kidney disease (PKD) mutation, common in Persians. The animals were classified according to the presence or not of HCM. Blood samples from experimental groups (20 cats without HCM and 22 cats with HCM) were subjected to DNA extraction, genotyping by PCR and sequencing of cardiac alpha-actin gene (exon 5 of ACTC1) and myosin binding protein C gene (exon 27 of MYBPC3) with subsequent correlation with the presence of mutations and HCM. In the evaluated population, HCM was more prevalent in older and male cats; it occurred in 22 animals; and the asymmetric hypertrophy at basal region of septum was the most common. The PKD mutation was more common in cats with left ventricular hypertrophy, despite they presenting normal blood pressure and renal function. One single nucleotide polymorphism (SNP) at position 890 of exon 5 of the gene ACTC1 and three SNP in intron 5-6 of the same gene were identified. No polymorphism, addition or deletion was observed in other regions of the gene ACTC1 or MYBPC3 gene. Despite the SNP observed in the study, they do not fit the criteria of HCM causal mutation because they do not cause changes in amino acids and do not occurred exclusively in animals with HCM. Thus, a causal mutation of HCM in Persians cat has not been elucidated and mutations in these two exons of cardiac genes do not seem to be the cause of HCM in this breed. Additional screening of cardiac genes is necessary to identify the molecular cause of this feline disease in Persian cats. Regarding the results founded in PKD positive cats, it is important for further studies to evaluate the genetic association or causal relationship (PKD and hypertrophy). The cardiologic evaluation of PKD positive cats is necessary, and the PDK must be included as a differential diagnosis of HCM in Persian.

Cardiac muscle contraction is responsible for pumping blood throughout the body. The cyclical, ATP-hydrolysis dependent interaction of the myosin motor protein with filamentous actin drives muscle contraction. During this process the α-helical neck region of myosin acts as a lever arm, transmitting contractile force between thick and thin filaments by amplifying small conformational changes in the myosin motor domain. The resulting relative displacement of thick and thin filaments causes muscle shortening. The regulatory light chain (RLC) of myosin mechanically supports the lever arm by binding to the myosin heavy chain neck region; this is a crucial interaction in maintaining myosin's ability to produce force and motion. We investigated the role of N-terminal modifications of the RLC in modulating actomyosin contractility at the molecular level. Phosphorylation of the RLC is a naturally occurring post-translational modification of the RLC N-terminus that is important for cardiac function and has been shown to enhance contractility at the cellular level. In contrast, genetic mutations of the RLC that lead to familial hypertrophic cardiomyopathy (FHC) disrupt cardiac function and trigger remodeling of the cardiac muscle structure. We studied two FHC-linked mutations, N47K and R58Q, located in the N-terminus of the RLC in close proximity to the phosphorylation site. Using in vitro motility assays we examined how RLC modifications affect the mechanochemical properties of cardiac β-myosin. We found that the FHC mutations reduced myosin force and power generation, in contrast to RLC phosphorylation which increased myosin force and power for WT and mutant myosins. Phosphorylation of mutant RLC resulted in a restoration of the mutation-induced decreases in contractility to WT dephosphorylated levels. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the fundamental contractile FHC-induced phenotype.

Or Man Wai. / "January 2002." / "glued" in title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

A cardiomiopatia hipertrófica (CMH) é a principal cardiopatia dos felinos e é caracterizada por hipertrofia miocárdica concêntrica, sem dilatação ventricular. Disfunções miocárdicas ocorrem em gatos com CMH, mas pouco se conhece a respeito destas alterações nos estágios iniciais da afecção. Em gatos da raça Maine Coon, a mutação no gene MyBPC-A31P está relacionada com a CMH de origem familial, porém, a correlação exata entre o genótipo e o fenótipo ainda é inconclusiva. A ecocardiografia tecidual é uma modalidade não invasiva que permite avaliação da função miocárdica e é mais sensível que a ecocardiografia convencional. Para avaliar a função sistólica e diastólica, antes ou após a ocorrência de hipertrofia ventricular, gatos da raça Maine Coon (n=57), geneticamente testados para a mutação, foram avaliados por meio de ecocardiografia convencional e tecidual (nas modalidades Doppler tecidual pulsado, Doppler tecidual colorido e strain). Posteriormente, foram fenotipicamente classificados em: normais (n=45), suspeitos (n=7) e acometidos pela CMH (n=5); e genotipicamente classificados em: negativos (n=28), heterozigotos (n=26) e homozigotos para a mutação (n=3). Valores de velocidades miocárdicas (Doppler tecidual pulsado e colorido) e valores de strain, medidos na região basal e média do septo interventricular (SIV), da parede livre do ventrículo esquerdo (PVE), da parede anterior do ventrículo esquerdo (PAVE), da parede posterior do ventrículo esquerdo (PPVE) e do segmento radial da PVE, foram comparados nos diferentes grupos. Observou-se que as velocidades longitudinais Em (Doppler tecidual pulsado) na região média da PVE foram menores nos gatos com CMH quando comparados com suspeitos e normais. Os valores de Em/Am (Doppler tecidual colorido), na região basal do SIV, foram inferiores nos gatos com CMH quando comparados com suspeitos e normais. A relação E/Em (Doppler tecidual colorido), na região basal do SIV, foi maior nos gatos com CMH em relação aos suspeitos e normais. E os valores de Sm (Doppler tecidual colorido), em região basal da PVE, foram menores nos gatos heterozigotos em relação aos negativos, ambos sem hipertrofia ventricular. Observou-se correlação positiva entre a ocorrência de fusão das ondas Em e Am e a frequência cardíaca; e correlação positiva entre valores de Sm e Em e a frequência cardíaca (Doppler tecidual pulsado e colorido). Enquanto à ecocardiografia convencional observou-se um estado de contratilidade aparentemente normal, os valores de strain (em região média do SIV) nos gatos com CMH foram inferiores aos dos gatos normais. Valores de strain (em região basal da PAVE) também foram menores nos gatos heterozigotos em relação aos negativos, antes mesmo da hipertrofia ventricular. Observou-se correlação negativa entre valores de strain e espessura miocárdica. A ecocardiografia tecidual é uma nova modalidade ecocardiográfica reprodutível em gatos que, isoladamente, não permite a identificação de gatos com mutação antes do desenvolvimento de hipertrofia. O strain possibilita a detecção de anormalidades sistólicas em gatos da raça Maine Coon, apesar da aparente normalidade à ecocardiografia convencional. Apesar da expectativa em relação ao uso da ecocardiografia tecidual para a identificação precoce de indivíduos portadores da CMH, ainda há necessidade de estudos mais extensos e com maior número de indivíduos. / Hypertrophic cardiomyopathy (HCM) is the most common feline heart disease and is characterized by increased cardiac mass with a hypertrophied nondilated left ventricle. Myocardial dysfunction occurs in cats with HCM but less is known about dysfunctions in initial stages of HCM. A mutation in MYBPC-A31P gene has been identified in a colony of Maine Coon cats with HCM. However, the close correlation between genotype and phenotype still be inconclusive. Myocardial analysis by tissue Doppler imaging (TDI) is a noninvasive echocardiographic method to assess systolic and diastolic function that is more sensitive than conventional echocardiography. To evaluate diastolic and systolic function in cats with mutation, with or without ventricular hypertrophy, Maine Coon cats (n=57) were screened for mutation and examined with both echocardiography and TDI (pulsed tissue Doppler, color tissue Doppler and Strain methods). Then, were phenotypically classified in: normal (n=45), suspects (n=7) and HCM group (n=5); and genotypically classified in: negative (n=28), heterozygous (n=26) and homozygous group (n=3). Myocardial velocities (by pulsed and color tissue Doppler imaging) and peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (IVS), left ventricular free wall (LVW), left ventricular anterior wall (LVAW), left ventricular posterior wall (LVPW) and radial segment of LVW, was compared among different groups. A decreased longitudinal Em velocities (pulsed tissue Doppler) at the mildventricular segment of LVW was observed in HCM cats compared with suspects and normal cats. A decreased longitudinal Em/Am (color tissue Doppler) at the basal segment of IVS was observed in HCM cats compared with suspects and normal cats. A significant increased longitudinal E/Em (color tissue Doppler) at the basal segment of IVS was observed in HCM cats compared with suspects and normal cats. And a significant decreased longitudinal Sm (color tissue Doppler) at the basal segment of the LVW was observed in heterozygous cats compared with negative cats, both without hypertrophy. There was a positive correlation between summated early and late diastolic velocities (EmAm) and heart rate; and a positive correlation between Sm and Em velocities and heart rate, both in pulsed and in color TDI. Whereas conventional echocardiography demonstrated an apparently normal contractile state, myocardial strain (at mildventricular segment of IVS) in HCM cats was decreased compared with normal group. Myocardial strain (at basal segment of LVAW) also was decreased in heterozygous cats compared with negative group; and was decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy. And there was a negative correlation between strain values and wall thickness. TDI analyses are a new, valuable and reproducible method in cats that alone is not able to identify cats with mutation before myocardial hypertrophy. Strain method allows noninvasive detection of abnormal systolic deformation in Maine Coons cats with HCM mutation despite apparently normal left ventricular systolic function. Despite high expectations regarding the use of TDI for early identification of individuals with HCM, there is still need for larger studies with greater numbers of individuals.

Background: Hypertrophic Cardiomyopathy (HCM) and Long QT Syndrome (LQTS) are genetic cardiovascular diseases that cause sudden cardiac death. When an individual is diagnosed with an inherited disease such as HCM/LQTS it is critical that their biological relatives are notified of their increased risk. Newly diagnosed individuals in turn notify other at-risk family members through a successive process called cascade screening. This facilitates screening of at-risk biological relatives through genetic testing and/or clinical testing, and treatment for HCM/LQTS prior to development of life-threatening complications. However, for cascade screening to detect all potential cases the disease risk must be effectively communicated to all at-risk relatives. The responsibility for notifying family members of this risk largely falls to the first person diagnosed in the family (proband). Empiric evidence suggests that around half of at-risk relatives are not screened in accordance with cascade screening recommendations, potentially due to information about HCM/LQTS risk not being communicated effectively in their families. Factors have been identified that influence communication about genetic risk in families with non-cardiac disease; however, it is not known if or how these factors apply in families with genetic cardiac disease. These include network factors, which describe characteristics of relationships between family members and non-network factors, which describe characteristics of individuals including individual factors, disease factors, and sociocultural factors. There is a critical need to understand communication in families with HCM/LQTS in order to facilitate effective genetic risk communication in families, improve adherence to cascade screening recommendations, and prevent death and complications from cardiovascular diseases. Objectives: The purpose of this study was to improve our understanding of the relationships among network and non-network factors and communication of genetic risk for HCM/LQTS between probands and their relatives. I proposed the following aims: Aim 1: Describe family social network structures and communication paths about risk for HCM/LQTS from probands to their relatives. Aim 2: Identify which network and non-network factors are associated with who is told about risk for HCM/LQTS. Methods: The sample for this study included individuals with HCM or LQTS recruited through the University of Iowa Cardiology Clinics (UI) and the University of Wisconsin Inherited Arrhythmia Clinic (UW). Data were collected using a structured interview, family pedigree, and survey. Analysis included egocentric social network analysis, descriptive, bivariate, and multilevel logit regression modeling. Results: Participants in this study had an average of 24 living at-risk relatives in their families. Overall, just over half (52%) of these at-risk relatives had been reported to have been told about their risk. However, within families, the percentage of relatives told about their risk ranged from 0%-100%. Ninety percent of first-degree relatives were told about their risk, 61% of second-degree relatives were told and 33% of third-degree relatives were told. Recruitment site affiliation was determined to be a confounder and so analyses were calculated separately for UI and UW. In both the UI and UW samples, network factors including closer geographic distance, increased emotional closeness, increased relationship quality, increased frequency of communication, higher betweenness centrality, and closer degree of biological relation were independently associated with increased odds of communication of risk. In the UI sample, non-network factors that were independently associated with increased odds of communication of risk included younger age at diagnosis; having LQTS; having positive genetic test results; having an ICD; younger current age; being female; having increased role limitations due to physical functioning; feeling anxious about telling family members about risk; feeling communication was a burden; feeling that communication was a responsibility or duty; being happy to be able to share important information; and identifying financial issues, pregnancies, or upcoming marriages as playing a role in communication. In a multivariate model, increased frequency of communication, closer degree of biological relation, having an ICD, and identifying financial issues and pregnancies as contributors to communication were significantly associated with communication of genetic risk information. In the UW sample, non-network factors that were independently associated with increased odds of communication of risk included younger age, decreased emotional wellbeing, increased role limitations due to emotional wellbeing, and decreased energy and fatigue. In a multivariate model, increased frequency of communication and closer degree of biological relation were significantly associated with communication. Although over half of at-risk relatives were told about their risk, just over half of those (53.8%) were reported to have screened for disease, which represents 27% of all at-risk relatives. Of those tested, 35% were reported as diagnosed with HCM/LQTS. Conclusion: Communication of genetic risk for HCM/LQTS in families is inadequate and contributes to the problem of relatives not being screened for disease. Insight on the factors that influence communication in families at risk of sudden cardiac death can guide development of interventions, policies, and future research aimed at improving genetic risk communication and cascade screening, and preventing death and complications from inherited cardiac diseases. This research is applicable for genetic conditions where population based screening methods are not effective and rely on families to communicate risk and need for screening.

Characterization of the RNA transcriptome by next-generation sequencing can produce an unprecedented yield of information that provides novel biologic insights. I describe four approaches for sequencing different aspects of the transcriptome and provide computational tools to analyze the resulting data. Methods that query the dynamic range of gene expression, low expressing transcripts, micro RNA levels, and start-site usage of transcripts are described.

No description available.