Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction

Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction Marta Andreia Ribeiro de Pinho Porto 2013 Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction Marta Andreia Ribeiro de Pinho Biologia Celular e Molecular Departamento de Biologia 2013 Orientador Doutora Ana Luísa Pires Investigadora Associada Departamento de Obstetrícia e Ginecologia Universidade de Cambridge, Reino Unido Todas as correções determinadas pelo júri, e só essas, foram efetuadas. O Presidente do Júri, Porto, ______/______/_________ Dissertação de candidatura ao grau de Mestre em Biologia Celular e Molecular submetida à Faculdade de Ciências da Universidade do Porto. O presente trabalho foi desenvolvido sob a orientação científica da Doutora Ana Pires, na Faculdade de Medicina das Universidades do Porto e de Coimbra. Dissertation for applying to a Master’s Degree in Cell and Molecular Biology, submitted to the Faculty of Sciences of the University of Porto. The present work was developed under the scientific supervision of Doctor Ana Pires and was done at the Faculty of Medicine of the Universities of Porto and Coimbra. List of Original Publications This dissertation contains results from the following published article: Pires AL, Pinho M, Alves BS, Pinho S, Sena C, Seica RM, Leite-Moreira AF. Reverse myocardial effects of intermedin in pressure overloaded rats: role of endothelial nitric oxide synthase activity. J Physiol 2012. “The rung of a ladder was never meant to rest upon, but only to hold a man's foot long enough to enable him to put the other somewhat higher.” Thomas Huxley Acknowledgements "Não é aos saltos que se sobe uma montanha, mas a passos lentos." São Gregório Magno Ao longo desta caminhada até esta etapa, muitos foram aqueles que, de uma ou de outra forma, me apoiaram e incentivaram! Sobretudo, estes companheiros de viagem estiveram sempre presentes em cada uma dessas pequenas passadas que fui dando. A todos eles, o meu sincero obrigado! Aos meus pais e avós agradeço a constante motivação. Não são precisos gestos grandes ou programados: é na simplicidade do dia-a-dia e no exemplo de vida que sempre me têm mostrado que a vida é uma luta constante pelo sucesso e pela felicidade! Aos meus irmãos, Marco, Sofia e Eva agradeço a boa-disposição, a alegria e a paciência constantes. O vosso apoio incondicional tornou a conclusão desta etapa mais fácil e possível! Ao Mano agradeço a paciência, a compreensão, o optimismo, a alegria e a escuta. Nos momentos menos bons, o teu abraço confortou-me; nos momentos bons ele foi expressão de alegria e significou sempre motivação para continuar. À Doutora Ana Luísa Pires agradeço a sinceridade com que sempre expressou as suas opiniões, a compreensão pelos erros que fui cometendo, a motivação, o apoio e a disponibilidade que sempre demonstrou. Consigo aprendi, cresci e tornei-me mais autónoma! À Doutora Cristina Sena agradeço os conhecimentos científicos que me transmitiu durante as experiências realizadas em Coimbra. À Dra. Bárbara Alves agradeço a companhia, as conversas, o trabalho e o esforço nas muitas horas que passamos juntas no laboratório. À Dra. Marta Oliveira agradeço o apoio laboratorial que sempre mostrou. Às funcionárias do Serviço de Fisiologia D. Francelina, D. Margarida e D. Rosinha agradeço o apoio prático que sempre me deram e a simpatia com que sempre me trataram. Ao Doutor Adelino Moreira agradeço o espaço e material disponibilizados sem os quais este trabalho não poderia ter sido concretizado. Por fim, mas igualmente importante, agradeço às minhas amigas Nádia Eusébio, Sofia Moreira e Marina Santos o apoio incondicional, os conselhos e a compreensão, especialmente nos momentos menos bons! A partilha de conhecimentos e experiências foi também essencial ao longo deste percurso. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction vii Resumo A insuficiência cardíaca afecta grande parte da população mundial de tal forma que é já considerada uma epidemia. Esta síndrome caracteriza-se por alterações estruturais e funcionais no coração, que passa a bombear o sangue de forma insuficiente. O estudo da progressão desta desordem cardíaca é portanto essencial e os estudos recentes visam identificar potenciais alvos terapêuticos que sejam mais eficazes. Recentemente, foi identificado um novo péptido chamado intermedina (IMD), cujos níveis de expressão estão aumentados em vários modelos de patologias cardíacas, incluindo hipertensão sistémica e isquemia. No entanto, o seu papel nestas patologias ainda não está completamente estabelecido. Assim, começamos por estudar o efeito miocárdico da IMD em dois modelos de insuficiência cardíaca induzida por sobrecarga de pressão: “TAC”, cuja sobrecarga resulta do aperto da aorta; o modelo que denominamos por “L-NAME”, cuja sobrecarga de pressão resulta da inibição da produção de óxido nítrico, por administração oral de LNAME, um inibidor das sintases de óxido nítrico. Nos seus respectivos controlos (“Sham” e “Ctrl”), a adição de IMD induziu um efeito inotrópico negativo. Apesar de semelhante em valor absoluto, o efeito da IMD nos modelos de doença foi totalmente oposto: verificamos um efeito inotrópico positivo acompanhado por um aumento da lusitropia. Ao nível basal, verificamos que os dois modelos de doença apresentam função sistólica inalterada, hipertrofia do ventrículo esquerdo e stress oxidativo aumentado. Ao nível proteico, verificamos que, nos controlos, a adição de IMD triplicou a quantidade de eNOS fosforilada no resíduo Ser-1177, sugerindo um aumento de actividade desta proteína. Além disso, também observamos um aumento da fosforilação da troponina I, possivelmente devida à activação da via de sinalização dependente de óxido nítrico e cGMP. Por outro lado, nos modelos de doença, verificamos que a fosforilação basal da eNOS estava aumentada e que a adição de IMD não alterou estes níveis. Verificamos, contudo, que houve um aumento da fosforilação da fosfolambam, possivelmente devida à activação da via de sinalização dependente de cAMP e PKA. Assim, mostramos pela primeira vez que a IMD induz efeitos opostos em miocárdios saudáveis e doentes, que se devem à incapacidade de a IMD aumentar a actividade da eNOS nos modelos de doença. Além disso, e dado que mecanismos distintos medeiam estes efeitos, este trabalho sugere que a disfunção endotelial cardíaca desempenha um papel preponderante na patofisiologia da insuficiência cardíaca por sobrecarga de pressão e, ao mesmo tempo, aponta a IMD como potencial marcador da disfunção endotelial cardíaca. Palavras-chave: insuficiência cardíaca, disfunção endotelial cardíaca, sintase endotelial de óxido nítrico, intermedina. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction viii Abstract Heart failure affects many people around the world and then it is considered an epidemic. This syndrome is characterized by structural and functional changes in heart, which inefficiently pumps blood. The study of progression of this cardiac disorder is thus crucial and recent studies aim to identify potential and more effective therapeutic targets. Recently, a new peptide called intermedin (IMD) was identified, whose expression levels are augmented in several models of cardiac pathologies, such as systemic hypertension and ischemia. However, its role in these pathologies is not yet completely established. Therefore, we started to study myocardial effects of IMD in two heart failure models induced by pressure-overload: “TAC”, whose overload results from aortic banding; the model which we named as “L-NAME”, whose pressure-overload results from inhibition of nitric oxide production, by oral administration of L-NAME, an inhibitor of nitric oxide synthases. In their respective controls (“Sham” and “Ctrl”), addition of IMD elicited a negative inotropic effect. Despite being similar in absolute value, effect of IMD in diseased models was totally opposite: we verified a positive inotropic effect, allied to an increase of lusitropy. At the baseline, we observed these two diseased models present preserved systolic function, left ventricle hypertrophy and increased oxidative stress. At the protein level, in controls, IMD tripled content of phosphorylated eNOS at the Ser-1177, which suggest an increase of eNOS activity. Moreover, we also verified an increase of phosphorylation of troponin I, possibly due to activation of signaling pathway dependent of nitric oxide and cGMP. On the other hand, in diseased models, basal phosphorylated eNOS was increased and IMD did not alter those levels of phosphorylation. However, IMD elicited phosphorylation of phospholamban, eventually due to activation of cAMP/PKA signaling pathway. Thereby, we showed for the first time IMD induces opposite effects in healthy and diseased myocardium, which are due to inability of IMD to increase eNOS activity in these latter models. Besides, and given that distinct mechanisms mediate those effects, this work suggests cardiac endothelial dysfunction plays a preponderant role in pathophysiology of pressure-overload-induced heart failure and, at the same time, points IMD as a potential marker of cardiac endothelial dysfunction. Key words: heart failure, cardiac endothelial dysfunction, endothelial nitric oxide synthase, intermedin. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction xv List of Abbreviations AM Adrenomedullin ATP Adenosine triphosphate BH4 Tetrahydrobiopterin CaM Calmodulin cAMP/cGMP Cyclic adenosine/guanosine monophosphate CGRP Calcitonin gene-related peptide CRLR Calcitonin receptor-like receptor cTnI Cardiac troponin I ECM Extracellular matrix eNOS Endotelial nitric oxide synthase FAD Flavin adenine dinucleotide FMN Flavin mononucleotide GTPCH1 Guanosine triphosphate cyclohydrolase 1 HF Heart failure IMD Intermedin L-NAME NG-nitro-L-arginine methyl ester LTCC L-type calcium channels LV Left ventricle/Left ventricular NADPH nicotinamide adenine dinucleotide phosphate NO Nitric oxide NOS Nitric oxide synthase PKA/PKB/PKG Protein kinase A/B/G PLB Phospholambam RAMP Receptor activity-modifying proteins ROS/RNS Reactive oxygen/nitrogen species Ser Serine SNS Sympathetic nervous system SR Sarcoplasmic reticulum TAC Transverse aortic constriction Thr Threonine Tyr Tyrosine β-ARs Beta adrenergic receptors FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 1 I. Introduction FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 2 1. Heart Physiology 1.1 Functional anatomy. Heart (Figure 1A) is a vital and muscular organ responsible for blood pumping to all body parts. Anatomically, it has four chambers: two auricles or atria, which are responsible for receiving blood, and two ventricles, whose main function is pumping blood. Heart is contained in a sac called pericardium, which is divided in fibrous and parietal pericardium. Next, there is the pericardial cavity containing the serous fluid and then there are the epicardium, the myocardium (composed entirely by muscle cells called cardiomyocytes) and endocardium (composed by endothelial cells). All these layers are illustrated in Figure 1B. The cardiac muscle is an involuntary contraction muscle with a type of response to a stimulus called “all-or-none response”. Figure 1 – A: Structure of the heart, from anterior perspective. B: Transversal section of the heart, showing its different layers. From http://www.guwsmedical.info/heart-failure/images/4415_27_47-pericardium-and-heart-wall.jpg . B A FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 3 Cardiomyocytes are short branching interconnecting cells, arranged as a syncytium, which is a multinucleated, protoplasmic mass of cells. In fact, myocardium functions like a syncytium since an applied stimulus in some part of the muscle results on contraction of whole muscle. However, anatomically, myocardium is not considered a syncytium since effectively cardiomyocytes are separated from each other: laterally, by their cellular membranes (called sarcolemmas) and at their ends by dense structures called intercalated disks (Figure 2). These disks have gap junctions (Figure 2), which facilitate the cardiac impulse conduction from a cell to another, and longer desmosomes (called fasciae adherens; Figure 2), which keep not only the cardiomyocytes together during contraction but also the normal electrical coupling between adjacent cardiomyocytes (Rasmussen, Palmfeldt et al., 2013). Cardiomyocytes contain a large number of mitochondria (called sarcossome; Figure 2) in order to allow a quick substrate oxidation and adenosine triphosphate (ATP) synthesis (Drago, De Stefani et al., 2012), which support the myocardial energetic requirements. In most species, each one of cardiomyocytes have two nuclei (Wright, Wu et al., 2012) (Figure 2) and, on average, each one is associated to one capilar, allowing a fast exchange and movement of important molecules between cardiomyocyte and capilar. This vascular network provides a suitable availability of oxygen and substrates in order to sustain the metabolic machinery. Figure 2 – Detailed view of disposition and structural composition of cardiomyocytes, showing gap juntions, desmossomes, intercalated discs, nucleus, sarcolemma, mitochondria and sarcoplasmic reticulum. From http://apbrwww5.apsu.edu/thompsonj/Anatomy%20&%20Physiology/2010/2010%20Exam%20Reviews/Exam%203%20 Review/18-11_AnatCardMusc.JPG FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 4 Inside cardiomyocytes, myofibrils are the contractile structures constituted by thousands of sarcomeres (Gregorio and Antin, 2000), which is defined as the section between two Z consecutive discs (Figure 3A). The sarcomere is divided into two main bands (Figure 3B): I band, which comprises the thin (actin) filaments segment which are not overlapped with thick (myosin) filaments; A band, which comprises the thick filaments, either those overlapped (zones of overlap) and those non-overlapped (H zone) with thin filaments. It’s because of those zones of overlap that cardiac muscle has a striated appearance (Cooper, 2000). The cross-connecting elements of cytoskeleton constitute the M line, which defines the middle line of the sarcomere (Figure 3A). As we can see in Figure 3B, thin and thick filaments are arranged in parallel (Krans, 2010). In addition to actin and myosin, sarcomere has other proteins such as titin and nebulin (Figure 3C), which are giant proteins that, among other functions, are responsible for protecting myofibrils when they are overstretched (Joseph, Stier et al., 2001) and for regulation of muscle contraction (Labeit, Ottenheijm et al., 2011). Troponin complex are another group of proteins of sarcomere, which is associated to tropomyosin and actin filaments and includes cardiac troponins I, C and T (cTnI, cTnC and cTnT). Particularly, cTnI is susceptible to be phosphorylated in its Threonine 144 (Thr-144), Serine 149 (Ser-149), Serine 43/45 (Ser-43/45) and Serine 23/24 (Ser-23/24) residues (relatively to human sequence) (Layland, Solaro et al., 2005). Phosphorylation of this latter is mediated by protein kinase A (PKA) and contributes for decreasing of myofilament Ca2+-sensivity (Kooij, Saes et al., 2010). A B FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 5 Figure 3 – A: Structure of a myofibril, showing its reference points (the M line and the Z discs) and the striated pattern of sarcomere. B: Detailed structure of a sarcomere, showing the titin filaments and the parallel alignment of thin (actin) and thick (myosin) filaments. Bands A and I, Z discs, M line and overlap and H zones are also represented. C: Protein composition of a sarcomere, showing tropomyosin, troponin complex (I, C and T), nebulin, C protein, myosin and titin. From http://www.revespcardiol.org/imatges/255/255v59n05/grande/255v59n05-13088668fig05.jpg and http://www.baileybio.com/plogger/images/anatomy___physiology/05._powerpoint_-_muscular_system/sarcomere.jpg . Myofibrils are surrounded by sarcoplasmic reticulum (SR; Figure 2), a smalldiameter sarcotubules network, which is responsible for storing intracellular Ca2+. Since its discovery, SR has always been touted as playing an important role in contraction/relaxation cycle (Fawcett, 1961), which was confirmed later. There is another tubular system inside cardiomyocytes which is composed by a large number of transversal tubules (Ttubules; Figure 4), which are invaginations of the sarcolemma into cardiomyocytes at the Z lines. This tubular system plays an important role in excitation-contraction coupling and provides to myofibrils and mitochondria a ready access to interstitial fluid. Figure 4 – Detailed view of cardiomyocyte, evidencing the T-tubular system. Adapted from http://www.courstudy.com/wp-content/uploads/2013/07/microscopic-anatomy-of-cardiac-muscle-sarcolemma-fasciaeadherens-intercalated-discs-gap-junctions-t-tubules.jpg . C FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 6 1.2 Excitation-contraction coupling Muscular contraction (Figure 5) begins when an excitation wave spreads quickly along the sarcolemma and gap junctions and into cardiomyocytes via T-tubules. At sarcolemmal level, permeability to Ca2+ increases, leading to Ca2+ entering on the cell through sarcolemmal Ca2+ channels, called L-type Ca2+ channels (LTCC), whose channel proteins are phosphorylated by cAMP, and T-tubules. This extracellular Ca2+ is not sufficient to set off myofibrils contraction; indeed, it is just a trigger for Ca2+ releasing by SR, which augments free Ca2+ in cytoplasm. In turn, these Ca2+ ions bind to cTnC, forming a complex which interacts with tropomyosin, unlocking active sites between actin and myosin filaments. Then, myofibrils contract and systole occurs. At the end of systole, Ca2+ influx stops and SR is no more stimulated. Cyclic adenosine monophosphate (cAMP) promotes phospholamban phosphorylation (p-PLB), which stimulates ATP-dependent Ca2+ pump (placed on SR), resulting in pumping of Ca2+ into SR. Besides, cAMP also phosphorylates cTnI, inhibiting the binding of Ca2+ to cTnC. So, cTnI binds to actin, keeping actin/tropomyosin complex and preventing the myosin connecting to the actin (Gomes, Potter et al., 2002). Diastole occurs (Figure 5) and extracellular Ca2+ is removed from cytoplasm through a Na+/Ca2+ exchanger and an ATP-dependent Ca2+ pump, both localized on sarcolemma. Figure 5 – Systole and diastole mechanisms. Numbers indicate the succession of events responsible for cardiac contraction and relaxation. From http://faculty.pasadena.edu/dkwon/chapter%2014/Chapter%2014_cardiology_files/slide0059_image021.jpg . Action potential enters from adjacent cells.  Voltage-gated Ca2+ channels open. Ca2+ enters cell. Ca2+ induces Ca2+ release through ryanodine receptor-channels (RyR).  Local release causes Ca2+ spark.  Summed Ca2+ sparks create a Ca2+ signal.  Ca2+ ions bind to troponin to initiate contraction.  Relaxation occurs when Ca2+ unbinds from troponin.  Ca2+ is pumped back into the sarcoplasmic reticulum for storage.  ca2+ is exchanged with Na+.  Na+ gradient is maintained by the Na+/K+ ATPase.           FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 7 2. Heart failure 2.1 Definition and general considerations Heart failure (HF), also called congestive heart failure (CHF), is defined as a clinical syndrome characterized by both structural and functional alterations in heart (Francis and Tang, 2003). That condition incapacitates heart, thus it does not pump sufficient blood to all parts of body, leading to a multisystem disorder. Nowadays, heart failure is considered an epidemic disease, given that many people around the world suffer it: in fact, in Europe, it is the major cause of death (Figure 6). This elevated rate is intimately associated with increased prevalence of risk factors, such as diabetes, hypertension and obesity (López-Sendón, 2011), arising from unhealthy lifestyles. While at the society it is increasingly important to raise awareness of the prevention of HF, at the scientific community there is an effort to unravel processes and mechanisms responsible for emergence and progression of heart failure. Figure 6 – Major causes of death in Europe. Cardiovascular disease is the major one in both genders. From http://www.micromedcv.com/eu/images/stories/major_causes_death_europe-big.jpg. Clinically, according the New York Heart Association (NYHA), progression of HF is divided into four different classes, characterized by severity of symptoms (Table 1) and quality of life. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 8 Table 1 – Classification of heart failure and their respective symptomatology, according with the New York Heart Association. HF may assume two different forms, since it can result from a disability of the ventricles both contract normally (called heart failure with reduced ejection fraction or systolic heart failure, HFrEF) or relax completely during diastole (called heart failure with preserved ejection fraction or diastolic heart failure, HFpEF) (Borlaug and Paulus, 2011; Gutierrez and Blanchard, 2004). Population studies have pointed out this latter dysfunction as the most popular among elderly people (Kitzman, 2000); moreover, HFpEF is considered a major cause of morbidity and mortality (Borlaug and Paulus, 2011). Despite not having systolic dysfunction, these patients have a stiff heart due to fibrosis, resulting in a decreasing of diastolic filling. Hence, the amount of blood pumped is lower during systole (Kitzman, 2000). Given its importance and since its pathophysiology is not very well understood yet (Borlaug and Paulus, 2011), diastolic HF has been studied by many groups. 2.2 Animal Models Although there is not an animal model able to completely mimic the human HF (Doggrell and Brown, 1998), investigators have tried to develop the best one. Currently, rat is the most used animal model since it presents numerous advantages, such as low costs and advanced technology, which has been optimized for this model (Patten and Hall-Porter, 2009). However, this animal model also presents some limitations especially concerning its myocardial function, which differs from human in some aspects, such as the size of action potential (Hasenfuss, 1998). Regarding HF, there are many different rat models, depending on the primary reason for that failure and what the investigators want to study (Table 2) (Doggrell and Brown, 1998; Hasenfuss, 1998). One of the most used models is the transverse aortic constriction (TAC), where HF results from a pressure overload-induced hypertrophy (Rockman, Ross et al., Class Patient Symptoms I (Mild) No limitation of physical activity. Ordinary physical activity does not cause undue fatigue, rapid/irregular heartbeat (palpitation) or shortness of breath (dyspnea). II (Mild) Slight limitation of physical activity. Comfortable at rest, but ordinary physical activity results in fatigue, rapid/irregular heartbeat (palpitation) or shortness of breath (dyspnea). III (Moderate) Marked limitation of physical activity. Comfortable at rest, but less than ordinary activity causes fatigue, rapid/irregular heartbeat (palpitation) or shortness of breath (dyspnea). IV (Severe) Unable to carry out any physical activity without discomfort. Symptoms of fatigue, rapid/irregular heartbeat (palpitation) or shortness of breath (dyspnea) are present at rest. If any physical activity is undertaken, discomfort increases. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 9 1991). According to deAlmeida et al, TAC is a more faithful model of cardiac hypertrophy and resembles to human HF because of its gradual development (deAlmeida, van Oort et al., 2010). This model consists in constriction of aorta, resulting in a pressure-overloaded heart. Table 2 – Some rat models of hypertension, hypertrophy and heart failure. This table was adapted from Doggrell et al (Doggrell and Brown, 1998). Syndrome Model Hypertension  Systemic  Spontaneously Hypertensive Rats (SHRs)  Stroke-prone SHR (SHR-SP)  Mineralocorticoids (DOCA–salt)  NO synthase inhibition (L-NAME administration)  Transgenics (TGR(mREN2)27 rats)  Diabetic hypertensive rats (STZ-SHR, Zucker) o Renal o Renal artery occlusion (1K1C, 2K1C)  Pulmonary  Monocrotaline  Hypoxia (normobaric, hypobaric) Hypertrophy - Spontaneously Hypertensive Rats (SHRs) - Renal artery occlusion - Pressure loading (aortic banding) - Catecholamines (noradrenaline, isoprenaline) - Transgenics Heart failure  Systemic hypertension  Spontaneously Hypertensive Rats (SHRs-F)  Hypertensive HF prone rats  Dahl/Rapp salt-sensitive rats o Ischaemic heart failure o Non-occlusive coronary ligation  Myocardial infarction  Coronary ligation  Microembolization of coronary vessels  Cardiomyopathy  Toxins (adriamycin, ethanol) o Myocarditis o Autoimmune o Chagas' disease  Pulmonary hypertension  Monocrotaline  Hypoxia  Miscellaneous  Aortacaval fistula (shunts) Another recognized pressure-overload model is L-NAME, which results from chronic administration of L-NAME (NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor) in drinking water, promoting systemic hypertension. This model is mostly used in human hypertension studies (Kopincova, Puzserova et al., 2012). Under these conditions, heart triggers a set of mechanisms resulting in molecular, cellular and FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 16 responsible for degradation and removal of ECM molecules (Nagase, Visse et al., 2006). In pigs with HF, MMPs abundance and activity are increased (Coker, Thomas et al., 1998); furthermore, under those conditions also TIMPs levels are altered, resulting in an imbalance between these two protein families (Moore, Fan et al., 2012). Hence, both MMPs and TIMPs are promising therapeutic targets for discovery of new drugs, which could be more effectively in treatment of HF (Li, McTiernan et al., 2000). 2.3.4 Oxidative stress Evidence has also demonstrated that in HF, oxidative stress, defined as an imbalance between ROS/RNS produced and cellular antioxidant capacity (Thannickal and Fanburg, 2000), is augmented and accompanied by an antioxidant deficit (Belch, Bridges et al., 1991; Hill and Singal, 1996; Hill and Singal, 1997; Mallat, Philip et al., 1998). Despite playing an important role in many biological cellular processes (Anathy, Roberson et al., 2012; Hancock, Desikan et al., 2001; Martinez and Andriantsitohaina, 2009; Simon, Haj-Yehia et al., 2000), both ROS and RNS cellular accumulation is deleterious. To the molecular level, oxidative stress is responsible for damaging of nucleic acids, lipids, proteins and other macromolecules (Martinez and Andriantsitohaina, 2009; Thannickal and Fanburg, 2000) however, it can also promotes specific, reversible/irreversible oxidative modifications on proteins, altering their activity or function (Finkel and Holbrook, 2000). Within the heart, cardiomyocytes, endothelial cells and neutrophils are sources of ROS, but inside each cardiomyocyte there are many different sources responsible for that production, namely mitochondria, nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidases and uncoupled NOS (Bevers, Braam et al., 2006; Tsutsui, Kinugawa et al., 2011) (Figure 10). Concerning mitochondria, recent data shows progression of HF is associated with a progressive loss of respiratory activity of mitochondria, decreasing ATP production and jeopardising cellular processes responsible for cardiac pump function (Huss and Kelly, 2005). Many differences have been discovered between mitochondria from normal and failing hearts, either at morphologic, functional and biochemical levels (Abel and Doenst, 2011; Rosca and Hoppel, 2010). Curiously, despite being a source of ROS, mitochondria are severely affected by them since ROS are responsible for mitochondrial DNA damage (Tsutsui, Kinugawa et al., 2011). Recently, also endothelial dysfunction was indicated as a potential strong contributor for pathophysiology and progression of HF (Lam and Brutsaert, 2012) and is also associated with HF severity (Marti, Gheorghiade et al., 2012). Given that and since endothelial dysfunction results from an imbalance between FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 17 NO and ROS production (Bauersachs and Schafer, 2004) and endothelial NOS (eNOS) is the most abundant form of NOS expressed in endothelium (Balligand, Feron et al., 2009), namely endocardial endothelium (Kelly, Balligand et al., 1996), eNOS might play a central role in endothelial dysfunction and therefore in progression of HF. Accordingly, some authors have reported the potential protective role of endotheliumderived NO in HF and other cardiac dysfunctions, such as arrhythmia (Albrecht, Stegeman et al., 2003; Burger and Feng, 2011; Fischer, Rossa et al., 2005; Nishida, Yu et al., 2009). Figure 10 – NADPH oxidase as the major source of ROS, under different stimuli including pressure-overload. Also mitochondria, xanthine oxidase and eNOS are sources of ROS, which contribute for an increasing of ROS/antioxidants (AO) ratio, resulting in oxidative stress. This redox cellular imbalance leads to heart failure, through a set of ROSmediated alterations including apoptosis and dysfunction of cardiomyocytes, fibrosis and hypertrophy. From Ocatavia et al (Octavia, Brunner-La Rocca et al., 2012). Thus, understand how eNOS works and is regulated under both physiologic and pathophysiologic conditions will be an important help for discovering of new efficient therapeutic targets. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 18 2.3.4.1 eNOS Endothelial NOS is constitutively expressed by endothelial cells and belongs to a family of enzymes called nitric oxide synthases (NOSs), which are responsible for NO synthesis (Stuehr, 1999). This family also comprises two other NOS isoforms: neuronal and inducible NOS, nNOS and iNOS respectively (Stuehr, 1999). In heart, both eNOS and nNOS are constitutively expressed by cardiomyocytes while iNOS is only expressed after an inflammatory stimuli (Mount, Kemp et al., 2007). Structurally, eNOS has an N-terminal oxygenase domain containing binding domains to heme, L-arginine and tetrahydrobiopterin (BH4), a central region enabling calmodulin (CaM) binding and finally a C-terminal reductase domain containing binding sites to NADPH, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) (Stuehr, 1997) (Figure 11). Endothelial NOS active form is an homodimer; however, since eNOS depends primarily on Ca2+/CaM presence (Rafikov, Fonseca et al., 2011), it should indeed be considered a tetramer (Alderton, Cooper et al., 2001). eNOS synthetizes NO and Lcitrulline from substrate L-arginine, requiring molecular oxygen, its dimer form and the cofactors: BH4, haem, FAD, FMN, CaM and NADPH (Forstermann and Munzel, 2006; Marletta, 1993). The cofactor BH4 is essential for eNOS normal function (Alp and Channon, 2004): decreasing of BH4 bioavailability, either by its oxidation or reduction of its synthesis, results in uncoupling of eNOS (Moens and Kass, 2006). Besides, BH4 is responsible for dimer stabilization and increase of substrate affinity of NOS, and has some antioxidant effects through scavenging of NO-derived RNS and ROS (Moens, Kietadisorn et al., 2010). Despite its exogenous administration was shown to be beneficial for cardiac function, BH4 has potential limitations in clinical use (Moens, Ketner et al., 2011). Figure 11 – Endothelial NOS structure, showing its oxygenase and reductase domains and its CaM binding site. This protein is able to be phosphorylated at tyrosine (Y), threonine (T) and serine (S) residues, which are numbered. Other modifications include myristoylation (M), palmitoylation (P), S-glutathionylation (G) and S-nitrosylation (N). Adapted from May (May, 2012). FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 19 De novo synthesis of BH4 is limited by guanosine triphosphate cyclohydrolase 1 (GTPCH1), whose transcription (Katusic, Stelter et al., 1998; Linscheid, Schaffner et al., 1998), activity (Werner-Felmayer, Werner et al., 1993) and phosphorylation (Lapize, Pluss et al., 1998) are controlled by several citokines, angiotensin-II and platelet-derived growth factor. Degradation of GTPCH1 was reported in cardiovascular diseases (Moens, Kietadisorn et al., 2010; Zhao, Zhu et al., 2012), such as diabetes mellitus (Xu, Wu et al., 2007) and hypertension (Xu, Wang et al., 2009). In addition, eNOS expression itself is regulated at transcriptional and posttranscriptional levels (Balligand, Feron et al., 2009; Searles, 2006). This latter includes posttranslational modifications such as S-nitrosylation, S-glutathionylation, binding of CaM, palmitoylation, acylation and phosphorylation (Chen, Wang et al., 2010; Dudzinski and Michel, 2007). Particularly, eNOS contains many potential phosphorylation sites such as tyrosine, serine and threonine residues (Alderton, Cooper et al., 2001) (Figure 11). Their phosphorylation regulates NO production, depending on not only residues type but also residue per se (Fulton, Church et al., 2005; Li, Ruan et al., 2007; Ritchie, Kohlhaas et al., 2010; Ruan, Torres et al., 2011). Another regulatory mechanism of NO production by eNOS include protein-protein interactions: for instance, the binding of heat shock protein 90 (Hsp90) to eNOS is needed for Akt (also called PKB)-mediated phosphorylation of eNOS at Ser-1177 (using the human sequence nomenclature) (Mount, Kemp et al., 2007) while the binding of caveolin-1 to eNOS is responsible for its inactivation (Ju, Zou et al., 1997). Also subcellular localization is important for eNOS activity (Sullivan and Pollock, 2003). At the baseline, eNOS is associated with plasma membrane (Church and Fulton, 2006) mainly due to its myristoylation (Busconi and Michel, 1993; Michel, 1999). However, in hypertensive rats, which present endothelial dysfunction, authors verified eNOS translocation from membrane to cytosol, suggesting that eNOS translocation may contribute for decreasing of NO bioavailability and thus for worsening of endothelial dysfunction and subsequently hypertension (Sullivan, Pollock et al., 2002). As stated above, in the failing heart there is an overproduction of ROS from various sources. Particularly, generated ●O2react with NO to form peroxynitrite (ONOO-), which then oxidizes BH4 (Balligand, Feron et al., 2009), diminishing its bioavailability. Consequently, eNOS suffers an enzymatic change through alteration of its dimer architecture as well as its catalytic activity, leading to the synthesis of ●O2and/or hydrogen peroxide in vitro (Moens and Kass, 2006) and eventually to the direct production of ONOO- (Balligand, Feron et al., 2009). This production of ●O2instead of NO is commonly designated as uncoupling of eNOS (Forstermann and Munzel, 2006; FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 20 Massion, Feron et al., 2003). At the protein level, ONOOreacts with tyrosine, generating 3-nitrotyrosine, which can be detected by specific antibodies (Daiber and Munzel, 2012). Indeed, nitrotyrosine is referred as a marker of both ONOOformation and presence of oxidative stress (Sun, Carretero et al., 2005). Accordingly, its levels are increased in CHF patients (Hryniewicz, Yasskiy et al., 2004). Endothelial NOS-derived NO is thought to be coupled to cyclic guanosine monophosphate/protein kinase G (cGMP/PKG) pathway (Hammond and Balligand, 2012). Activation of PKG is able to promote phosphorylation of various proteins such as PLB, LTCC and cTnI (Tsai and Kass, 2009). Throughout this 2.3 section, we mentioned some of the reasons that explain the transition between the compensated state and HF. However, for now, other mechanisms and intervenients are still under exhaustive study and investigators continue to search for answers (Kehat and Molkentin, 2010). Next years, this so complex transition could be more highlighted, bringing new potential and more efficient therapeutic targets. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 21 3. Intermedin The calcitonin/calcitonin gene-related peptide (CGRP) family peptide includes peptides such as calcitonin, CGRP, amylin and AM (Chang, Roh et al., 2004). But in 2004, the number of members of this family augmented, when a new peptide – IMD – was independently discovered by two distinct groups (Roh, Chang et al., 2004; Takei, Hyodo et al., 2004). It is for that reason IMD is also known as AM2. The human IMD preprotein is constituted by 148 amino acids (preproIMD) but, through proteolytic cleavage at the N-terminus and amydation of C-terminus (Yang, Jia et al., 2005), it is able to generate two distinct mature forms constituted by 47 (IMD1-47 or IMDL) (Bell and McDermott, 2008) or 40 amino acids (IMD8-47 or IMDS). In addition, it is thought there is another IMD form with 53 amino acids (IMD1-53) resultant from cleavage of preproIMD at the Arginine 93-Argine 94 sites (Yang, Jia et al., 2005). Eventually, IMD1-53 would be degraded endogenously but might have an important bioactive action (Yang, Jia et al., 2005). Alignment of mature IMD1-47 shows that human and rodent aminoacid sequences are 87% identical (Roh, Chang et al., 2004) (Figure 12). Figure 12 – Alignment of amino acids of IMD long form (IMD 1-47) from human (hIMD), mouse (mIMD) and rat (rIMD). Adapted from Roh et al (Roh, Chang et al., 2004). IMD is distributed all over the mammalians body, namely kidneys, gastrointestinal tract, brain, lungs, spleen, ovaries, pituitary, among many others (Takahashi, Morimoto et al., 2011). In rat heart, IMD is found in cardiomyocytes, with greater levels are found in those neonatal when compared with adult ones (Pan, Yang et al., 2005), which suggests IMD plays an important role during embryonic development (Bell and McDermott, 2008). The biological actions of IMD are mediated by three different receptors, which are heterodimers constituted by a calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMP1-3) (Naot and Cornish, 2008). Moreover, these RAMP proteins are essential for ligand selectivity since CRLR alone shows a decreased affinity to its ligands (Pfeil, Aslam et al., 2009). The CRLR/RAMP1 complex (or CGRP receptor) forms the CGRP receptor while the CRLR/RAMP2-3 complexes (also called AM1 and AM2 receptors, respectively) form the AM receptors FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 22 (Hay, Walker et al., 2011). IMD binds non-specifically to all three CRLR/RAMP complexes (Takahashi, Morimoto et al., 2011). Despite that, there is a degree of binding affinity (Figure 13), since, for example, AM is also capable to bind to CGRP receptor (Zhao, Bell et al., 2006). Figure 13 – Binding affinity of CGRP, AM and IMD (AM2) to CGRP, AM1 and AM2 receptors and their constitution. Light grey cylinders represent calcitonin receptor-like receptor (CRLR). ECD means extracellular domain; TM means transmembrane; ICD means intracellular domain. Adapted from Hay et al (Hay, Walker et al., 2011). 3.1 Cardiovascular effects Concerning cardiovascular effects, IMD1-47 elicits an increased heart rate in rat mediated by activation of SNS (Taylor, Bagley et al., 2005). Besides, its intravenous administration promotes a negative inotropic action suggested by a decline of blood pressure (Bell and McDermott, 2008). However, a positive inotropic effect elicited by IMD1-53 and IMD1-47 was reported both in isolated perfused heart from rat (Yang, Jia et al., 2005) and ventricular cardiomyocytes from mouse (Dong, Taylor et al., 2006), respectively. This apparent contradiction was recently clarified by Pires et al, who established a negative inotropic effect of IMD on myocardium, using rat left ventricular (LV) papillary muscles (Pires, Pinho et al., 2012). Since AM also has different inotropic actions, dependent on both species and methodology used (Fontes-Sousa, Pires et al., 2009; Ihara, Ikeda et al., 2000; Ikenouchi, Kangawa et al., 1997; Szokodi, Kinnunen et al., 1998), these evidences show and reinforce the complex biological action of members of this peptide family. R A M P 1 R A M P 2 R A M P 3 FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 23 Behind IMD myocardial effects, there is a set of signalling pathways, messengers and mediators involved. However, there is no direct evidence proving that and conclusions have often been drawn by extrapolating of data from other members of this peptide family and from studies about IMD vascular effects. Nevertheless, it is thought IMD negative inotropic effect and vasodilator action is endothelium-dependent and mediated by NO/cGMP pathway, whereas positive inotropic effect is mediated by cAMP/PKA pathway (Burak Kandilci, Gumusel et al., 2006; Dong, Taylor et al., 2006; Pires, Pinho et al., 2012). But most importantly, IMD is now recognized as a potential important peptide in cardiac pathology since its expression levels are much greater in several models of failing hearts than in healthy ones (Bell, Zhao et al., 2008; Hirose, Totsune et al., 2008; Zeng, Yuan et al., 2009; Zhang, Jiang et al., 2009; Zhao, Bell et al., 2006). Furthermore, some studies have reported the potential protective role of IMD (Holmes, Campbell et al., 2013) as for example in prevention of myocardial apoptosis mediated by mitochondria via Akt/glycogen synthase kinase 3 beta (GSK-3β) signalling pathway (Song, Teng et al., 2009) and induction of autophagy of cardiomyocytes via cAMP/PKA and mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signalling pathways, preventing both apoptosis and hypertrophy of cardiomyocytes (Chen, Wang et al., 2013). Nevertheless, there is much work to do in order to explain the pathophysiologic impact of the augmented levels of IMD. 4. Aims The general goal of this thesis was to study the myocardial response to IMD in chronic pressure-overloaded rats, its intracellular mechanisms and impact of cardiac endothelial dysfunction. In order to facilitate the understanding of the work, we divided it into three different points: 1) To clarify and show differences between the two well-known pressure-overload models TAC and L-NAME, regarding their morphometric, haemodynamic and contractile parameters. 2) To study myocardial effects of IMD in both control and pressure-overload animal models and unveil the potential intracellular signalling pathways involved. 3) To investigate the influence of eNOS in differential response of myocardium to IMD. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 24 II. Materials & Methods FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 25 1. Ethical approval The animal handling and experiments were performed in accordance with Portuguese law animal welfare and conform to the guidelines established by the Faculty of Medicine of the University of Porto (Porto, Portugal), animal welfare committee and principles of UK regulations, as described by Drummond (Drummond, 2009). 2. Animals 2.1 TAC rats Transverse aortic constriction (TAC) was performed similarly to Litwin et al (Litwin, Katz et al., 1995). Seven weeks-old male Wistar-Han rats (Rattus norvegicus) were anesthetized with sevoflurane (2%) and placed on a temperature-controlled surgical table. After tracheal intubation, rats were connected to a rodent ventilator (Topo, small animal ventilator, Kent Scientific). A small incision at the level of the second left intercostal space was performed, the different muscle layers were cut and thoracic cavity was accessed. After identification and isolation of the aortic arch, a 3-0 non-absorbable silk suture was placed around aorta between the brachiocephalic and left carotid arteries (Figure 14) and tied together with a 25G needle, in order to standardize the constriction. After tying the suture, the needle was immediately removed to produce an aorta with a stenotic lumen. Then the thoracic cavity was closed using a 4-0 polypropylene suture, all layers of muscle were repositioned and skin was closed with 4-0 silk suture (TAC, n=14). The control animals are called “Sham” and they were subjected to a similar surgery without the tightening of aorta (Sham, n=12). Twenty one weeks after surgery, rats underwent hemodynamic evaluation or papillary muscles protocols, as described in Figure 15A. Figure 14 – Diagram of aorta adapted from an online quiz (http://quizlet.com/14064108/mini2-anatomy-flash-cards/). The blue boxes identify the brachiocephalic and left common carotid arteries and the black line in arch of aorta represents the local of the constriction. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 32 9. Chemicals and solutions All chemicals were obtained from Sigma Chemical Co, St Louis, with the exception of: IMD, which corresponds to the active form of 47 amino-acid (IMD1-47), that was obtained from Bachem (Bubendorf, Switzerland); eNOS activity kit, obtained from Stratagene (La Jolla, California, USA); [3H]-arginine, obtained from Perken Elmer (Massachusetts, USA). Antibodies anti-eNOS and anti-cTnI were obtained from Cell Signaling Technology Inc., (Massachusetts, USA) and antibodies anti-PLB were obtained from Abcam (Cambridge, UK). The stock solutions, including IMD, were prepared in distilled water and stored as frozen aliquots at -20ºC until use. 10. Statistical analysis GraphPad Prism 5.0 software was used for data analysis. Values are presented as mean ± standard error of mean (SEM) and n represents the number of animals or papillary muscles. Differences in treatment effects among the different animal groups were evaluated using a two-way ANOVA. When significant differences were detected with any of the ANOVA tests, the Dunnet test was selected to perform pairwise multiple comparisons, with P<0.05 being considered significant. In all other experiments, statistical significance was assessed using a two-tailed Student's unpaired t test with P<0.05 being considered significant. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 33 III. Results FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 34 1. Characterization of animal models In order to characterize the animal models, we performed morphometric and hemodynamic measures, which are summarized in Table 4. Both TAC and L-NAME animals presented a significant increase in heart weight/body weight ratio, compared to their respective controls (Sham and Ctrl). These results suggest TAC and L-NAME animals developed LV hypertrophy. Similarly, left ventricular systolic pressure (LVSP) is significantly increased in those models relatively to their controls; however a significantly higher left ventricular end-diastolic pressure (LVEDP) was recorded just in the TAC animals. Furthermore, these animals showed a slower left ventricular relaxation, suggested by a greater tau value. Table 4 – Summary of morphometric (7-8 animals) and hemodynamic (4-7 animals) measures performed in Sham, TAC, Ctrl and L-NAME animals. All data are presented as mean SEM (standard error mean). HW means heart weight; BW means body weigh; LVSP means left ventricular systolic pressure; LVEDP means left ventricular end-diastolic pressure; dPdt max means maximum velocity of pressure rise; dPdt min means maximum velocity of pressure decline; means tau. * P<0.05 vs Sham; #P<0.05 vs Ctrl. Sham TAC Ctrl L-NAME HW/BW (g/kg) 2.9±0.1 3.4±0.2* 2.3±0.1 2.7±0.2# Heart Rate (bpm) 403±11 386±17 410±19 378±21 LVSP (mmHg) 120±5 158±9* 110±3 147±12# LVEDP (mmHg) 4.0±0.4 10.0±2.0* 3.7±0.4 5.6±1.0 dPdt max (mmHg/sec) 7559±143 7234±629 6612±430 7131±900 dPdt min (mmHg/sec) -9436±474 -8065±948 -8714±2 -9382±1583 8±0.3 12±1.0* 9±2.0 10±1.0 FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 35 2. Effect of IMD on contractile and relaxation parameters Before the beginning of IMD protocol, contractile parameters of stabilized left ventricular papilary muscles were recorded and are summarized in Table 5. Results indicate there are not any statistical differences between the different animal models except for time to half relaxation (tHR), which is significantly higher in L-NAME animals. This particular result was already expected since it is known NO promotes hastening of relaxation (Paulus, Vantrimpont et al., 1994). Table 5 – Summary of contractile and relaxation parameters at baseline of LV papillary muscles from the different animal models. All data are presented as mean SEM (standard error mean). AT means active tension; dT/dtmax means maximum velocity of tension rise; dT/dtmin means maximum velocity of tension decline; PS means passive tension; dL/dtmax means maximum velocity of shortening; dL/dtmin means maximum velocity of lengthening; tHR means time to half relaxation. #P<0.05 vs Ctrl. Sham (n=6) TAC (n=7) Ctrl (n=6) L-NAME (n=5) AT (mN/mm2) 15±2 16±4 18±3 23±8 dT/dtmax ( mN/mm2/sec) 141±35 198±53 199±20 230±81 dT/dtmin (mN/mm2/sec) -71±10 -99±20 -112±9 -115±39 PS (%Lmax) 0.1±0.01 0.08±0.01 0.08±0.01 0.1±0.01 dL/dtmax (Lmax/sec) 1.5±0.1 1±0.1 1±0.1 1±0.1 dL/dtmin (Lmax/sec) -1.7±0.3 -1.3±0.2 -1.2±0.2 -1.2±0.2 tHR (msec) 292±34 257±16 265±21 318±19# Concerning IMD protocols, we verified that IMD addition in LV papillary muscles from Sham and Ctrl rats induced a similar decrease in AT (11±5% and 12±4%, respectively) and dT/dtmax (5±4% and 7±4%, respectively) while relaxation parameters did not present any statistical changes (Figure 17, A, B and C). On the other hand, in left ventricular papillary muscles from TAC and L-NAME rats, IMD induced an increase of FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 36 AT (11±3% and 13±3%, respectively) and dT/dtmax (20±3% in both). Furthermore, in these animal groups, IMD induced an increase in the rate of relaxation (dT/dtmin; 23±4% and 31±6%, TAC and L-NAME rats respectively), suggesting an hastening effect on relaxation. In short, these data showed a different IMD myocardial effect, promoting a negative inotropic effect in both control groups (Sham and Ctrl) but the opposite effect in TAC and L-NAME rats, in which it induced a positive inotropic and lusitropic effects. Figure 17 – Effect of increasing concentrations (10-10 to 10-6) of IMD on isolated left ventricular papillary muscles from Sham (n=6), TAC (n= 7), Ctrl (n= 6) and L-NAME (n= 5) rats, in A) Active Tension (AT), B) maximum velocity of tension rise (dT/dtmax) and C) maximum velocity of tension decline (dT/dtmin). *P<0.05 vs Sham; #P<0.05 vs Ctrl. A B C # * # * # * # * # * # * # * # * FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 37 3. Cardiac endothelial dysfunction in TAC rats Since IMD has an eNOS-mediated effect on myocardium, and so it is dependent on NO bioavailability (Pires, Pinho et al., 2012), we searched for endothelial dysfunction in LV hipertrophy rat models (Figure 18). Figure 18– Search for endotelial dysfunction on left ventricle hipertrophy animal models. A) Western blot analysis of expression of total eNOS (t-eNOS), normalized to beta-actin (β-actin), whose graphic representation shows a statistical similarity between Sham and TAC rats. B) Immunohistochemical staining of left ventricle samples from Sham, Ctrl, TAC and L-NAME rats (n= 5-6). The graphic representation shows both TAC and L-NAME rats have an increased level of nitrotyrosine relatively to their respective controls, Sham and Ctrl. C) GTPCH-1 mRNA expression in Sham and TAC rats, whose levels are statistically similar in both groups. D) Levels of conversion of L-arginine to Lcitruline, which are significantly greater in Sham rats than in TAC rats, suggesting a diminished eNOS activity in these latter. *P<0.05 vs Sham; #P<0.05 vs Ctrl. A C B # * D * 140 KDa 45 KDa TAC L-NAME S C Sham Ctrl FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 38 To do that, we started by accessing eNOS protein expression in TAC rats, which was statiscally similar to that in Sham rats (Figure 18A). Since oxidative stress is a main cause of endothelial dysfunction, we immunohistochemically stained left ventricle samples from all animal models to detect nytrotirosine, whose levels were increased in TAC and L-NAME rats (Figure 18B), suggesting the presence of high levels of ONOO-, a resultant product of reaction between superoxide and NO. On the other hand, decrease of BH4 is another possible cause for eNOS uncoupling so we investigated transcript levels of GTPCH1, whose expression translates in production of the rate-limiting enzyme of de novo synthesis of BH4. However, those transcript levels are not statistically different in TAC model (Figure 18C), indicating that BH4 production is unchanged. Despite that, TAC rats presented a lower conversion of L-arginine into Lcitruline, showing a diminished baseline eNOS activity, relatively to Sham rats (Figure 18D). FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 39 4. Activation of eNOS by IMD1-47 Phosphorylation of eNOS at Ser-1177, which is associated with activation of eNOS, is statistically greater in TAC rats when compared with Sham rats, at baseline (Figure 19A). In papillary muscles from Sham rats, IMD addition tripled the eNOS phosphorylation levels, while in those from TAC rats IMD addition was not able to enhance those levels (Figure 19A). A similar trend was observed in papillary muscles from Ctrl and L-NAME rats (Figure 19B). Thus, in TAC and L-NAME rats, there is an impairment of IMD-induced eNOS activation, since IMD is not able to increase baseline levels of phosphorylated eNOS in those animal models. 5. IMD treatment-induced phosphorylation of regulatory proteins According with our previous report (Pires, Pinho et al., 2012), addition of IMD increasing concentrations in papillary muscles from Sham and Ctrl rats increased c-TnI phosphorylation relatively to those muscles without IMD addition (Figure 20, A and C). However, IMD treatment in TAC and L-NAME rats had no statistical effect in p-cTnI * * Figure 19 – Western blot analysis of phosphorylated eNOS at Ser-1177 (p-eNOS) and total eNOS (t-eNOS) levels in papillary muscles from A) Sham and TAC rats and B) Ctrl and L-NAME rats. Papillary muscles from Sham and Ctrl rats showed increasing levels of p-eNOS upon IMD addition (IMD) relatively baseline levels (control), while in TAC and L-NAME rats IMD addition failed to increase those levels. *P<0.05 vs Sham control; #P<0.05 vs Ctrl control. A B # # * * 140 KDa 140 KDa 45 KDa 140 KDa 140 KDa 45 KDa FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 40 levels (Figure 20, A and C). Concerning p-PLB, its levels increased in both L-NAME and TAC rats but it was statistically significant only in these latter (Figure 20, B and D). These results suggest that IMD activates different signalling pathways in control and pressure-overloaded animal models, with phosphorylation of different proteins. Figure 20 – Western blot analysis of phosphorylated cardiac troponin I (p-cTnI) and phosphorylated phospholambam (p-PLB) in papillary muscles from Sham and TAC rats (A and B) and Ctrl and L-NAME rats (C and D), without (control) and with IMD addition (IMD). Upon IMD addition, in both Sham and Ctrl rats, cTnI phosphorylation increased, while in TAC (A) and L-NAME (C) rats this effect was blunted. Concerning p-PLB, statistical increasing was found only in TAC rats upon IMD addition (B), despite a similar trend is verified in L-NAME rats (D). *P<0.05 vs Sham control; **P<0.05 vs TAC control; #P<0.05 vs Ctrl control. A B * ** C D # 28 KDa 28 KDa 6 KDa 6 KDa FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 41 IV. Discussion FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 48 contributing to elevation of both active tension and maximum velocity of force generation (Figure 23). Interestingly, in HF rats, velocity of force generation is decreased due to both downregulation of α-MHC (myosin heavy chain) and upregulation of β-MHC mRNA expression levels (Huang, Liu et al., 2001). Therefore, IMD seems to play an important role at MHC expression levels, reversing α/β ratio and contributing for increasing of force generation velocity. In addition, particularly in TAC rats, we cannot exclude the involvement of neurohumoral activation-related mechanisms, since progression of heart failure is intimately associated with neurohumoral activation (Middlekauff and Mark, 1998), namely increasing of plasma catecholamines levels (Viquerat, Daly et al., 1985). This releasing results in a chronic catecholamine stress, which leads to a desensitization and downregulation of β1-AR and β2-AR/adenylyl cyclase signaling pathway (Okumura, Vatner et al., 2007), accompanied with upregulation of β3-AR (Moniotte, Kobzik et al., 2001; Morimoto, Hasegawa et al., 2004; Zhao, Wu et al., 2007), which mediates a NO-dependent negative inotropic effect (Gauthier, Leblais et al., 1998). However, it seems IMD elicits activation of both β1 and β2-ARs, which are able to induce cAMP/PKA pathway, leading to phosphorylation of LTCC (Kamp and Hell, 2000). This modification promotes the Ca2+ entering on the cell (Zipes and Jalife, 2009), contributing to the positive inotropic effect (by enhancing active tension) and to the increasing of velocity of tension rise. Also the involvement of cyclic nucleotide phosphodiesterases (PDEs) cannot be excluded since there are 11 mammalian PDE families responsible for specific cAMP and/or cGMP hydrolysis (Omori and Kotera, 2007). Some of them are reported as playing important role in heart failure: for example, PDE3A expression is decreased in human samples (Ding, Abe et al., 2005) while PDE5 inhibition is not only prescribed for treatment of erectile dysfunction and pulmonary hypertension (Kumar, Francis et al., 2009) but recently also for therapeutic management of HF (Guazzi, 2008). Concerning L-NAME rats, phosphorylation of PLB presents a positive trend, however it was not statistically significant. This result suggests there are other possible regulatory proteins involved, so more researches are needed. Therefore, we are aware that cascade of events which mediates IMD myocardial effects in pressure-overload rats is much more complex. Nevertheless, we constructed Figure 23 where we include both our results and what is already known from literature, creating a simple illustration with what we think that is the most likely sequence of events which leads to IMD myocardial effects, under physiologic and pathophysiologic conditions. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 49 peNOS cGMP PKG activation pTnI Ca2+ sensitivity Negative inotropic effect A Legend: IMD IMD receptors NO SERCA2a complex Ca2+ PLB Oxidative stress FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 50 Figure 23 – Cascade of events which mediated myocardial effects of IMD both under physiological (A) and pathophysiological (B) conditions. In presence of NO (A), cGMP/PKG signaling pathway is activated, resulting in phosphorylation of cTnI, decrease of myofilament Ca2+-sensitivity and thereby negative inotropic effect. On the other hand, in presence of endothelial dysfunction (B) there is no NO production, thus IMD directly activates cAMP/PKA signaling pathway, leading to positive inotropic and lusitropic effects mediated by phosphorylation of PLB and increase of SR Ca2+ uptake. This figure was done using Servier Medical Art. cAMP PKA activation pPLB Positive inotropic and lusitropic effects SR Endothelial dysfunction B FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 51 V. Conclusion FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 52 The present work provides new insights about the myocardial effects of IMD and mechanisms behind these effects, in pressure-overload-induced HF. Besides, the study of involvement of cardiac endothelial dysfunction in those effects point IMD as a potential marker of cardiac endothelial dysfunction. Firstly, haemodynamic and morphometric analysis showed that the two disease models TAC and L-NAME are quite similar, with both showing LV hypertrophy and a preserved systolic function. Concerning contractile and relaxation parameters at the baseline, also both models did not present any statistical difference, except for tau which was increased in L-NAME, as we expected. Despite these similarities, for the subsequent analysis, we took into account differences between the two models, since pressure-overload is obtained through very different ways: one by aortic constriction (TAC), the other one by systemic hypertension (L-NAME). Next, administration of IMD in LV papillary muscles from the four different animal groups showed, for the first time, that IMD elicits two completely opposite effects: in diseased myocardium it promotes positive inotropic and lusitropic effects, whereas in normal myocardium data suggests a decrease of inotropy. This observation emphasizes the putative cardioprotective role of IMD in HF. One of the reasons which could explain these so different behaviors is endothelial dysfunction, since IMD myocardial effects are mediated by endothelium and HF is associated to its dysfunction. Despite eNOS expression was unchanged at the baseline, activity assay showed eNOS function was diminished. This fact allied to the presence of oxidative stress suggests eNOS uncouling thus endothelial dysfunction, which is not due to the depletion of BH4 but probably is partly due to a decrease of its bioavailability, since GTPCH1 expression remains unaltered. Interestingly, we verified phosphorylation of eNOS at Ser-1177 was augmented at the baseline in diseased myocardium and IMD was not able to increase those levels, contrary to what we observed in normal myocardium. We hypothesize this increased phosphorylation levels are likely due to Akt chronic activation. Taken together, results suggest that phosphorylation of eNOS at Ser-1177 1) is a great contributor for endothelial dysfunction in TAC rats, by promoting the “pathologic” activity of eNOS and 2) explains the opposite myocardial effects of IMD. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 53 Finally, and based on expression results for regulatory proteins, we hypothesize IMD effects are mediated by 1) activation of cAMP/PKA signaling pathway and increase of phosphorylation of cTnI, in diseased myocardium and 2) NO-dependent activation of cGMP/PKG signaling pathway and increase of phosphorylation of PLB, in normal myocardium. Since IMD administration elicits different effects which are mediated by distinct mechanisms, this work also suggest that 1) endothelial dysfunction plays a central role in pathophysiology of HF and 2) IMD is a potential marker of cardiac endothelial dysfunction. FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 54 VI. Future Perspectives FCUP Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction 55 This work provides new insights about myocardial effects of intermedin and signaling pathways involved, reinforcing its putative cardioprotective role in cardiovascular disease. However, more studies are needed in order to prove this hypothesis. Firstly, it would be interesting draw a protocol for IMD chronic administration on chronic pressure-overloaded rats, namely in TAC model, since it is the most closer model of human HF. Recently, IMD chronic administration was done in SHR model and results seem to be very promising (Yuan, Wang et al., 2012). Another interesting topic would be to investigate the role of other posttranslational modifications of eNOS such as S-glutathionylation in HF. Indeed, recently Chen et al showed that under oxidative stress eNOS is S-glutathionylated in endothelial cells and this modification is responsible for production of ●O2by eNOS (Chen, Wang et al., 2010). Given the importance of study of heart failure, many authors have investigated different players in this so complex cardiac dysfunction. One of those players is oxidative stress. Contrary to expected, treatments using antioxidants have shown no clinical benefits (Hennekens, Buring et al., 1996; Yusuf, Dagenais et al., 2000). For this reason, studies have focus on sources of ROS: eNOS recoupling and increase endogenous levels of antioxidants are potential solutions (Takimoto and Kass, 2007). One of these studies recoupled eNOS through administration of both BH4 and apocynin, an inhibitor of NADPH oxidases, but the study was performed only in hypertensive aortas (Cai, Laude et al., 2006). Finally, in my opinion, the most interesting study line is miRNAs. 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