Multiscale Modeling and Simulation of Human Heart Failure

Tesis por compendio[EN] Heart failure (HF) constitutes a major public health problem worldwide. Operationally it is defined as a clinical syndrome characterized by the marked and progressive inability of the ventricles to fill and generate adequate cardiac output to meet the demands of cellular metabolism that may have significant variability in its etiology and it is the final common pathway of various cardiac pathologies. Much attention has been paid to the understanding of the arrhythmogenic mechanisms induced by the structural, electrical, and metabolic remodeling of the failing heart. Due to the complexity of the electrophysiological changes that may occur during heart failure, the scientific literature is complex and sometimes equivocal. Nevertheless, a number of common features of failing hearts have been documented. At the cellular level, prolongation of the action potential (AP) involving ion channel remodeling and alterations in calcium handling have been established as the hallmark characteristics of myocytes isolated from failing hearts. At the tissue level, intercellular uncoupling and fibrosis are identified as major arrhythmogenic factors. In this Thesis a computational model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of the late sodium current (INaL) in order to study the arrhythmogenic processes due to failing phenotype. Experimental data from several sources were used to validate the model. Due to extensive literature in the subject a sensitivity analysis was performed to assess the influence of main ionic currents and parameters upon most related biomarkers. In addition, multiscale simulations were carried out to characterize this pathology (transmural cardiac fibres and tissues). The proposed model for the human INaL and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. An enhanced INaL appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of calcium homeostasis of failing myocytes. Our strand simulation results illustrate how the presence of M cells and heterogeneous electrophysiological remodeling in the human failing ventricle modulate the dispersion of action potential duration (APD) and repolarization time (RT). Conduction velocity (CV) and the safety factor for conduction (SF) were also reduced by the progressive structural remodeling during heart failure. In our transmural ventricular tissue simulations, no reentry was observed in normal conditions or in the presence of HF ionic remodeling. However, defined amount of fibrosis and/or cellular uncoupling were sufficient to elicit reentrant activity. Under conditions where reentry was generated, HF electrophysiological remodeling did not alter the width of the vulnerable window (VW). However, intermediate fibrosis and cellular uncoupling significantly widened the VW. In conclusion, enhanced fibrosis in failing hearts, as well as reduced intercellular coupling, combine to increase electrophysiological gradients and reduce electrical propagation. In that sense, structural remodeling is a key factor in the genesis of vulnerability to reentry, mainly at intermediates levels of fibrosis and intercellular uncoupling.[ES] La insuficiencia cardíaca (IC) constituye un importante problema de salud pública en todo el mundo. Operacionalmente se define como un síndrome clínico caracterizado por la incapacidad marcada y progresiva de los ventrículos para llenar y generar gasto cardíaco adecuado para satisfacer las demandas del metabolismo celular, que puede tener una variabilidad significativa en su etiología y es la vía final común de varias patologías cardíacas. Se ha prestado mucha atención a la comprensión de los mecanismos arritmogénicos inducidos por la remodelación estructural, eléctrica, y metabólica del corazón afectado de IC. Debido a la complejidad de los cambios electrofisiológicos que pueden ocurrir durante la IC, la literatura científica es compleja y, a veces equívoca. Sin embargo, se han documentado una serie de características comunes en corazones afectados de IC. A nivel celular, se han establecido como las características distintivas de los miocitos aislados de corazones afectados de IC la prolongación del potencial de acción (PA), que implica la remodelación de los canales iónicos y las alteraciones en la dinámica del calcio. A nivel de los tejidos, el desacoplamiento intercelular y la fibrosis se identifican como los principales factores arritmogénicos. En esta tesis se propuso un modelo celular computacional para la insuficiencia cardíaca utilizando una versión modificada del modelo de potencial de acción ventricular humano de Grandi y colaboradores que incorpora la formulación de la corriente tardía de sodio (INaL) con el fin de estudiar los procesos arritmogénicas debido al fenotipo de la IC. Los datos experimentales de varias fuentes se utilizaron para validar el modelo. Debido a la extensa literatura en la temática se realizó un análisis de sensibilidad para evaluar la influencia de las principales corrientes iónicas y los parámetros sobre los biomarcadores relacionados. Además, se llevaron a cabo simulaciones multiescala para caracterizar esta patología (en fibras y tejidos transmurales). El modelo propuesto para la corriente tardía de sodio y la remodelación electrofisiológica de los miocitos de corazones afectados de IC reprodujeron con precisión las observaciones experimentales. Una INaL incrementada parece ser un importante contribuyente al fenotipo electrofisiológico y la desregulación de la homeostasis del calcio de los miocitos afectados de IC. Nuestros resultados de la simulaciones en fibra ilustran cómo la presencia de células M y el remodelado electrofisiológico heterogéneo en el ventrículo humano afectado de IC modulan la dispersión de la duración potencial de acción (DPA) y el tiempo de repolarización (TR). La velocidad de conducción (VC) y el factor de seguridad para la conducción (FS) también se redujeron en la remodelación estructural progresiva durante la insuficiencia cardíaca. En nuestras simulaciones transmurales de tejido ventricular, no se observó reentrada en condiciones normales o en presencia de la remodelación iónica de la IC. Sin embargo, determinadas cantidades de fibrosis y / o desacoplamiento celular eran suficientes para provocar la actividad reentrante. En condiciones donde se había generado la reentrada, el remodelado electrofisiológico de la IC no alteró la anchura de la ventana vulnerable (VV). Sin embargo, niveles intermedios de fibrosis y el desacoplamiento celular ampliaron significativamente la VV. En conclusión, niveles elevados de fibrosis en corazones afectados de IC, así como la reducción de acoplamiento intercelular, se combinan para aumentar los gradientes electrofisiológicos y reducir la propagación eléctrica. En ese sentido, la remodelación estructural es un factor clave en la génesis de la vulnerabilidad a las reentradas, principalmente en niveles intermedios de fibrosis y desacoplamiento intercelular. El remodelado electrofisiológico promueve la arritmogénesis y puede ser alterado dependi[CA] La insuficiència cardíaca (IC) constitueix un important problema de salut pública arreu del món. A efectes pràctics, es defineix com una síndrome clínica caracteritzada per la incapacitat marcada i progressiva dels ventricles per omplir i generar el cabal cardíac adequat, per tal de satisfer les demandes del metabolisme cel·lular, el qual pot tenir una variabilitat significativa en la seua etiologia i és la via final comuna de diverses patologies cardíaques. S'ha prestat molta atenció a la comprensió dels mecanismes aritmogènics induïts per la remodelació estructural, elèctrica, i metabòlica del cor afectat d'IC. A causa de la complexitat dels canvis electrofisiològics que poden ocórrer durant la IC, trobem que la literatura científica és complexa i, de vegades, equívoca. No obstant això, s'han documentat una sèrie de característiques comunes en cors afectats d'IC. A nivell cel·lular, com característiques distintives dels miòcits aïllats de cors afectats d'IC, s'han establert la prolongació del potencial d'acció (PA), que implica la remodelació dels canals iònics, i les alteracions en la dinàmica del calci. A nivell dels teixits, el desacoblament intercel·lular i la fibrosi s'identifiquen com els principals factors aritmogènics. Per tal d'estudiar els processos aritmogènics a causa del fenotip de la IC, es va proposar un model cel·lular computacional d'IC utilitzant una versió modificada del model de potencial d'acció ventricular humà de Grandi i els seus col·laboradors, el qual incorpora la formulació del corrent de sodi tardà (INaL). Amb l'objectiu de validar el model es van utilitzar dades experimentals de diverses fonts. A causa de l'extensa literatura en la temàtica, es va realitzar una anàlisi de sensibilitat per tal d'avaluar la influència de les principals corrents iòniques i els paràmetres sobre els biomarcadors relacionats. A més, es van dur a terme simulacions multiescala per a la caracterització d'aquesta patología (fibres i teixits transmurals). El model proposat per al corrent de sodi tardà i la remodelació electrofisiològica dels miòcits de cors afectats d'IC van reproduir amb precisió les observacions experimentals. Una INaL incrementada sembla contribuir de manera important al fenotip electrofisiològic i a la desregulació de l'homeòstasi del calci dels miòcits afectats d'IC. Els resultats de les nostres simulacions en fibra indiquen que la presència de cèl·lules M i el remodelat electrofisiològic heterogeni en el ventricle humà afectat d'IC modulen la dispersió de la durada del potencial d'acció (DPA) i el temps de repolarització (TR). La velocitat de conducció (VC) i el factor de seguretat per a la conducció (FS) també es van reduir en la remodelació estructural progressiva durant la IC. A les nostres simulacions transmurals de teixit ventricular, no s'observà cap reentrada ni en condicions normals ni en presència de la remodelació iònica de la IC. No obstant això, amb determinades quantitats de fibrosi i/o desacoblament cel·lular sí que es provocà l'activitat reentrant. I amb les condicions que produïren la reentrada, el remodelat electrofisiològic de la IC no va alterar l'amplada de la finestra vulnerable (FV). Tanmateix, nivells intermedis de fibrosi i el desacoblament cel·lular sí que ampliaren significativament la FV. En conclusió, nivells elevats de fibrosi en cors afectats d'IC, així com la reducció d'acoblament intercel·lular, es combinen per augmentar els gradients electrofisiològics i reduir la propagació elèctrica. Per tant, la remodelació estructural és un factor clau en la gènesi de la vulnerabilitat a les reentrades, principalment en nivells intermedis de fibrosi i desacoblament intercel·lular.Gómez García, JF. (2015). Multiscale Modeling and Simulation of Human Heart Failure [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52389TESISCompendi

Atrial remodelling in hypertensive heart disease: role of Na+ homeostasis and contractility

Arterial hypertension causes hypertensive heart disease. Constant mechanical stress and activation of neurohormonal systems cause structural and functional changes in the myocardium termed “remodelling”. Remodelling is beneficial in the beginning of the disease development; however, with time it becomes detrimental and impairs cardiac function. Remodelling of the myocardium occurs in hypertension, atrial fibrillation and heart failure. These cardiac diseases are tightly linked by the mechanisms of pathological remodelling and induce development and maintenance of one another. Ventricular remodelling has been studied intensively in hypertensive heart disease, however, atrial remodelling has been studied much less and is only poorly understood. Physiology of cardiac myocytes relies on balanced intracellular Na+ homeostasis. Na+ is involved in many cellular processes, such as action potential initiation, Ca2+ homeostasis, intracellular pH, metabolism and contractility. In the first part of the thesis I investigated ionic (Na+ homeostasis) and functional (contractility) atrial remodelling in an animal model of hypertensive heart disease – spontaneously hypertensive rats (SHR). In early hypertension, SHR exhibited elevated blood pressure and isolated left ventricular hypertrophy. The atria were not hypertrophied. Contractility of atrial myocytes and intracellular Na+ concentration ([Na+]i) were both unaltered. Expression of most Na+-handling proteins was unaffected in the atria of SHR. In advanced hypertension, SHR exhibited further progression of left ventricular hypertrophy and signs of heart failure. Left atria were hypertrophied. The contractility of atrial myocytes was reduced. [Na+]i was significantly decreased together with increased expression of the α 1 subunit of Na+/K+-ATPase. Expression of Na+/H+-exchanger was increased, suggesting activation of pro-hypertrophic pathways. Comparison of SHR with and without signs of heart failure (i.e. increased lung weight) revealed development of right ventricular hypertrophy and progression of bi-atrial hypertrophy in SHR with heart failure. Moreover, the impairment of atrial myocyte contractility progressed. However, [Na+]i and the expression of major Na+-handling proteins were not changed during the transition to heart failure. In addition to studies on atrial myocytes, we performed measurements of [Na+]i and contractility of ventricular myocytes from old SHR. In contrast to our findings in the atria, no impairment of contractility or changes in [Na+]i were observed in the ventricular myocytes, indicating atria-specific remodelling. Taken together, the presented results indicate that in early hypertension no significant signs of atrial remodelling in terms of contractility and Na+ homeostasis were found. However, in advanced hypertensive heart disease there was atria-specific functional atrial remodelling, which might contribute to the transition from compensated left ventricular hypertrophy to heart failure. Atrial ionic remodelling is an important factor in the development and maintenance of atrial fibrillation. The role of intracellular Na+ homeostasis in these processes is not understood. In the second part of the thesis, I investigated expression of Na+-handling proteins in right atrial tissue of patients suffering from paroxysmal and chronic atrial fibrillation compared to patients with sinus rhythm. The results indicated that the expression of Na+-handling proteins, including Na+ channels, Na+/H+ exchanger, alpha subunits of Na+/K+-ATPase, phospholemman, was not altered in either paroxysmal or chronic atrial fibrillation. The expression of β 1 subunit of Na+/K+-ATPase was significantly reduced in chronic atrial fibrillation. However, the functional consequences of this change require further investigation. Endothelin-1 plays an important role in the regulation of blood pressure and cardiac physiology. Enhancement of endothelin-1 system activity contributes to cardiac maladaptive remodelling, including disturbances in Ca2+ and Na+ homeostasis in cardiac myocytes. At the age of 7 months, SHR exhibit enhanced endothelin-1 signalling and altered Ca2+ handling. Therefore, in the third part of the thesis we investigated the effect of endothelin-1 receptor blockage on blood pressure and expression and phosphorylation of Ca2+-handling proteins, as well as the expression of proteins involved in endothelin-1 signalling in the atria of SHR. The results revealed that the blockage of endothelin receptors by 8 weeks treatment with macitentan (novel dual endothelin A and endothelin B receptor antagonist) did not lower blood pressure in SHR. Expression and phosphorylation of major Ca2+-handling proteins and endothelin-1 signalling proteins were both unaffected. Thus, the blockage of endothelin receptors did not cause any major changes in atrial Ca2+ remodelling in SH

β-Adrenergic Receptor Stimulation and Alternans in the Border Zone of a Healed Infarct: An ex vivo Study and Computational Investigation of Arrhythmogenesis

Background: Following myocardial infarction (MI), the myocardium is prone to calcium-driven alternans, which typically precedes ventricular tachycardia and fibrillation. MI is also associated with remodeling of the sympathetic innervation in the infarct border zone, although how this influences arrhythmogenesis is controversial. We hypothesize that the border zone is most vulnerable to alternans, that β-adrenergic receptor stimulation can suppresses this, and investigate the consequences in terms of arrhythmogenic mechanisms.Methods and Results: Anterior MI was induced in Sprague-Dawley rats (n = 8) and allowed to heal over 2 months. This resulted in scar formation, significant (p < 0.05) dilation of the left ventricle, and reduction in ejection fraction compared to sham operated rats (n = 4) on 7 T cardiac magnetic resonance imaging. Dual voltage/calcium optical mapping of post-MI Langendorff perfused hearts (using RH-237 and Rhod2) demonstrated that the border zone was significantly more prone to alternans than the surrounding myocardium at longer cycle lengths, predisposing to spatially heterogeneous alternans. β-Adrenergic receptor stimulation with norepinephrine (1 μmol/L) attenuated alternans by 60 [52–65]% [interquartile range] and this was reversed with metoprolol (10 μmol/L, p = 0.008). These results could be reproduced by computer modeling of the border zone based on our knowledge of β-adrenergic receptor signaling pathways and their influence on intracellular calcium handling and ion channels. Simulations also demonstrated that β-adrenergic receptor stimulation in this specific region reduced the formation of conduction block and the probability of premature ventricular activation propagation.Conclusion: While high levels of overall cardiac sympathetic drive are a negative prognostic indicator of mortality following MI and during heart failure, β-adrenergic receptor stimulation in the infarct border zone reduced spatially heterogeneous alternans, and prevented conduction block and propagation of extrasystoles. This may help explain recent clinical imaging studies using meta-iodobenzylguanidine (MIBG) and 11C-meta-hydroxyephedrine positron emission tomography (PET) which demonstrate that border zone denervation is strongly associated with a high risk of future arrhythmia

Cardiac Arrhythmias

The most intimate mechanisms of cardiac arrhythmias are still quite unknown to scientists. Genetic studies on ionic alterations, the electrocardiographic features of cardiac rhythm and an arsenal of diagnostic tests have done more in the last five years than in all the history of cardiology. Similarly, therapy to prevent or cure such diseases is growing rapidly day by day. In this book the reader will be able to see with brighter light some of these intimate mechanisms of production, as well as cutting-edge therapies to date. Genetic studies, electrophysiological and electrocardiographyc features, ion channel alterations, heart diseases still unknown , and even the relationship between the psychic sphere and the heart have been exposed in this book. It deserves to be read

Tissue engineering of the human atrium : approaching mechanisms of genesis and control of atrial fibrillation

Cardiovascular disease is prevalent across the western world and is a major cause of morbidity and mortality, accounting for approximately a third of all fatalities. Investigating the heart by simulating its electrophysiology via the aid of mathematical models has advanced significantly over the past 60 years and is now a well established field. While much of the research focus is placed on the ventricles, the study of the atria is in comparison neglected. Therefore this Thesis is focused on the genesis and maintenance of atrial fibrillation (AF). A series of case studies are performed whereby established biophysically detailed mathematical models are implemented and modified to incorporate electrophysical alterations of atrial cells resulting from a variety of external conditions. The opening section of this Thesis is dedicated to developing a background to the field, including a discussion into the clinical aspect of the diagnosis and management of AF. The suitability of two atrial cell models is discussed and the development of single cell, 1D, 2D, and 3D multi-scale simulation protocols are described in detail. In addition measurements taken to quantify the arrhythmogenic properties of the cells susceptibility to AF are outlined. The second section is focused on the incorporation of conditions thought to enhance atrial tissues ability to initiate and maintain the genesis of AF. Included is a case study into the missence S140G gene mutation, and elevated physiological levels of the hormone Homocystein. The third section investigates the effectiveness of well established and widely used pharmacological treatments such as Beta-Blockers. In addition possible avenues of investigations for the development of atrial specific drugs are explored. These include blocking of the ultra rapid potassium channel and a more novel target for therapy via the targeting of 5HT4 receptors; which is transcribed solely in the atria and alters the electrophysical properties of the L-type Calcium current. The final part of this Thesis is dedicated to the development of a 2D atrial sheet model which includes electrical and spatial heterogeneities via the inclusion of multiple cell types and basic fiber orientation respectively. This allows for an investigation into the role that heterogeneities play in role genesis and maintenance of AF. The main finding of this Thesis is that alterations to the electrophysiology of atrial cells, due to external factors, can be successfully simulated via the implementation of mathematically detailed atrial cell models. It is concluded that simulations of the KENQ1 mutation and elevated levels of Homocystein successfully reproduce conditions which increase the onset of AF. Established treatments such as Beta-Blockers are found to have limited effectiveness. Possible theoretical treatments, such as the blocking of IKur, are found to provide a small amount of therapeutic benefit. In contrast, investigations into the effects of Serotonin were inconclusive. The study into the 2D atria indicated the importance that heterogeneities play in atria. The conclusions show that models provide a powerful tool when investigating how changes to electrophysiology of cells are manifested at a multi-scale level. The models also have their limitations and require further advancement to improve their accuracy.EThOS - Electronic Theses Online ServiceEPSRCGBUnited Kingdo

Aberrant intracellular calcium cycling in the heart : mechanistic insights into catecholaminergic polymorphic ventricular tachycardia and heart failure

Heart disease is the biggest killer world-wide, causing a quarter of all deaths. During the past two decades, it has also risen above infectious diseases as the leading cause of years of life lost. Heart failure, characterized by weak pump function of the heart, and disturbances in heart rhythm (arrhythmias) are common and interrelated mechanisms underlying cardiac mortality. Intracellular calcium ions are crucial to contraction and relaxation of the heart muscle, as well as to control of its rhythm. How calcium is handled and regulated is thus essential to normal cardiac function, and disturbances in these processes can have catastrophic consequences. Understanding the mechanisms of these disturbances is important for improving disease prevention, diagnosis, and management. The studies in this thesis focus on two conditions where cardiac calcium handling is impaired. Studies I - III examine the mechanisms of a genetic arrhythmia disease named catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by stress-induced ventricular tachycardia in a structurally normal heart. Study IV investigates cardiac function in a model of another genetic disease, autosomal dominant polycystic kidney disease (ADPKD). This systemic disease mainly affects the kidneys, and the mechanisms of the concomitant decline in cardiac function have thus far remained underinvestigated. We evaluated clinical data on cardiac function of CPVT patients, including 24h electrocardiograms, intracardiac monophasic action potential recordings, and exercise stress tests. We used cell models to study the underlying disease mechanisms in detail. During conditions of stress, CPVT cells showed increased spontaneous and irregular release of calcium from within the intracellular stores through the cardiac ryanodine receptors. These receptors, which function as intracellular calcium release channels, harbor the disease-causing mutation. The spontaneous release of calcium led to changes in the membrane potential of the cells, manifested as afterdepolarizations during the resting phase of the cardiac cycle. These afterdepolarizations were reproduced in the clinical monophasic action potential recordings of CPVT patients, and were shown to trigger arrhythmias in these patients. Changes in intracellular calcium alter the membrane potential, and these changes are reflected on the electrocardiogram. Thus, irregularities that might correspond to those observed in the cell model were then investigated in 24h electrocardiograms of CPVT patients. Increased irregularity of cardiac repolarization was found in the CPVT patients. Such irregularity was greater in the electrocardiograms of CPVT patients with a history of more severe arrhythmic events. Additionally, we found slowed depolarization in response to stress in CPVT cells and patients, suggesting reduced conduction velocity might contribute to an arrhythmic substrate in these patients. Cardiac function in ADPKD caused by mutations in polycystin-2, another intracellular calcium channel, was investigated in a zebrafish model lacking expression of the polycystin-2 protein. The zebrafish lacking polycystin-2 showed signs of heart failure, including reduced cardiac output, edema, and arrhythmias. Hearts, which were then examined in more detail ex vivo, showed impaired cycling of intracellular calcium, which is likely to underlie the cardiac dysfunction observed in vivo. The association of ADPKD with idiopathic dilated cardiomyopathy (IDCM) was examined using the Mayo ADPKD Mutation Database, which contains data on genotyped ADPKD patients. Examination of the ADPKD Database showed IDCM to be very common among ADPKD patients. IDCM was most prevalent in patients with mutations in polycystin-2, suggesting impaired calcium cycling as a potential pathomechanism. Studies I-III shed new light on mechanisms of arrhythmias in CPVT and related conditions, opening the way for future studies on arrhythmia risk and therapeutic evaluation. Furthermore, the results encourage pursuing the novel stem cell models for studying pathomechanisms and therapeutics. Study IV showed an association between ADPKD and IDCM. The zebrafish model suggested impaired calcium cycling as an underlying mechanism, highlighting the usefulness of zebrafish as a model in cardiac research.Sydänsairaudet ovat maailmanlaajuisesti yleisin kuolinsyy, aiheuttaen neljänneksen kuolemista. Kahden viime vuosikymmenen aikana ne ovat ohittaneet tartuntataudit suurimpana menetyttyjen elinvuosien aiheuttajana. Sydämen vajaatoiminta ja rytmihäiriöt ovat sydänkuolleisuuden taustalla olevia yleisiä ja toisiinsa liittyviä mekanismeja. Solunsisäiset kalsiumionit ovat elintärkeitä sekä sydämen supistuvuuden ja relaksaation, että sen rytmin säätelyn kannalta. Kalsiumin tiukka säätely on siten sydämen normaalin toiminnan edellytys, ja tämän säätelyn häiriöillä voi olla vakavia seuraamuksia. Näiden häiriöiden mekanismien ymmärtäminen on tärkeää sairauksien ehkäisyn, diagnostiikan ja hoidon kannalta. Tämän väitöskirjan osatyöt paneutuvat kahteen sairauteen, joissa sydämen solunsisäisen kalsiumin säätely on häiriintynyt. Osatyöt I - III tutkivat katekoliaminergisen monimuotoisen kammiotiheälyöntisyyden (CPVT) mekanismeja. Tämä perinnöllinen rytmihäiriösairaus aiheuttaa henkeä uhkaavaakin kammiotiheälyöntisyyttä stressin yhteydessä rakenteellisesti terveessä sydämessä. Osatyössä IV tutkitaan sydämen toimintaa liittyen autosomissa vallitsevasti periytyväään munuaisten monirakkulatautiin (ADPKD). Tämä perinnöllinen systeeminen sairaus vaikuttaa oleellisesti munuaisiin, mutta samanaikainen sydämen toiminnan heikkeneminen on jäänyt vähemmälle huomiolle. Arvioimme sydämen toimintaa CPVT-potilailla käyttäen menetelminä sydänsähkökäyrän pitkäaikaisnauhoitusta, sydämen rasituskoetta ja elektrofysiologista tutkimusta, johon sisältyi oikean kammion aktiopotentiaalimittaus. Lisäksi käytimme solumalleja mekanismien yksityiskohtaiseen selvittämiseen. CPVT:n taustalla ovat mutaatiot sydämen ryanodiinireseptorissa, joka on kalsiumin säätelyssä oleellinen solunsisäinen kalsiumkanava. Stressin yhteydessä CPVT-soluissa esiintyi lisääntynyttä spontaania ja epäsäännöllistä kalsiumin vapautumista solunsisäisistä varastoista mutatoituneiden ryanodiinireseptorien läpi. Tämä johti solukalvon jännitteen muutoksiin, jotka havaittiin sydämen lepovaiheen aikana ilmenevinä jälkidepolarisaatioina. Vastaavia jälkidepolarisaatioita havaittiin myös CPVT-potilaiden aktiopotentiaalimittauksissa, missä ne ajoittain laukaisivat kammiolisälyöntejä. Solunsisäisen kalsiumpitoisuuden muutokset vaikuttavat solukalvon jännitteeseen, mikä taas heijastuu sydänsähkökäyrään. Tämän vuoksi solumallissa todettua kalsiumpitoisuuden vaihtelua vastaavia muutoksia tutkittiin CPVT-potilaiden sydänsähkökäyrissä. Vastaavasti, myös sydämen repolarisaation vaihtelu oli lisääntynyt CPVT-potilailla. Tämä vaihtelu oli suurempaa niiden CPVT-potilaiden sydänsähkökäyrissä, joilla oli aiemmin todettuja vakavia rytmihäiriötapahtumia. Tämän lisäksi löysimme CPVT-soluilla ja -potilailla stressin yhteydessä hidastuvaa depolarisaatiota, viitaten hidastuneen sähköisen johtumisen mahdolliseen osuuteen rythmihäiriölttiutta lisäävänä tekijänä näillä potilailla. Tutkimme sydämen toimintaa polykystiini-2:n mutaatioista aiheutuvassa ADPKD-mallissa. Käytetyltä seeprakalamallilta puuttuu polykysteiini-2-proteiini, joka normaalisti muodostaa solunsisäisen kalsiumkanavan. ADPKD:n yhteyttä tuntemattomasta syystä johtuvaan laajentavaan sydänlihassairauteen (IDCM) tarkasteltiin Mayo-klinikan ADPKD tietokannasta, joka sisältää tietoa genotyypatuista ADPKD-potilaista. Seeprakalamallissa todettiin sydämen vajaatoimintaan viittaavina löydöksinä alentunut sydämen minuuttitilavuus, turvotusta ja rytmihäiriöitä. Kalojen sydämiä tutkittiin tarkemmin kuvantamalla solunsisäistä kalsiumia, jonka säätelyn havaittiin olevan heikentynyttä ADPKD-kaloilla sopien havaitun sydämen vajaatoiminnan syyksi. Mayo-klinikan tietokannassa IDCM oli yleinen diagnoosi ADPKD-potilaiden keskuudessa. Vallitsevuus etenkin polykystiini-2-mutaatioita kantavien ADPKD-potilaiden keskuudessa oli yleistä, viitaten heikentyneeseen kalsiumin säätelyyn mahdollisena tautimekanismina. Osatyöt I III tuovat uutta tietoa CPVT:n ja senkaltaisten tautitilojen yhteydessä esiintyvien rytmihäiriöiden mekanismeista, avaten samalla tien tuleville rytmihäiriöriskiin ja hoidon tehon arviointiin liittyville tutkimuksille. Lisäksi tulokset kannustavat jatkamaan uusien solumallien käyttöä tautimekanismeihin ja hoitoihin liittyvissä tutkimuksissa. Osatyö IV:ssä havaittiin yhteys ADPKD:n ja IDCM:n välillä. Tutkimukset seeprakalamallilla viittaavat heikentyneeseen solunsisäisen kalsiumin säätelyyn taustalla olevana mekanismina, samalla alleviivaten seeprakalamallin käyttökelpoisuutta sydäntutkimuksessa

Role of glycogen synthase kinase 3 (GSK-3) and its substrate proteins in the development of cardiomyopathy associated with obesity and insulin resistance

Thesis (MScMedSc)--University of Stellenbosch, 2011.ENGLISH ABSTRACT: INTRODUCTION: Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase that was first discovered as a regulator of glycogen synthase thus playing a role in glycogen synthesis (Embi et al. 1980). GSK-3 has also been shown to down regulate the expression of SERCA-2a (a calcium ATPase pump) thus playing a role in myocardial contractility (Michael et al. 2004). However, SERCA-2a activity is regulated by phospholamban (PLM) and sarcolipin (SLN) (Asahi et al. 2003). GSK-3 is constitutively active in cells and can be acutely inactivated by insulin through phosphorylation by PKB/Akt. However, GSK-3 is known to phosphorylate and inhibit IRS-1 protein, thus disrupting insulin signaling (Eldar-Finkelman et al. 1996). In addition, abnormally high activities of GSK-3 protein has been implicated in several pathological disorders which include type 2 diabetes, neuron degenerative and affective disorders (Eldar-Finkelman et al 2009). This led to the development of new generations of inhibitors with specific clinical implications to treat these diseases (Martinez 2008). GSK-3 inhibition has been shown to improve insulin and blood glucose levels and to be cardioprotective during ischemia/reperfusion (Nikoulina et al. 2002; Kumar et al. 2007). AIMS: To determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether a specific GSK-3 inhibitor can prevent or reverse the cardiovascular pathology found in obese and insulin resistant animals. OBJECTIVES: To correlate the alterations in expression and activation of GSK-3 protein in a well characterised rat model of obesity coupled to insulin resistance with: i) myocardial contractile dysfunction and an inability of hearts to withstand ischemia/reperfusion, ii) the activation and expression of phospholamban and SERCA-2a in the sarcoplasmic reticulum, iii) the activation of intermediates (IRS-1, IRS-2 and PKB/Akt) that lie upstream in the activation pathway of GSK-3 and iv) to determine the effects of inhibition of GSK-3 on the abovementioned parameters. METHODS: Age and weight matched male Wistar rats (controls and diet induced obese (DIO) animals) were used in the present study. Controls were fed normal rat chow, while DIOs were fed a rat chow diet supplemented with sucrose and condensed milk, for 8 or 16 weeks. Half of each group of animals were treated with the GSK-3 inhibitor for 4 weeks (from 12 to 16 weeks). After the feeding and treatment period, animals were weighed, sacrificed, hearts removed and freeze clamped immediately or perfused with Krebs-Henseleit buffer and subjected to low flow ischemia (25 min) followed by 30 min reperfusion. Biometric (body weight, intraperitoneal fat, ventricular weight and tibia length) and biochemical (fasting blood glucose and insulin levels) parameters were determined. Expression of GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a and Phospholamban were determined by Western blotting. Ca2+ ATPase activity was determined spectrophotometrically. RESULTS: At both 8 and 16 weeks DIO animals were significantly bigger than control animals and this was associated with increased intraperitoneal fat in DIOs. In DIO animals: IRS-1 was downregulated at 8 weeks and both IRS-1 and IRS-2 as well as PKB/Akt at 16 weeks. There was an increased tendency of GSK-3 expression at both 8 and 16 weeks in DIO animals while SERCA-2a was severely downregulated from 8 weeks onwards and associated with lower Ca2+-ATPase activity. PLM expression was upregulated but its phosphorylation was attenuated. At 16 weeks, baseline heart rate (225 vs 275 in control, P<0.0001, n=6) and rate pressure product (21000 vs 30000 in control, P=0.019, n=6) were significantly lower in hearts from DIO animals. Functional recovery was unchanged but the time to ischemic contracture development was increased (11.6±0.4 control vs 16.2±0.5 min DIO, P<0.01, n=6). Treatment had no effect on total GSK-3 expression. However, GSK-3 phosphorylation was significantly increased in treated controls, while there was no significant difference in DIO animals. However, there was a tendency for an increased GSK-3 phosphorylation in treated DIO animals. GSK-3 inhibitor, improved hypertrophy in DIO animals, while it led to its development in control animals. GSK-3 inhibitor improved IRS-2 expression in both control and DIO animals while it had no effect on IRS-1 and SERCA-2a expression and activity. However, GSK-3 inhibition increased PKB/Akt and phospholamban phosphorylation in DIO animals. CONCLUSION: These findings show that high calorie diet as well as imbalance between energy intake and expenditure lead to the development of obesity and insulin resistance in male Wistar rats. We showed that GSK-3 and its substrate proteins are dysregulated in obesity and insulin resistance. The reduced SERCA-2a expression at baseline may have a negative impact on cardiac function. By treating the animals with GSK-3 inhibitor, we showed that GSK-3 protein may not be responsible for changes seen at baseline. The decreased IRS-1 and SERCA-2a expression may have been caused by a different mechanism other than the actions of GSK-3. However, according to this study, GSK-3 may play a role in regulation of IRS-2 expression but not in IRS-1. Increased PKB/Akt phosphorylation may contribute to the GSK-3 inhibition. In addition, GSK-3 inhibition may reverse cardiac hypertrophy in DIO animals, thus acting as a negative regulator of hypertrophy.AFRIKAANSE OPSOMMING: Inleiding: Glikogeen sintase kinase-3 (GSK-3), 'n serien/threonien proteïen kinase, is oorspronklik ontdek as 'n rolspeler in glikogeen sintese, aangesien dit 'n reguleerder van glikogeen sintase is (Embi et al.1980). Intussen is dit ook bevind dat GSK-3 die uitdrukking van SERCA-2a ('n kalsium ATPase pomp) kan afreguleer en dus sodoende 'n rol speel in miokardiale kontraktiliteit (Michael et al. 2004). Die aktiwiteit van SERCA-2a kan egter ook gereguleer word deur fosfolamban (PLM) en sarkolipin (Asahi et al. 2003). GSK-3 is deurgaans aktief, maar kan tydelik geïnaktiveer word onder kondisies van insulien stimulasie deur PKB/Akt gemedieerde fosforilering. Aan die ander kant is dit bekend dat GSK-3 die IRS-1 proteïen kan fosforileer om dus sodoende insulien sein-transduksie af te reguleer (Eldar-Finkelman et al. 1996). Daarmee saam is abnormaal hoë vlakke van GSK-3 aktiwiteit geassosieer met verskeie patologiese versteurings, insluitend tipe 2 diabetes, neuron degeneratiewe en affektiewe versteurings (Eldar-Finkelman et al. 2009). Daar is dus nuwe generasies GSK-3 inhibitore ontwikkel met die kliniese potensiaal om hierdie patologieë te behandel (Martinez 2008). Dit is al bevind dat GSK-3 inhibisie geassosieer kan word met beide die normalisering van plasma insulien- en glukose vlakke, asook kardiobeskerming in die konteks van iskemie/herperfusie (Nikoulina et al. 2002; Kumar et al. 2007). Doelwitte: Om te bepaal of GSK-3 proteïen en sy substraat proteïene gedisreguleer is onder kondisies van obesiteit en insulien weerstandigheid, asook om vas te stel of 'n spesifieke GSK-3 inhibitor die kardiovaskulêre patologie wat gevind word in obese en insulien weerstandige diere kan verhoed of omkeer. Mikpunte: Om veranderinge in uitdrukking en aktiwiteit van GSK-3 proteïen in 'n goed gekarakteriseerde rotmodel van obesiteit, gekoppel aan insulien weerstandigheid, te korreleer met die volgende: i) miokardiale kontraktiele disfunksie en onvermoë om kardiale iskemie/herperfusie besering te weerstaan, ii) aktivering en uitdrukking van PLM en SERCA-2a in die sarkoplasmiese retikulum, iii) die aktivering van intermediêres wat proksimaal geleë is in die insulienseintransduksiepad van GSK-3 (IRS-1, IRS-2 en PKB/Akt) en iv) om die effek van behandeling met 'n spesifieke inhibitor van GSK-3 op die bogenoemde punte te bepaal. Metodes: Ouderdoms- en gewigsgepaarde manlike Wistar rotte (kontrole en dieet geïnduseerde obees (DIO) diere) is in die studie gebruik. Kontrole diere was normale rotkos gevoer, terwyl die DIO diere op 'n dieet van rotkos aangevul met sukrose en kondensmelk geplaas is vir 'n periode van 8 of 16 weke. Helfte van die diere van elke groep is behandel met die GSK-3 inhibitor vir 4 weke (vanaf week 12 tot 16). Na afloop van die voer- en behandelingsperiode is die diere geweeg, doodgemaak en die harte verwyder om dan of onmiddelik gevriesklamp te word, of retrograad geperfuseer te word met Krebs-Hensleit buffer. Ex vivo geperfuseerde harte is dan blootgestel aan 25 minute lae vloei iskemie gevolg deur 30 minute herperfusie. Biometriese (liggaamsgewig, intraperitoneale vet, ventrikulêre gewig en tibia lengte) en biochemiese (vastende bloedglukose en -insulien vlakke) parameters is telkens bepaal. Western klad tegnieke is gebruik om die uitdrukking en fosforilering van GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a en PLM te bepaal. Ca2+-ATPase aktiwiteit is spektrofotometries bepaal. Resultate: Na beide 8 en 16 weke was die DIO diere beduidend swaarder as die kontrole diere. Hierdie gewigstoename was geassosieer met meer intraperitoneale vet in die DIO diere. Verder, in die DIO diere was IRS-1 afgereguleer na 8 weke, terwyl beide IRS-1 en IRS-2 asook PKB/Akt afgereguleer was na 16 weke. GSK-3 uitdrukking het 'n neiging getoon om toe te neem na beide 8 en 16 weke in die DIO diere, terwyl SERCA-2a beduidend afgereguleer was reeds vanaf 8 weke, geassosieer met laer Ca2+-ATPase aktiwiteit. PLM uitdrukking het toegeneem en die fosforilering daarvan was verlaag. Op 16 weke was die basale harttempo (225 vs 275 in die kontrole groep, P<0.0001, n=6) en tempo druk produk (21000 vs 30000 in die kontrole groep, P=0.019, n=6) betekenisvol laer in die DIO diere. Funksionele herstel het onveranderd gebly, alhoewel die tyd tot iskemiese kontraktuur toegeneem het in die DIO groep (kontrole: 11.6±0.4 min vs DIO: 16.2±0.5 min, P<0.01, n=6). Toediening van die inhibitor het geen effek op totale GSK-3 uitdrukking gehad nie. Fosforilering van GSK-3 was egter wel beduidend verhoog in die behandelde kontrole diere, terwyl daar geen verskille in die DIO groep was nie. Die fosforilering van GSK-3 het wel geneig na 'n toename in die behandelde DIO diere. Die GSK-3 inhibitor het kontrasterende effekte op hipertrofie gehad: dit het dit omgekeer in die DIO groep, maar veroorsaak in die kontrole diere. Daarmee saam het die inhibitor die uitdrukking van IRS-2 in beide DIO en kontrole diere gestimuleer, maar geen effek op IRS-1 en SERCA-2a uitdrukking en aktiwiteit gehad nie. GSK-3 inhibisie het wel PKB/Akt en PLM fosforilering in die DIO diere verhoog. Gevolgtrekking: Hierdie bevindinge toon dat 'n hoë kalorie dieet, tesame met 'n wanbalans tussen energie inname en verbruiking, lei tot die ontwikkeling van obesiteit en insulien weerstand in manlike Wistar rotte. Die studie het ook getoon dat GSK-3 en sy substraat proteïene wel gedisreguleer is in obesiteit en insulien weerstandigheid. Die verlaagde basale uitdrukking van SERCA-2a mag dalk 'n negatiewe impak hê op kardiale funksie. Behandeling van die diere met 'n GSK-3 inhibitor het getoon dat GSK-3 moontlik nie verantwoordelik is vir die basislyn veranderinge nie. Die afname in IRS-1 en SERCA-2a uitdrukking kan moontlik toegeskryf word aan ander meganismes buiten die effekte van GSK-3. Hierdie studie toon wel dat GSK-3 moontlik 'n rol speel in die regulering van die uitdrukking van IRS-2, maar nie IRS-1 nie. Verhoogde PKB/Akt fosforilering mag dalk bydra tot die inhibisie van GSK-3. Daarmee saam blyk dit dat GSK-3 inhibisie hipertrofie kan omkeer in DIO diere, om dan sodoende op te tree as 'n negatiewe reguleerder van hipertrofie, maar in normale kontrole diere, hipertrofie in die hand werk.South African Medical Research CouncilUniversity of Stellenbosch, Dept. of medical Physiolog