Heart failure is a debilitating condition in which the heart cannot meet the metabolic demands of the body. Chronic β-adrenergic (β-AR) stimulation causes pathological myocardial remodeling that leads to heart failure, in part, by promoting apoptosis of cardiac myocytes. Work from our laboratory has shown that β-AR stimulated apoptosis is dependent on reactive oxygen species (ROS), but the molecular targets by which ROS mediate apoptosis is not known. One target of ROS that may contribute to activating the apoptosis pathway is the sarco-endoplasmic reticulum ATPase (SERCA2). SERCA2 is responsible for moving the large majority of intracellular calcium in the cardiac myocyte. We have identified that SERCA2 can undergo oxidative post-translational modification (OPTM) of cysteine C674: Low ROS increase activity while high ROS decreases. Since SERCA is the primary calcium transporter and is located in close proximity of the mitochondria, it is possible SERCA activity may affect the level of calcium in mitochondria, which in excess is a known activator of the intrinsic mitochondrial apoptosis pathway. Progressive loss of myocardial cells in ischemia and heart failure likely contributes to the pathogenesis of cardiomyopathy. We hypothesized that oxidation of SERCA2 at C674 increases mitochondrial calcium, thereby activating the mitochondrial apoptosis pathway. To address this thesis, we used a novel redox-insensitive SERCA2 mutation in which C674 is replaced by serine (C674S) to determine the role of oxidative inhibition of SERCA in H2O2-stimulated apoptosis in vitro. We tested our hypothesis using adult rat ventricular myocytes (ARVM) that overexpress wild type or SERCA C674 and assessed intra-organelle calcium content, mitochondrial function and activation of the apoptosis pathway. To measure mitochondrial calcium, we optimized the use of an ultrasensitive genetically-encoded calcium indicator (GECI) targeted to the mitochondria which was expressed in ARVM via adenovirus infection. Redox-insensitive SERCA C674S expressing ARVM displayed less sensitivity to H2O2-stimulated mitochondrial calcium uptake which was confirmed by measuring calcium sensitive pyruvate dehydrogenase phosphorylation status. Furthermore, SERCA C674S ARVM were protected from H2O2 -mediated apoptosis, indicated by a reduction in cytochrome c release and annexin V staining. Lastly, H2O2 treatment decreased the cytosolic ATP/ADP ratio and depolarized the mitochondrial membrane potential, however this was independent of SERCA C674 oxidation. Taken together, these experiments elucidate a novel role for SERCA2 activity in cardiac myocytes and provide a potential therapeutic target for reducing cardiac myocyte apoptosis, potentially improving cardiac function during heart failure.

No description available.

Nos études ont démontrées que la formation de la cicatrice et la guérison sont associées avec l’apparition de cellules de type myocytes cardiaques nestine(+) dans la région péri-infarcie. Présentement, l’étude examine le mécanisme, tel que l’hypoxie ou les hormones neuronales, possiblement impliqué dans leur recrutement et de dévoiler leur origine cellulaire. La présence de ces cellules a été détectée dans les coeurs infarcies d’une semaine et maintenue après neuf mois suite à une sujétion coronaire complète. Aussi, ces cellules de type myocytes cardiaques nestine(+) ont été observées dans le coeur infarci humain. L’hypoxie représente un événement prédominant suite à un infarctus de myocarde, mais l’exposition des rats normaux à un environnement hypoxique n’a pas pu promouvoir l’apparition de ces cellules. Autrement, l’infusion de l’agoniste -adrénergique non-sélectif isoprotérénol (ISO) dans les rats adultes Sprague-Dawley a augmenté la protéine nestine dans le ventricule gauche et a été associé avec la réapparition de cellules de type myocytes cardiaques nestine(+). Cela représente possiblement un effet secondaire suite à la nécrose des myocytes cardiaques par l’administration d’isoprotérénol. Dernièrement, on a identifié une sous-population de cellules nestine(+) dans le coeur normal du rat qui co-exprime les marqueurs de cellules cardiaques progénitrices Nkx-2.5 et GATA-4. Cette sous-population de cellules nestine/Nkx-2.5/GATA-4 pourrait représenter des substrats cellulaires qui puissent se différentier en cellules de type myocytes cardiaques nestine(+) suite à une ischémie. Mots clés: nestine, isoprotérénol, nécrose, cellule souche, cellule progénitrice, myocyte cardiaque / Studies from our lab demonstrated that scar formation and healing was associated with the appearance of nestin(+) cardiac myocyte-like cells predominantly at the peri-infarct region. The focus of the present study was to identify the underlying mechanism(s) (e.g. hypoxia, neurohormones) implicated in their recruitment and their cellular origin. The presence of these cells was detected as early as 1-week post-myocardial infarction (MI) and persisted 9 months after complete coronary artery ligation. Furthermore, nestin(+) cardiac myocyte-like cells were also detected in the infarcted human heart. Hypoxia represents a predominant stimulus following MI, however the exposure of normal rats to a hypoxic environment failed to promote the re-appearance of nestin(+) cardiac myocyte-like cells. By contrast, the infusion of the non-selective -adrenergic agonist isoproterenol (ISO) in the normal adult Sprague-Dawley rat increased nestin expression in the left ventricle and was associated with the reappearance of nestin(+) cardiac myocyte-like cells. However, the reappearance of nestin(+) cardiac myocyte-like cells may not represent a direct effect but was apparently secondary to cardiac myocyte necrosis mediated by isoproterenol. Lastly, we identified a subpopulation of nestin-immunoreactive cells in the normal rat heart that coexpressed cardiac progenitor cell markers Nkx-2.5 and GATA-4. This subpopulation of nestin/Nkx-2.5/GATA-4 cells may represent the progenitor pool that differentiates to a nestin(+) cardiac myocyte-like cell following an ischemic insult. Key words: nestin, isoproterenol, cardiac myocyte, cardiac progenitor, necrosis

Nos études ont démontrées que la formation de la cicatrice et la guérison sont associées avec l’apparition de cellules de type myocytes cardiaques nestine(+) dans la région péri-infarcie. Présentement, l’étude examine le mécanisme, tel que l’hypoxie ou les hormones neuronales, possiblement impliqué dans leur recrutement et de dévoiler leur origine cellulaire. La présence de ces cellules a été détectée dans les coeurs infarcies d’une semaine et maintenue après neuf mois suite à une sujétion coronaire complète. Aussi, ces cellules de type myocytes cardiaques nestine(+) ont été observées dans le coeur infarci humain. L’hypoxie représente un événement prédominant suite à un infarctus de myocarde, mais l’exposition des rats normaux à un environnement hypoxique n’a pas pu promouvoir l’apparition de ces cellules. Autrement, l’infusion de l’agoniste -adrénergique non-sélectif isoprotérénol (ISO) dans les rats adultes Sprague-Dawley a augmenté la protéine nestine dans le ventricule gauche et a été associé avec la réapparition de cellules de type myocytes cardiaques nestine(+). Cela représente possiblement un effet secondaire suite à la nécrose des myocytes cardiaques par l’administration d’isoprotérénol. Dernièrement, on a identifié une sous-population de cellules nestine(+) dans le coeur normal du rat qui co-exprime les marqueurs de cellules cardiaques progénitrices Nkx-2.5 et GATA-4. Cette sous-population de cellules nestine/Nkx-2.5/GATA-4 pourrait représenter des substrats cellulaires qui puissent se différentier en cellules de type myocytes cardiaques nestine(+) suite à une ischémie. Mots clés: nestine, isoprotérénol, nécrose, cellule souche, cellule progénitrice, myocyte cardiaque / Studies from our lab demonstrated that scar formation and healing was associated with the appearance of nestin(+) cardiac myocyte-like cells predominantly at the peri-infarct region. The focus of the present study was to identify the underlying mechanism(s) (e.g. hypoxia, neurohormones) implicated in their recruitment and their cellular origin. The presence of these cells was detected as early as 1-week post-myocardial infarction (MI) and persisted 9 months after complete coronary artery ligation. Furthermore, nestin(+) cardiac myocyte-like cells were also detected in the infarcted human heart. Hypoxia represents a predominant stimulus following MI, however the exposure of normal rats to a hypoxic environment failed to promote the re-appearance of nestin(+) cardiac myocyte-like cells. By contrast, the infusion of the non-selective -adrenergic agonist isoproterenol (ISO) in the normal adult Sprague-Dawley rat increased nestin expression in the left ventricle and was associated with the reappearance of nestin(+) cardiac myocyte-like cells. However, the reappearance of nestin(+) cardiac myocyte-like cells may not represent a direct effect but was apparently secondary to cardiac myocyte necrosis mediated by isoproterenol. Lastly, we identified a subpopulation of nestin-immunoreactive cells in the normal rat heart that coexpressed cardiac progenitor cell markers Nkx-2.5 and GATA-4. This subpopulation of nestin/Nkx-2.5/GATA-4 cells may represent the progenitor pool that differentiates to a nestin(+) cardiac myocyte-like cell following an ischemic insult. Key words: nestin, isoproterenol, cardiac myocyte, cardiac progenitor, necrosis

Orientadores: Jose Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-09T15:25:46Z (GMT). No. of bitstreams: 1 Soriano_DiogoCoutinho_M.pdf: 3276695 bytes, checksum: c47b4ce326955b65ff10f5c2891b25c2 (MD5) Previous issue date: 2007 / Resumo: No presente trabalho foi desenvolvido um instrumento capaz de quantificar de forma simultânea o encurtamento celular e a concentração citosólica de Ca2+ ([Ca2+]i), utilizando o indicador fluorescente fluo-3. A instrumentação foi aplicada para estudar o papel da ATPase de Ca2+ do RS (SERCA, principal transportador responsável pelo relaxamento celular e pela reposição do estoque de Ca2+ intracelular) na remoção de Ca2+ do citosol em miócitos cardíacos isolados de rato, o que exigiu o desenvolvimento de um protocolo experimental específico. O protocolo experimental desenvolvido consistiu em tornar inoperante o principal competidor da SERCA pelo Ca2+ citosólico, o trocador Na+/Ca2+ (NCX), pela perfusão da célula com solução sem Ca2+ e sem Na+. Nesta situação, considera-se que os demais transportadores de Ca2+ (ATPase de Ca2+ do sarcolema e uniporter mitocondrial de Ca2+) transportem o íon a uma taxa baixa demais para competir com a SERCA. A liberação do Ca2+ do RS foi induzida por pulsos rápidos de cafeína, e foram medidos a amplitude (?[Ca2+]i) e tempo para 50 % de queda (t1/2) dos transientes de [Ca2+]i na ausência e na presença de fármacos que afetam a taxa de captação de Ca2+ pela SERCA. Foram analisados os efeitos do agonista de receptores beta-adrenérgicos isoproterenol (ISO), que promove estimulação da SERCA, e de 2,5-di-(tert-butil) hydroquinona (tBQ), que atua como inibidor da enzima. ISO causou redução significativa do t1/2 de queda do [Ca2+]i (p< 0,01), sem alteração significativa de ?[Ca2+]i (p> 0,05). Já no caso do tBQ, foi observado um aumento significativo do t1/2 de queda do [Ca2+]i (p< 0,01) e redução da ?[Ca2+]i, (p< 0,05). Um modelo teórico da literatura foi adaptado para descrever matematicamente o modelo experimental proposto. As simulações com alterações nos parâmetros cinéticos da SERCA pelos fármacos (de acordo com dados da literatura) foram razoavelmente bem sucedidas em reproduzir os dados experimentais com relação ao tempo de remoção do Ca2+ do citosol. Portanto, foi apresentada aqui uma nova ferramenta experimental e teórica para estudar a captação de Ca2+ pela SERCA em miócitos cardíacos intactos / Abstract: The goal of this work was to develop an instrument for simultaneous measurement of cell shortening and cytosolic Ca2+ concentration ([Ca2+]i), by using the fluorescent Ca2+ indicator fluo-3. The instrument was applied to study the uptake of cytosolic Ca2+ by the sarcoplasmic reticulum (RS) Ca2+- ATPase (SERCA, the main transporter responsible for cell relaxation in intact isolated rat ventricular myocytes). For this purpose, the development of a specific experimental protocol was required. In this protocol, the main competitor of SERCA by cytosolic Ca2+, the Na+/Ca2+ exchanger (NCX), was disabled by removal of extracellular Ca2+ and Na+. In this situation, it may be assumed that the other Ca2+ transporters (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter) are too slow to compete with SERCA. SR Ca2+ release was induced by short, rapid caffeine pulses, and the amplitude (?[Ca2+]i) and time for 50% [Ca2+]i decline (t1/2) of the resulting [Ca2+]I transients were measured in the absence and in the presence of drugs that affect the rate of SR Ca2+ uptake by SERCA, namely the beta-adrenergic receptor agonist isoproterenol (ISO), which causes increase in SERCA activity, and 2,5-di-(tert-butyl) hydroquinone (tBQ), which inhibits the enzyme. ISO caused significant reduction of t1/2 (p< 0,01), without any significant change in ?[Ca2+]i (p> 0,05). In case of tBQ, significant increase of t1/2 (p< 0,01) and reduction of ?[Ca2+]I (p< 0,05) were observed. A theoretical model (Tang & Othmer, 1994) was adapted for mathematical description of the experimental model proposed. Simulation of the effects of the drugs, in which SERCA kinetic parameters were changed according to data obtained from literature, were reasonably successful at reproducing the cytosolic [Ca2+]i kinetics observed experimentally / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Abstract Molecular oxygen is a prerequisite for essential metabolic processes in multicellular organisms. However, the supply of oxygen can be disturbed and tissue aerobic metabolism becomes compromised in several pathophysiological conditions. In prolonged hypoxia, cells initiate cell type-specific adaptation processes, which are typically mediated by alterations in gene expression. Changes are mainly driven by a transcription factor called hypoxia-inducible factor 1 (HIF-1). Heart muscle is a highly oxidative tissue and HIF-1 activation turns on myocardial adaptation mechanisms for enhanced survival in oxygen-deprived conditions. The aim of this study was to characterize myocardial gene expression changes during chronic hypoxia and couple the adaptational changes to cardiomyocyte function. The role of hypoxia and HIF-1 activation was studied by using in vitro mouse and rat heart cell culture models, tissue perfusions and in vivo infarction models. In this study, apelin, sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and G protein-coupled receptor 35 (GPR35) were characterized as novel functionally important myocardial HIF-1 target genes. Apelin and GPR35 were induced in hypoxia, while SERCA2a expression was reduced HIF-1 dependently. HIF-1 activation also altered cardiac myocyte contractility through modulation of SERCA2a and GPR35 expression, leading to impairment of the cellular calcium metabolism. Reduced contractility was suggested to serve as an adaptive mechanism for reduced aerobic ATP production in hypoxic conditions. This study presents novel information about the plasticity of myocardial adaptation to prolonged hypoxia. The role of a conserved transcription factor, HIF-1, was shown to be essential in the adaptation process in the myocardial cells. / Tiivistelmä Riittävä hapensaanti on välttämätöntä monisoluisten eliöiden elintoiminnoille. Hapensaanti voi kuitenkin häiriintyä erilaisissa tautitiloissa, jolloin happea käyttävät prosessit estyvät. Hapenpuutteen (hypoksia) pitkittyessä elimistön solut aloittavat sopeutumisen tilanteeseen muuttamalla toimintaansa geenien ilmentymismuutosten kautta. Adaptaatiota ohjaa pääasiassa hypoksia-indusoituva tekijä 1 (HIF-1). Sydän käyttää runsaasti happea energiantuotannossaan. Hapenpuutteen aikana HIF-1-transkriptiotekijä muuttaa sydämen geenien ilmentymistä siten, että sydänsolut selviävät paremmin happivajaissa olosuhteissa. Tämän tutkimuksen tavoitteena oli määrittää sydämen geenien ilmentymisen hapenpuutevasteita ja yhdistää muutokset sydänsolujen toiminnallisiin muutoksiin. Hapenpuutteen ja HIF-1:n merkitystä sopeutumisessa tutkittiin käyttäen malleina rotan ja hiiren sydänsoluviljelmiä, in vitro-kudosperfuusiomalleja sekä in vivo-sydäninfarktimalleja. Tässä työssä havaittiin apeliinin, sarkoplasmisen kalvoston Ca2+-ATPaasin (SERCA2a) sekä G-proteiinikytketyn reseptori 35:n olevan toiminnallisesti tärkeitä HIF-1:n säätelemiä geenejä sydämessä. Apeliinin sekä GPR35:n ilmentyminen lisääntyi hypoksian aikana, mutta SERCA2a:n ilmentyminen sen sijaan väheni HIF-1 –aktivaation seurauksena. HIF-1 –aktivaation osoitettiin myös vähentävän sydänsolujen supistustoimintaa muuttuneiden SERCA2a:n ja GPR35:n ilmentymisten kautta. Heikentynyt supistustoiminta sopeuttaa soluja vähentyneeseen aerobiseen ATP:n tuottoon hapenpuutteen aikana. Tämä tutkimus antaa lisätietoa sydämen sopeutumiskyvyn mukautumisesta pitkittyneeseen hapenpuutteeseen. Lisäksi tutkimus osoittaa HIF-1:n roolin olevan oleellinen myös sydänsolujen hypoksia-adaptaatioprosesseissa.

Cardiovascular diseases (CVD) have been the leading cause of death worldwide for many years, making it a devasting and increasing concern across the globe. The risk factors of CVD include postnatal factors and prenatal factors. For the prenatal CVD risk factors study, we focused on maternal hypothyroidism (MH), which is a common clinical condition. Studies have shown MH progeny have increased susceptibility to both acquired cardiovascular disease in adulthood and congenital heart disease, but the underlying mechanisms are not well understood. The goal of the present experiments was to test the hypothesis that MH reduces early postnatal cardiac myocyte proliferation in the progeny so that their adult hearts have a smaller complement of cardiac myocytes, which leads to adverse cardiac disease responses. MH model was induced by thyroidectomy (TX) with total thyroxine (TT4) under 1ng/dl after surgery. The progeny from mice that underwent Sham or TX surgery was termed WT (wild type) or MH (maternal hypothyroidism) progeny, respectively. Hearts were collected from WT and MH progeny to determine heart weight (HW), CM size, CM proliferation, and cell culture. RNA-seq was performed on heart tissue at postnatal day 5 (P5) and P60. Transverse Aortic Constriction (TAC) was performed to cause pressure overload-induced cardiac hypertrophy and/or heart failure (HF) in adult WT and MH progeny. ECHO (in-vivo) and histological (ex-vivo) studies were performed at specific times after TAC. Thyroid hormone treatment (levothyroxine, T4) for MH mother was administered. The results showed that the Heart weight (HW) to body weight (BW) ratio at P60 was no difference between groups, but the MH progeny had a larger CM size, consistent with fewer CM numbers. MH progeny had lower EdU+, Ki67+, and PH3+ CMs, and fewer mononucleated CMs, which shows they had a decreased CM proliferation capacity. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH progeny, including Bmp10. Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation in the presence of thyroid hormone. In adult progeny, RNA-seq data showed that MH mice had genes upregulated in the inflammatory response before TAC surgery. Six weeks after TAC, the MH progeny had a greater HW/BW ratio, larger CM size, and more severe LV fibrosis consistent with more severe cardiac pathological remodeling compared with WT progeny. T4 supplemented treatment for MH mothers preserved progeny’s early postnatal CM proliferation capacity and the excessive pathological remodeling after TAC. Concluding, CM proliferation during the early postnatal development stage was significantly attenuated in MH progeny, which results in fewer CMs and CM hypertrophy in adult MH progeny. These changes are associated with worse cardiac disease responses under pressure overload in adult MH progeny. For the postnatal CVD risk factors study, we focused on calcium overload and metabolic disorder, which play a critical role in heart failure with preserved ejection fraction (HFpEF). HFpEF is defined as HF with an EF ≥50% and elevated cardiac diastolic filling pressures. The underlying causes of HFpEF are multifactorial and not well-defined. A transgenic mouse with low levels of cardiomyocyte (CM)-specific inducible Cavβ2a expression (β2a-Tg mice) showed increased cytosolic CM Ca2+, and modest levels of CM hypertrophy and fibrosis. This study aimed to determine if β2a-Tg mice develop an HFpEF phenotype when challenged with two additional stressors, a high-fat diet (HFD) and L-NAME (LN). Four-month-old wild-type (WT) and β2a-Tg mice were given either normal chow (WT-N, β2a-N) or HFD and/or L-NAME (WT-HFD, WT-LN, WT-HFD-LN, β2a-HFD, β2a-LN, and β2a-HFD-LN). Some animals were treated with the HDAC (hypertrophy regulators) inhibitor suberoylanilide hydroxamic acid (SAHA) (β2a-HFD-LN-SAHA). Echocardiography was performed monthly. After four months of treatment, terminal studies were performed, including invasive hemodynamics and organ weight measurements. Cardiac tissue was collected. Our results showed that four months of HFD plus L-NAME treatment did not induce a profound HFpEF phenotype in FVB WT mice. β2a-HFD-LN (3-Hit) mice developed features of HFpEF, including increased natriuretic peptide (ANP) levels, preserved EF, diastolic dysfunction, robust CM hypertrophy, increased M2 macrophage population, and myocardial fibrosis. SAHA reduced the HFpEF phenotype in the 3-Hit mouse model by attenuating these effects. Concluding, the 3-Hit mouse model induced a reliable HFpEF phenotype with CM hypertrophy, cardiac fibrosis, and an increased M2 macrophage population. This model could be used for identifying and preclinical testing of novel therapeutic strategies. / Biomedical Sciences

Um die Kontraktion einer Herzmuskelzelle durch den Kalziumeinstrom zu ermöglichen, ist die Kopplung von Erregung und Kontraktion (ECC) von zentraler Bedeutung. Durch das elektrische Signal einer Nachbarzelle wird die Depolarisation des Sarkolemmas verursacht, wodurch sich die L-Typ-Kalziumkanäale (LKK) öffnen und der Amplifizierungsprozess eingeleitet wird. Letzterer ist bekannt als Kalzium induzierte Kalzium Freisetzung (CICR). Durch die LKK wird ein Kalziumeinstrom in die Zelle ermöglicht, welcher zur Öffnung der Ryanodinrezeptoren (RyR) des Sarkoplasmatischen Retikulums (SR) führt. Durch die Kalziumfreisetzung des SR wird dieses im Cytoplasma akkumuliert. Modelle für diese Prozesse werden seit mehreren Jahrzenten entwickelt. Bisher fehlte jedoch die Kombination aus räumlich aufgelösten Kalziumkonzentrationen der dyadischen Spalte mit stochastischen Simulationen der einzelnen Kalziumkanäle und die Kalziumdynamiken in der ganzen Zelle mit einem Elektrophysiologiemodell einer ganzen Herzmuskelzelle. In dieser Arbeit entwickleten wir ein neues Modell, in welchem die Konzentrationsgradienten von einzelnen Kanälen bis zum Ganzzelllevel räumlich aufgelöst werden. Es wurde der quasistatische Ansatz und die Finite-Elemente-Methode zur Integration partieller Differentialgleichungen verwendet. Es wurden Simulationen mit unterschiedlichen RyR Markow-Kette-Modellen, verschiedenen Parametern für die Bestandteile des SR, verschiedenen Konditionen des Natrium-Kalzium-Austauschers und unter Einbindung der Mitochondrien durchgeführt. Ziel war es, das physiologische Verhalten einer Kaninchen-Herzmuskelzelle zu simulieren. In dem neu entwickelten Multiskalenmodell wurden Hochleistungsrechner verwendet, um detaillierte Informationen über die Verteilung, die Regulation und die Relevanz von den im ECC involvierten Komponenten aufzuzeigen. Zukünftig soll das entwickelte Modell Anwendung bei der Untersuchung von Herzkontraktionen und Herzmuskelversagen finden. / Excitation contraction coupling (ECC) is of central importance to enable the contraction of the cardiac myocyte via calcium in ux. The electrical signal of a neighbouring cell causes the membrane depolarization of the sarcolemma and L-type Ca2+ channels (LCCs) open. The amplifcation process is initiated. This process is known as calcium-induced calcium release (CICR). The calcium in ux through the LCCs activates the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR). The Ca2+ release of the SR accumulates calcium in the cytoplasm. For many decades models for these processes were developed. However, previous models have not combined the spatially resolved concentration dynamics of the dyadic cleft including the stochastic simulation of individual calcium channels and the whole cell calcium dynamics with a whole cardiac myocyte electrophysiology model. In this study, we developed a novel approach to resolve concentration gradients from single channel to whole cell level by using quasistatic approximation and finite element method for integrating partial differential equations. We ran a series of simulations with different RyR Markov chain models, different parameters for the SR components, sodium-calcium exchanger conditions, and included mitochondria to approximate physiological behaviour of a rabbit ventricular cardiac myocyte. The new multi-scale simulation tool which we developed makes use of high performance computing to reveal detailed information about the distribution, regulation, and importance of components involved in ECC. This tool will find application in investigation of heart contraction and heart failure.

No description available.