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Research has shown that autologous progenitor-like cardiac spheroids, when delivered to an infarcted heart, are able to restore mechanical function. These spheroids are made by isolating and expanding autologous cardiac progenitor cells. Though these results are promising, the process for creating cardiac spheroids is inefficient and time consuming, requiring a large amount of cardiac tissue. For every 10,000 cardiac myocytes in the heart there is only one cardiac progenitor cell; requiring a large amount of initial tissue. This clinical limitation could be overcome if cardiac myocytes, which are more abundant than cardiac progenitor cells, could be used to make cardiac spheroids. Research has shown that mesenchymal stem cells when co-cultured with adult cardiac myocytes cause the cardiac myocytes to behave like a progenitor cell. We found that, when co-cultured with mesenchymal stem cells, cardiac mycoytes could be made to form cardiac spheroid bodies. This was done by isolating adult myocytes from rat hearts and co-culturing them with human mesenchymal stem cells. After two weeks, cultures were observed to form spheroid bodies and the number of spheroids formed were compared to a pure myocyte control. Cardiac specific staining confirmed that the spheroids were made from the myocytes. It was also found that the mesenchymal stem cells, when co-cultured in the same well with the myocytes, form significantly more spheroids than myocytes treated with stem cell conditioned media. Further, no other cell type present in the co-cultures are able to create spheroid bodies when co-cultured with mycoytes or stem cells. The ability to create cardiac spheroid like bodies from adult myocytes offers a way to overcome the limitations of the time needed and the large quantity of autologous cardiac tissue required to currently make these types of bodies.

In heart, Ca2+ plays an important role in maintenance of normal cardiac functions. Regulation of Ca2+ is mainly through L-type Ca2+ channel (LTCC), Ryanodine receptor (RyR) and sarcoplasmic reticulum calcium ATPase pump (SERCA) on sarcoplasmic reticulum (SR), Na+-Ca2+ exchanger (NCX), plasma membrane Ca2+ ATPase (PMCA). It has been well-accepted that PMCA plays a minor contribution to elevation of Ca2+ compared to SERCA and NCX and in regulation of cytosolic Ca2+ homeostasis. There are four isoforms of PMCA, PMCA1-4, and PMCA1 is a house-keeping gene, and abundantly distributed in heart. However, the role of PMCA1 in the murine heart has not been fully explored. With a cardiac specific knockout mouse model, the electrophysiological characteristics of PMCA1 in murine hearts, particularly in atria under normal physiological and stress conditions ([Ca2+]o overload and pacing conditions) are investigated. Firstly the complete deletion of PMCA1 in the atria in PMCA1cko mice was confirmed by Western blotting and immunostaining, also the membrane localisation of PMCA1 in the atria in PMCA1loxP/loxP mice was demonstrated. Then the phenotypes of ex vivo whole hearts between PMCA1loxP/loxP and PMCA1cko mice under physiological conditions and [Ca2+]o overload condition and with different frequencies by programmed electrical stimulation (PES) were explored. Further more, the Ca2+ handling process in single atrial myocytes between the PMCA1 deletion mice and control mice under normal physiological conditions and [Ca2+]o overload condition and stimulation with different frequencies was investigated. Finally the Ca2+ handling process in single ventricular myocytes between the PMCA1 deletion mice and control mice under normal physiological condition was investigated. At the whole heart level, the PMCA1cko hearts became more susceptible to arrhythmias with PES under physiological conditions compared with the PMCA1loxP/loxP hearts, and such arrhythmic events occurred more often and had longer pacing durations under Ca2+ overload conditions and higher frequency of pacing. At the single cellular level, the NCX current decay was significantly prolonged in PMCA1cko atrial myocytes under physiological conditions. This was further increased under Ca2+ overload conditions. With frequency-dependent stimulation, the PMCA1cko atrial myocytes showed few EAD- or DAD-type APs under physiological conditions in contrast to PMCA1loxP/loxP atrial myocytes that showed no arrhythmic events. The occurrence increased significantly under Ca2+ overload condition and/or at higher frequency of stimulation. Similar findings were observed in isolated ventricular myocytes. To conclude, the role of PMCA1 in maintaining Ca2+ homeostasis and electrical function in atrial myocytes under physiological conditions is minor. ii) PMCA1 has a critical role in maintaining Ca2+ homeostasis and electrical function in the atrium under stress conditions. This is particularly important during fast efflux of Ca2+ which is required under stress conditions.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in models of cardiovascular disease. We previously suggested sphingosine-1-phosphate (S1P) activates volume-sensitive Cl- current (ICl,swell) by ROS-dependent signaling. S1P and its analog, FTY720 (fingolimod), primarily act via G-protein coupled receptors (S1PR; S1PR1-3 in heart), but several intracellular S1P ligands are known. We investigated how these agents regulate ICl,swell. ICl,swell was elicited by bath S1P (500 nM), FTY720 (S1PR1,3 agonist; 10 μM), and SEW2871 (S1PR1 agonist; 10 μM) and was fully inhibited by DCPIB, a specific blocker. These data suggested role of S1PR in activation of ICl,swell. Surprisingly, neither CAY10444 (S1PR3 antagonist; 10 μM) nor VPC23019 (S1PR1,3 antagonist; 13 μM) blocked FTY720-induced ICl,swell. Also, gallein a pan Gbeta-gamma inhibitor, failed to block the S1P-induced current. Moreover, 100 nM FTY720 applied via the pipette evoked a larger, faster activating current than 10 μM bath FTY720. Similarly, 500 nM S1P gave larger, faster activating ICl,swell when added to the pipette than when added in the bath. In contrast to FTY720, bath S1P-induced ICl,swell was blocked by CAY10444, but a 3-fold higher concentration failed to eliminate the response to pipette S1P, and VPC23019 failed to suppress bath and pipette S1P-induced currents. Taken together, inconsistencies in the responses to S1PR agents and the greater sensitivity to pipette than bath S1P and FTY720 support the notion that intracellular ligands rather than sarcolemmal S1PR activated ICl,swell. Next we tested if S1P and FTY720, like osmotic swelling, require both NADPH oxidase and mitochondrial ROS production to evoke ICl,swell. S1P- and FTY720-induced ICl,swell were blocked by rotenone but were insensitive to gp91ds-tat, suggesting only mitochondrial ROS production was needed. One possibility is that S1P and FTY720 elicit ICl,swell by binding to mitochondrial prohibitin-2, an S1P ligand whose knockdown augments mitochondrial ROS productions. These data suggest ICl,swell may be activated by S1P accumulation in ischemia-reperfusion and CHF. Understanding S1P-signaling that elicits ICl,swell may provide insight into electrophysiological mechanisms of cardiac pathology and help identify novel targets for therapy.

The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth. / Engineering and Applied Sciences

Orientador: Orientador : Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T12:58:21Z (GMT). No. of bitstreams: 1 Cardoso_AlissonCampos_D.pdf: 4487510 bytes, checksum: eec48cf16c52d89f7324a6a56476ca59 (MD5) Previous issue date: 2012 / Resumo: Durante os primeiros dias do desenvolvimento pós-natal, os miócitos cardíacos perdem a capacidade de proliferação, sendo o crescimento adicional do coração decorrente de hipertrofia e não hiperplasia dos miócitos cardíacos. No entanto, em situações de estresse os miócitos cardíacos diferenciados podem apresentar desdiferenciação e reestabelecimento do ciclo celular. Os mecanismos envolvidos nesse fenômeno são ainda pouco compreendidos. No presente estudo, demonstramos que a ativação do fator de transcrição MEF2C (Myocyte Enhancer Factor 2-C) tem papel crítico no processo de desdiferenciação de miócitos cardíacos. Essa conclusão foi obtida por meio de experimentos de ganho de função pela superexpressão de MEF2C em miócitos ventriculares de ratos neonatos em cultura (MVRNs). Demonstramos que a superexpressão de MEF2C em MVRNs induziu a desdiferenciação e a ativação de mecanismos envolvidos na progressão do ciclo celular. Esses resultados foram obtidos por meio de experimentos de microarranjo de DNA, PCR em tempo real, western blotting e análise do fenótipo celular por microscopias de luz, confocal e eletrônica de transmissão. Esses fenômenos foram atenuados pela superexpressão da quinase de adesão focal (FAK), uma proteína que reconhecidamente exerce efeitos pró-hipertróficos em miócitos cardíacos adultos. Experimentos in vivo e in vitro demonstraram a interação direta entre o fator de transcrição MEF2C e a FAK. Estudos com base em ensaios de reação cruzada associada à espectrometria de massas, dinâmica molecular, espalhamento de raios-X a baixos ângulos e mutação sítio dirigida, demonstraram que as hélices 1 e 4 do domínio FAT da FAK interagem diretamente com a domínio de ligação ao DNA do dímero de MEF2C. Estudos de afinidade e de gel shift demonstraram que a porção FAT da FAK desloca a interação MEF2C/DNA in vitro. Ensaios de gene repórter demonstraram que a FAK, mediada pela região C-terminal, diminui a atividade transcricional de MEF2C em células C2C12. O conjunto de dados demonstra que a ativação do fator de transcrição MEF2C em MVRNs induz a desdiferenciação e ativação de mecanismos de progressão do ciclo celular e que a FAK impede esses efeitos através da interação inibitória no domínio de ligação de MEF2C ao DNA / Abstract: During the first days of postnatal development, cardiac myocytes lose their ability to proliferate, and the further growth of the heart is due to hypertrophy and not hyperplasia of cardiac myocytes. However, in response to stress, cardiac myocytes may have dedifferentiation and re-establishment of the cell cycle. The mechanisms involved in this phenomenon are still poorly understood. In the present study, we demonstrated that activation of the transcription factor MEF2C (myocyte enhancer factor 2-C) plays a critical role in the process of dedifferentiation of cardiac myocytes. This conclusion was obtained by gain-of-function experiments through overexpressing MEF2C in neonatal rat ventricular myocytes in culture (NRVMs). We also showed that overexpression of MEF2C in NRVMs induced the dedifferentiation and activation of mechanisms involved on cell cycle progression. These results were obtained by DNA microarray experiments, real time PCR, western blotting and cell phenotype analysis by light microscopy, confocal and electronic transmission. These effects were attenuated by overexpression of focal adhesion kinase (FAK) protein known to exert pro-hypertrophic effects on adult cardiac myocytes. In vivo and in vitro experiments demonstrated the direct interaction between the transcription factor MEF2C and FAK. A model based on crosslinking technology coupled with mass spectrometry, small angle X-ray scattering and the site directed mutation analyses indicated that alpha-helices 1 and 4 of FAK FAT domain interacts directly with the DNA binding domain of MEF2C dimer. Affinity studies and gel shift assay demonstrated that the FAK FAT domain displaces the MEF2C/DNA interaction in vitro. Reporter gene assays demonstrated that FAK, mediated by the C-terminal region, decreases the transcriptional activity of MEF2C in C2C12 cells. The data set shows that the activation of the transcription factor MEF2C in MVRNs induces dedifferentiation and activation of cell cycle progression and that FAK prevents these effects by inhibitory interaction with DNA binding domain of MEF2C / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

Mūsų tyrymų tikslas – ištirti baltymų kinazės A, baltymų kinazės C, Src šeimos nereceptorinės baltymų tirozino kinazės ir jų signaliniuose keliuose dalyvaujančių molekulių įtaką L tipo kalcio srovei (ICa,L) fermentiniu būdu izoliuotuose iširdies miocituose. Šis tikslas buvo įgyvendintas sprendžiant keturias užduotis: 1) ištiriant varlės ir žiurkės skilvelių bei žmogaus prieširdžių miocitų β-adrenerginių receptorių signalinės grandinės elementų: β-adrenerginių receptorių, adenilatciklazės, fosfodiesterazių, baltymų kinazės A, baltymų fosfatazių (baltymų fosfatazės 1 ir baltymų fosfatazės 2A) bei įtampos valdomų L tipo kalcio kanalų bazinį aktyvumą; 2) ištiriant β3-adrenerginių receptorių įtaką žmogaus prieširdžių ICa,L ir susitraukimo jėgai; 3) nustatant Src šeimos nereceptorinės baltymų tirozino kinazės įtaką žmogaus prieširdžių ICa,L, taip pat jos aktyvinimo būdą ir veikimo vietą β-adrenerginių receptorių signalinėje grandinėje; 4) ištiriant baltymų kinazės C įtaka žmogaus prieširdžių ICa,L. / The objective of study was to investigate the role of protein kinase A, protein kinase C, Src family nonreceptor protein tyrosine kinases and other signaling molecules involved in pathways regulating the L-type calcium current (ICa,L) in enzymatically isolated cardiac myocytes. This objective was realized by resolving four tasks: 1) Examination of the basal activity of β-adrenergic receptor (β-AR) signaling cascade involving β-ARs, adenylyl cyclases, phosphodiesterases, protein kinase A, protein phosphatases (protein phosphatase 1 and protein phosphatase 2A) and L-type voltage-dependent calcium channels in frog and rat ventricular myocytes and human atrial myocytes; 2) Investigation of the role of β3-ARs in regulation of ICa,L and force of contraction in human atrium; 3) Exploration of the role of Src family nonreceptor tyrosine kinases in regulation of ICa,L, determining the route of their activation and site of action in β-AR signaling cascade of human atriual myocytes; 4) Probing of the impact of protein kinase C on basal and β-AR stimulated ICa,L in human atrial myocytes. The experiments were performed using whole-cell configuration of the pach-clamp technique.

Orientadores: Jose Wilson Magalhaes Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-15T01:38:31Z (GMT). No. of bitstreams: 1 Fonseca_AlexandraValenzuelaSantelicesda_M.pdf: 1311974 bytes, checksum: ab061a6f8d63a1175d6a3c9281e0704e (MD5) Previous issue date: 2009 / Resumo: O procedimento mais efetivo para reverter arritmias cardíacas consiste na aplicação de choques elétricos de alta intensidade, como e o caso da desfibrilação. Estimulação com campos elétricos (E) elevados, entretanto, exerce efeitos deletérios sobre o músculo cardíaco, podendo causar disfunções elétrica e contrátil e até a morte celular. Privilegiar a estimulação na direção longitudinal, para qual o limiar de excitação das células cardíacas e menor, seria uma forma de se reduzir a amplitude do estimulo sem perder a efetividade da estimulação. Para isto, foi desenvolvido e testado, em miócitos ventriculares orientados de maneira aleatória, um sistema de estimulação multidirecional automática que permite o chaveamento controlado de estímulos sequênciais para três diferentes pares de eletrodos (cada um correspondendo a uma direção) em um intervalo de tempo inferior a duração do potencial de ação (período em que a célula se encontra eletricamente refrataria). A estimulação multidirecional com uma intensidade de E 20% acima do limiar estimulatório (1,2× ETM) dobrou o recrutamento (excitação) de células (80 vs. 40% com estimulação unidirecional, p<0,001). Adicionalmente, o recrutamento com a estimulação multidirecional automática foi maior (p< 0,001) do que a soma dos recrutamentos obtidos com a estimulação em cada direção individualmente (sem intersecção), o que sugere que a estimulação sublimiar durante o procedimento automático pode aumentar a excitabilidade celular. Foi observado também que, para uma dada amplitude do estimulo, o uso da forma de onda bipolar (para a qual o valor de ETM foi menor que para pulsos monopolares: 3,2 ± 0,1 vs. 3,9 ± 0,1 V/cm; p< 0,001) promoveu um recrutamento maior do que com o pulso monopolar (recrutamento de 50% das células foi obtido com 2,97 ± 0,04 e 4,18 ± 0,05 V/cm para pulsos bipolares e monopolares, respectivamente; p< 0,05). A combinação da estimulação multidirecional automática com o uso da forma de onda bipolar permitiu, portanto, uma redução de cerca de 50% no valor do E absoluto (3,8 vs. 7,8 V/cm com estimulação unidirecional e pulso monopolar) para um recrutamento de ~80% das células. A aplicação destes procedimentos na estimulação cardíaca (marcapasso e desfibrilação) pode otimizar o processo, levando a uma melhor eficiência e uma menor incidência de lesão. / Abstract: The most effective procedure to revert cardiac arrhythmias consists in the application of high intensity electric discharge, such as in cardiac defibrillation. Nevertheless, stimulation using high electric fields (E) may cause injury to the cardiac muscle, generating electric and contractile dysfunctions and even cell death. A possible way to reduce the stimulus intensity while maintaining the stimulation effectiveness would be stimulate cardiac cells with E applied parallel to the cell major axis, in which case the stimulation threshold is lower. To test this possibility, a multidirectional stimulation system was developed and tested on randomly-oriented rat ventricular myocytes. The system allows the controlled switching of sequential stimuli delivered to three different pairs of electrodes (each one corresponding to one direction), in a period shorter than the action potential duration (when cell is electrically refractory). The multidirectional stimulation with E intensity 20% above the stimulation threshold (1.2× ETM) doubled the percentage of recruited (excited) cells (~80 vs. ~40 % with unidirectional stimulation, p<0.001). Additionally, recruitment with automatic multidirectional stimulation was greater (p< 0.001) than the sum of recruitments obtained from stimulation of each direction individually (without intersection), which is suggestive that subthreshold stimulation during the automatic procedure might enhance cell excitability. Moreover, it was observed that for a given absolute stimulus amplitude, the use of biphasic waveforms (for which ETM was lower than for monophasic pulses: 3.2 ± 0.1 vs. 3.9 ± 0.1 V/cm; p< 0.001) promoted higher recruitment than monophasic stimuli (50% recruitment was attained with 2.97 ± 0.04 and 4.18 ± 0.05 V/cm with biphasic and monophasic pulses, respectively; p< 0.05). Thus, the association of automatic multidirectional stimulation and biphasic waveform enabled a 50% reduction of the absolute E value (3.8 vs. 7.8 V/cm with unidirectional stimulation and monopolar pulse) to evoke excitation in ~80% of the cells. The application of these procedures to cardiac stimulation (pacemaker and defibrillation) might optimize the process, leading to greater efficiency and lower injury incidence. / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-27T15:52:15Z No. of bitstreams: 1 joaogabrielrochasilva.pdf: 4504745 bytes, checksum: 5d371d913693d1bf3a9425d887ab2ed1 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-03-27T17:49:33Z (GMT) No. of bitstreams: 1 joaogabrielrochasilva.pdf: 4504745 bytes, checksum: 5d371d913693d1bf3a9425d887ab2ed1 (MD5) / Made available in DSpace on 2018-03-27T17:49:33Z (GMT). No. of bitstreams: 1 joaogabrielrochasilva.pdf: 4504745 bytes, checksum: 5d371d913693d1bf3a9425d887ab2ed1 (MD5) Previous issue date: 2018-02-23 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A simulação da atividade eletromecânica do coração é uma ferramenta relevante para a interpretação e estudos de medidas fisiológicas e diversos fenômenos cardíacos. Entretanto, modelos computacionais para este propósito podem ser computacionalmente custosos. Assim, são propostos neste trabalho três modelos simplificados, a nível celular, que foram capazes de reproduzir de forma quantitativa o fenômeno da contração de miócitos cardíacos. Para obtenção destes modelos um ajuste de parâmetros foi realizado via algoritmos genéticos. Os modelos propostos com parâmetros ajustados apresentaram resultados satisfatórios para reprodução da força ativa do coração com a vantagem de serem baseados em apenas duas equações diferenciais ordinárias. Além disso, o modelo final foi validado utilizando simulações envolvendo extra-sístoles, sendo capaz de reproduzir o fenômeno de alternância na força ativa. / The simulation of the heart electromechanical activity is a relevant tool for the interpretation and studies of physiological measures and various cardiac phenomena. However, computational models for this purpose may be computationally costly. Thus, three simplified models which were able to quantitatively reproduce the phenomenon of cardiac myocyte contraction were proposed in this work. At the cellular level, they were able to quantitatively reproduce the phenomenon of cardiac myocyte contraction. A parameter adjustment via genetic algorithm was performed to obtain these models. The proposed models with adjusted parameters presented satisfactory results for the reproduction of the active force of the heart with the advantage of being based on only two ordinary differential equations. In addition, the final model was validated using simulations involving extra-systoles, being able to reproduce the phenomenon of alternation in the active stress.

Les deux nucléotides cycliques, AMPc et GMPc, sont des seconds messagers importants qui régulent une grande variété de fonctions cellulaires, en particulier la fonction contractile cardiovasculaire, la croissance des cardiomyocytaires et la prolifération des cellules musculaires lisses vasculaires. Les phosphodiestérases (PDE) dégradent les nucléotides cycliques et exercent un contrôle local de leur concentration intracellulaire. Une altération de la voie de signalisation des nucléotides cycliques est impliquée dans plusieurs situations pathologiques telles que l’hypertension artérielle systémique ou pulmonaire, l’athérosclérose et l'hypertrophie cardiaque. Ainsi, les PDE constituent de puissantes cibles thérapeutiques pour restaurer un contrôle correct des nucléotides cycliques. Onze familles de PDEs sont actuellement décrites, les PDE1-6 étant les plus étudiées et les PDE 7-11 représentant de nouvelles familles.L'objectif de cette thèse était d'étudier le rôle respectif de 4 familles de PDEs, la PDE1, famille stimulée par le complexe Ca2+/calmoduline, les PDE5 et PDE9 spécifiques du GMPc, et la PDE8 spécifique de l'AMPc, dans le contrôle des concentrations intracellulaires d'AMPc ([AMPc]i) et de GMPc ([GMPc]i) dans les cellules musculaires lisses aortiques de rat (CMLARs) et les myocytes cardiaques de rat en utilisant une approche pharmacologique facilitée par le développement de nouveaux inhibiteurs sélectifs de PDEs. Les activités d'hydrolyse d’AMPc et de GMPc ont été mesurées par dosage enzymatique, tandis que les [AMPc]i et [GMPc]i ont été suivies sur cellules isolées, in situ, en temps réel, grâce à l'utilisation de l'imagerie FRET (Fluorescence Resonance Energy Transfer). Dans les CMLARs en culture, une activité d'hydrolyse des nucléotides cycliques via les PDE1, PDE5 et PDE9 a été observée. Nous avons montré un rôle fonctionnel de la PDE1 non stimulée dans le contrôle de l’augmentation de la [GMPc]i induite par le peptide natriurétique de type C (CNP). Il est intéressant de noter que, lors de l’élévation de la concentration intracellulaire en Ca2+, la PDE1 exerce également un contrôle de la réponse GMPci induite par le monoxyde d’azote (NO) et de la réponse AMPc médiée par la stimulation des récepteurs β-adrénergiques (β-AR). La PDE5 exerce un rôle majeur dans la réponse GMPc provoquée par l'activation de la guanylyl cyclase (GC) soluble par le NO ou des GC membranaires par les peptides natriurétiques, CNP et ANP. En revanche, la PDE9 ne régule que la réponse GMPc induite par le NO dans les RASMC cultivées. Aucune activité ou fonction hydrolytique de l'AMPc n'a été révélée avec l'inhibiteur de la PDE8 dans les CMLARs ou les cardiomyocytes de rat. Dans ces cellules cardiaques, l'activité d'hydrolyse médiée par la PDE1 n'a été détectée que sur la réponse GMPc et uniquement en présence de Ca2 +/Calmoduline. L'inhibiteur de la PDE1 n'a que légèrement affecté la réponse AMPc médiée par les récepteurs β-AR, par augmentation du pic du signal FRET.En conclusion, notre travail démontre que dans les cellules musculaires lisses vasculaires, les PDE1, PDE5 et PDE9 exercent une régulation spécifique et locale des [AMPc]i et [GMPc]i, renforçant le rôle clé des PDEs dans la compartimentation subcellulaire de la signalisation des nucléotides cycliques. / The two cyclic nucleotides cAMP and cGMP are important second messengers that regulate a large variety of cellular functions, in particular cardiovascular contractile function, cardiomyocyte cell growth and vascular smooth muscle cell proliferation. Phosphodiesterases (PDEs) degrade cyclic nucleotides, and exert a fine local control of their intracellular concentration. Alteration of cyclic nucleotides signaling pathway is involved in several pathological situations such as systemic and pulmonary arterial hypertensions, atherosclerotic lesions and cardiac hypertrophy. Thus, PDEs constitute potent therapeutic targets to restore a right cyclic nucleotide function. Eleven families of PDEs are now described, PDE1-6 being the most studied and PDE 7-11 representing the new families.The aim of the present thesis was to investigate the respective role of 4 PDE families, the Ca2+/calmodulin-stimulated PDE1, the cGMP-specific PDE5 and PDE9, and the cAMP-specific PDE8, in controlling intracellular cAMP ([cAMP]i) and intracellular cGMP ([cGMP]i) concentrations in both rat aortic smooth muscle cells (RASMCs) and cardiac myocytes by using a pharmacological approach taken advantage of the development of new selective PDE inhibitors. Cyclic AMP- and cGMP-hydrolyzing activities were measured by enzymatic assay on cell lysate, whereas real-time [cAMP]i and [cGMP]i were followed in situ in isolated cells using Fluorescence Resonance Energy Transfer (FRET) imaging. In cultured RASMCs, PDE1, PDE5 and PDE9 hydrolyzing activities were observed. We showed a functional role of basal PDE1 in controlling [cGMP]i increased by the C-type Natriuretic Peptide (CNP). Interestingly, upon high intracellular Ca2+ concentration, PDE1 also regulated the Nitric Oxide (NO)-mediated [cGMP]i response and the β-adrenoceptor (β-AR)-mediated [cAMP]i response. PDE5 exerted a major role in degrading [cGMP]i produced by the activation of either the soluble guanylyl cyclase (GC) elicited by NO or the particulate GCs by the natriuretic peptides, CNP and ANP. By contrast, PDE9 only regulated NO-induced [cGMP]i increase in cultured RASMCs. No cAMP-hydrolyzing activity or function was revealed with the PDE8 inhibitor in RASMCs or cardiac myocytes. In rat cardiomyocytes, PDE1-mediated hydrolyzing activity was only detected on cGMP in the presence of Ca2+/calmodulin. Unexpectedly, PDE1 inhibition slightly affected the β-AR-mediated [cAMP]i response by increasing the peak of FRET signal.In conclusion, our work underscores the distinct role of PDE1, PDE5, and PDE9 in locally regulating the [cAMP]i and [cGMP]i, in vascular smooth muscle cells, strengthening the concept of PDEs as key actors of cyclic nucleotide subcellular compartmentation.