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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.

Teas and infusions from C. spiralis leaf have largely been used by folk medicine as diuretic, hypotensor, cytotoxic, immunomodulator, antilithiasic, antidiarrheic, antispasmodic, antiurolitic, antimicrobian, antifungic, antioxidant, antileishmania activity, antiinflamatory, and antiedematogenic activity. In spite of these biological effects attributed to the extracts of C. spiralis, nothing so far could be found in the scientific literature dealing with its effects on the mammalian myocardium.The present study aimed to describe the inotropic effects produced by extracts from the C. spiralis leaf on isolated guinea pig atrium, as well as to contribute for a better understanding about its mechanism of action in that tissue. In isolated mouse cardiomyocytes, the effect produced by those extracts on the intracellular calcium transient and on the sarcolemal L-type calcium current were also measured. Experiments performed to evaluate the contractile effects were carried out on isolated atrium from guinea pig (Cavia porcellus). Firstly, our purpose was to determine the most potent fraction obtained from the C. spiralis leaf. This was done by comparing the hydroalchoolic crude extract with the following ones: aqueous, chloroform, and ethyl acetate. A phytochemical analysis was performed on the fraction exhibiting the greater potency. This evaluation followed the procedures proposed by Matos (1997). The content of sodium and potassium in the most potent fraction was determined by flame photometry. In the contractile experiments, the atrial force was measured isometrically. Biological signals were captured, amplified, and then stored in computer to be processed off line. Intracellular calcium transients were studied by confocal microscopy with laser scanning by using the fluorescent dye FLUO 4AM. Calcium inward currents were measured in mouse cardiomyocytes by using patch clamp technique in the whole cell configuration. Yield percentage of the aqueous fraction (AqF) was 69,40%. This fraction showed the most potent depressor effect on the myocardial contractility (EC50 = 305 ± 41,00 mg/L, Hill constant = 1,46 ± 0,19). The following metabolites were found in the AqF: tannins, saponins, and polifenols (flavonol, flavononol, flavone, xanthone, phenol, and flavonoid). The potassium and sodium contents in 1 g/L of AqF were 1,91 and 0,15 mM, respectively. This was not enough to change the myocardial inotropism, even in the highest concentration of AqF used in the experiments. The contraction and the relaxation time, as well as the time related to the excitation-contraction coupling (stimulus-response) were not modified by adding AqF to the organ bath. However, AqF reduced the Efficiency Index for the contraction and relaxation phases. The Neyler & Merrillees protocol was employed to evaluate the AqF effect on the calcium inward current in myocardial cells. Our results showed that AqF is able to completely abolish the Bowditch phenomenon, suggesting that it could be acting by reducing the sarcolemal calcium current. Supported by those experimental evidences, experiments were proposed to better understand the relationship between AqF and calcium mechanisms in cardiac cells. The following results were obtained with 1,5 g/L AqF: 1) AqF completely abolished the positive inotropic effect induced by isoproterenol (10-1 to 103 pM); 2) AqF shifted rightwardly the concentration-effect curve for CaCl2 (0.5 to 7.0 mM) and increased the EC50 from 1.12 ± 0.07 (Hill = 1.5) to 7.23 ± 0.47 mM (Hill = 7.4) (n = 3; p < 0.05); 3) AqF completely abolished the positive inotropic effect of (-) BAY K8644 (5 to 2000 nM); 4) AqF reduced the intracellular fluorescence from 4.66 ±1.17 to 3.74 ± 1.0 a.u. (n = 30 cells, 4 mice, p < 0.05); 5) AqF did not modify the decay rate of the fluorescent signal (892 ± 37 to 930 ± 30 ms, n = 30 cells, 4 mice, p > 0.05), indicating that it does not interfiere with the calcium removal from the sarcoplasm; 6) AqF reduced the calcium inward current through L-type calcium channels from 6,29 ± 0,34 to 4,9 ± 0,2 A/F (23% , n = 5 animals, p < 0,05). This study brought us unto the following conclusions: 1) AqF is the most potent fraction obtained from C. spirallis leaves; 2) AqF contains the following secondary metabolites: tannins, saponins, and poliphenols; 3) AqF reduces the contraction force of the guinea pig left atrium; 4) AqF acts on the myocardium contractility by reducing the calcium entry in myocardial cells during contraction. / Preparados de Costus spiralis têm sido usados pela medicina popular (diurético, hipotensor, citotóxico, imunomodulador, antilitiásico, antidiarréico, antiespasmódico, antiurolítico, antimicrobiano, antifúngico, antioxidante, antileishmânia, anti-inflamatório e antiedematogênico). Apesar da gama de ações a eles atribuídas, nada pôde ser encontrado na literatura científica com respeito ao possível efeito dos Este trabalho visou determinar os efeitos inotrópicos obtidos das folhas de C. spiralis, que apresentava maior potência, bem como contribuir para o mecanismo de ação desse preparado no miocárdio de mamíferos. Os experimentos sobre contração foram realizados em átrio esquerdo de cobaia (Cavia porcellus), enquanto que as medidas de transiente de cálcio intracelular e de corrente de membrana foram feitas em cardiomiócitos de camundongo. A investigação fitoquímica do preparado mais ativo foi conduzida segundo Matos (1997). Os teores de sódio e de potássio presentes na fração mais potente, foram determinados por fotometria de chama. A força de contração atrial foi captada isometricamente e, depois amplificada, foi armazenada em computador. O transiente de cálcio intracelular foi avaliado com microscopia confocal de varredura a laser. As correntes de cálcio sarcolemais foram medidas em cardiomiócitos submetidos à técnica do patch clamp ( whole cell ). A fração aquosa (FAq) foi a que apresentou maior rendimento (69,40%) e a que exerceu maior efeito inotrópico negativo (CE50 = 305 ± 41,00 mg/L, Hill = 1,46 ± 0,19). Na sua constituição foram detectadas as seguintes classes de metabólitos secundários: taninos e saponinas, com reação fortemente positiva, e os polifenóis, com reação positiva (flavonóis, flavononóis, flavonas, xantonas, fenóis e flavonóides). Em 1 g/L de FAq foram encontrados 1,91 mM de potássio e 0,15 mM de sódio. A adição de FAq ao Tyrode não modificou significativamente a concentração desses íons. Os tempos de contração e de relaxamento, bem como o tempo de acoplamento eletromecânico não foram alterados pela FAq. Contudo, ela reduziu os Índices de Eficiência da contração e do relaxamento. A FAq aboliu completamente o fenômeno de Bowditch induzido por alta frequência de estimulação, indicando que ela reduz a entrada desse íon nas células. Com base nessa evidência, foram realizados protocolos para aprofundar o conhecimento sobre a participação das correntes de cálcio no mecanismo cardiodepressor da FAq. Esta fração produziu os seguintes resultados: 1) aboliu completamente o efeito inotrópico positivo do isoproterenol (10-1 a 103 pM); 2) deslocou para a direita a curva concentração-efeito para o CaCl2 (0,5 a 7,0 mM), aumentando a CE50 de 1,12 ± 0,07 (Hill = 1,5) para 7,23 ± 0,47 mM (Hill = 7,4) (n = 3; p < 0,05); 3) aboliu completamente o efeito inotrópico positivo do (-) BAY K8644 (5 a 2000 nM); 4) reduziu em cerca de 20% o pico da fluorescência intracelular correspondente ao transiente de cálcio citoplasmático (controle: n = 30 células; teste: n = 27 células; 4 animais); 5) não modificou a velocidade de decaimento do sinal de fluorescência, o que significa que ela não interfere com o funcionamento da bomba de cálcio do retículo sarcoplasmático; 6) reduziu em 23% a densidade de corrente de cálcio tipo-L que variou de -6,29 ± 0,34 para -4,9 ± 0,2 A/F (n = 5 animais, p < 0,05). 1) a FAq foi a fração com maior potência inotrópica; 2) os principais metabólitos secundários presentes na FAq foram taninos, saponinas e polifenóis; 3) a FAq reduz a força de contração do átrio; 4) o mecanismo da ação cardiodepressora da FAq sobre a contratilidade miocárdica se deve à diminuição da disponibilização do cálcio durante a contração.

Une accélération de la fréquence cardiaque (FC) au repos est observée chez les femmes enceintes. Au dernier trimestre, la FC accélère en moyenne de 15%, ce qui représente un facteur de risque dans le développement d’arythmies de novo ou dans l’exacerbation d’arythmies cardiaques préexistantes. Ceci est dangereux pour la mère ainsi que pour le fœtus. Cependant, les mécanismes responsables de ce changement cardiovasculaire restent peu connus. Notre laboratoire a récemment démontré que la grossesse était associée à une augmentation de la densité du courant pacemaker (If) et du courant calcique de type L (ICaL), ainsi qu’à des changements de l’homéostasie calcique dans les cellules de nœud sinusal (NS) de souris. Sachant que les concentrations plasmatiques en œstrogènes sont significativement augmentées pendant la grossesse et que ces hormones sexuelles féminines ont la capacité de modifier les propriétés électrophysiologiques du cœur, l’hypothèse de ce projet de recherche est que les œstrogènes jouent un rôle important dans l’augmentation de la FC associée à la grossesse et régulent les propriétés électrophysiologiques du NS. Les objectifs de ce projet de recherche sont de déterminer le rôle du 17β-œstradiol (E2) dans l’augmentation de la FC, d’examiner si ces effets sont régulés par les récepteurs aux œstrogènes alpha (ERα) et/ou bêta (ERβ) ainsi que d’évaluer les différents mécanismes de régulation de l’E2 sur l’électrophysiologie du NS. Des souris femelles adultes non-gestantes (2-4 mois) déficientes en ERα (ERKOα) ou en ERβ (ERKOβ) ont reçu un traitement chronique à l’E2 (30 μg deux fois par jour pendant quatre jours) simulant les concentrations plasmatiques en E2 retrouvées en fin de grossesse (23,3 ± 5,0 nM) chez la souris. L’analyse des électrocardiogrammes de surface montrent que la FC des souris ERKOβ (ERKOβ : 511 ± 15 bpm; ERKOβ +E2 : 580 ± 10 bpm, n = 10, p < 0,001) est significativement accélérée suivant le traitement à l’E2. Toutefois, la FC demeure inchangée chez les souris ERKOα (ERKOα : 520 ± 16 bpm; ERKOα +E2 : 530 ± 21 bpm, n = 7, p = 0,114). La méthode du patch-clamp en mode courant-imposé a permis de démontrer une accélération de l’automaticité des cellules du NS des souris ERKOβ suivant le traitement à l’E2, se traduisant par une augmentation de la fréquence des potentiels d’action spontanés (ERKOβ : 284 ± 24 bpm, n = 8; ERKOβ +E2 : 354 ± 23 bpm, n = 15, p = 0,0395) et par une pente de dépolarisation diastolique plus rapide (ERKOβ : 82 ± 12 mV/s, n = 8; ERKOβ +E2 : 140 ± 14 mV/s, n = 15, p < 0,003). En lien avec ces résultats, le patch-clamp en mode voltage-imposé a permis de démontrer que la densité de If est augmentée suivant un traitement à l’E2 (à -90 mV : ERKOβ : -6,6 ± 0,7 pA/pF, n = 12-15; ERKOβ +E2 : -11 ± 1 pA/pF, n = 9-11, p < 0,05). Cependant, If est similaire chez les souris ERKOα traitées ou non à l’E2. De plus, des cardiomyocytes humains dérivés de cellules souches pluripotentes induites de type nodal (N-hiPSC-CM) ont une accélération de la fréquence des potentiels d’action (CTL : 69 ± 5 bpm, n = 12; +E2 : 99 ± 6 bpm, n = 14, p < 0,001) ainsi qu’une augmentation de la densité de If (à -90 mV : CTL : -0,95 ± 0,14 pA/pF, n = 7-10; +E2 : -1,62 ± 0,17 pA/pF, n = 13-14, p < 0,05) suivant le traitement à l’E2. L’administration d’E2 ne modifie pas la fréquence des transitoires calciques des cellules de NS des souris ERKOα (139 ± 15, n = 13-14; +E2 : 142 ± 14, n = 15-16, p = ns) et ERKOβ (142 ± 11, n = 14-15; +E2 : 147 ± 13, n = 15-16, p = ns). En lien avec ces résultats, le courant ICaL des N-hiPSC-CM est inchangé suivant le traitement d’E2 (à 0 mV : CTL : -14,0 ± 1,3 pA/pF, n = 12-13; +E2 : -14,5 ± 1,4 pA/pF, n = 22, p = ns). En conclusion, l’accélération de l’automaticité cardiaque associée à la grossesse est, entre autres, expliquée par une augmentation de la densité de If, régulée par la voie de signalisation E2-ERα. Cependant, les changements de l’homéostasie calcique observés pendant la grossesse sont indépendants des niveaux élevés en œstrogènes. Les résultats obtenus sur les N-hiPSC-CM concordent avec ce qui est observé dans les cellules de NS de souris, ce qui démontre l’applicabilité humaine des résultats. Notre étude contribue à élucider l’influence de la grossesse et le rôle des hormones sexuelles féminines sur la fonction du NS et l’automaticité cardiaque. Ultimement, notre travail pourrait aider à développer une meilleure gestion des arythmies associées aux fluctuations hormonales féminines et/ou à la grossesse. / An increased heart rate (HR) is observed in pregnant women. In fact, in the last trimester, in average, the HR increases by 15%, which is a known risk factor to developing cardiac arrhythmias or exacerbating pre-existing arrhythmias. This can lead to major consequences for both the mother and fetus. However, the mechanisms underlying this increased HR remain largely unexplored. Our laboratory recently demonstrate that pregnancy is associated with an increased density of the pacemaker current (If) and the L-type calcium current (ICaL) as well as changes in calcium homeostasis of mouse sinoatrial node (SAN) cells. Knowing that estrogens are increased during pregnancy and that these sex hormones can modify cardiac electrophysiological properties, we hypothesized that estrogens play a key role in the pregnancy-induced increased HR and regulate the SAN electrophysiological properties. Our research project aims to determine the role of 17β-estradiol (E2) on the pregnancy-induced increased HR, to determine if these effects are regulated through estrogen receptor alpha (ERα) and/or beta (ERβ) and to study the E2 underlying mechanisms on SAN electrophysiology. Non-pregnant female mice (2-4 months) lacking ERα (ERKOα) or ERβ (ERKOβ) received a chronic E2 treatment (30 μg twice daily for four days) mimicking E2 concentrations found in late pregnancy (23.3 ± 5.0 nM). Surface electrocardiogram analysis showed a significant increased HR in ERKOβ mice (ERKOβ: 511 ± 15 bpm; ERKOβ +E2: 580 ± 10 bpm; n = 10; p<0.001) following E2 administration. However, the HR remains unchanged in ERKOα mice (ERKOα: 520 ± 16 bpm; ERKOα +E2: 530 ± 21 bpm, n = 7, p = 0.114). Following E2 treatment, current-clamp method demonstrates an increase SAN cells automaticity in ERKOβ mice, resulting in an increase in the spontaneous action potential frequency (ERKOβ : 284 ± 24 bpm, n = 8; ERKOβ +E2 : 354 ± 23 bpm, n = 15, p = 0.0395), associated with a steeper diastolic depolarization slope (ERKOβ : 82 ± 12 mV/s, n = 8; ERKOβ +E2 : 140 ± 14 mV/s, n = 15, p < 0.003), a major determinant of cardiac automaticity. In line with these results, voltage-clamp data showed an increased If density in SAN cells of ERKOβ mice treated with E2 (at -90 mV: ERKOβ: -6.6 ± 0.7 pA/pF, n = 12-15; ERKOβ +E2: -11.0 ± 1.3 pA/pF, n = 9-11, p < 0.05). Nevertheless, If density was similar in E2-treated ERKOα mice. E2-treated nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM) also showed an increased spontaneous action potential frequency (CTL : 69 ± 5 bpm, n = 12; +E2 : 99 ± 6 bpm, n = 14, p < 0.001) and If density (at -90 mV: CTL: -0.95 ± 0.14 pA/pF, n = 7-10; +E2: -1.62 ± 0.17 pA/pF, n = 13-14, p < 0.05). Following E2 administration, the rate of calcium transient was similar in SAN cells from ERKOα (139 ± 15, n = 13-14; +E2 : 142 ± 14, n = 15-16, p = ns) and ERKOβ (142 ± 11, n = 14-15; +E2 : 147 ± 13, n = 15-16, p = ns) mice. In line with these results, no modification was seen on ICaL density in E2-treated N-hiPSC-CM (at 0 mV: CTL: -14.0 ± 1.3 pA/pF, n = 12-13; +E2: -14.5 ± 1.4 pA/pF, n = 22, p = ns). In conclusion, the increased cardiac automaticity observed during pregnancy is, in part, explained by an increased If density. This mechanism is mediated by the E2-ERα pathway. In the other hand, calcium homeostasis changes detected during pregnancy appear to be mediated by an E2-independent mechanism. Finally, results obtained on N-hiPSC-CM are consistent with our observations on mouse SAN cells, demonstrating the human applicability of our results. This study provides novel insight on the effects of female sex hormones on the SAN functions. Ultimately, this information can lead to improved management of arrhythmias associated with female hormone fluctuations and/or pregnancy-induced arrhythmias.