Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

Les mécanismes sous-jacents aux effets pathologiques cardiaques de l’angiotensine II dans le remodelage électrique et contractile entre les sexes

L’insuffisance cardiaque (IC) est associée à un taux de mortalité et d’hospitalisations élevé causant un fardeau économique important. Les deux causes majeures de décès de l’IC sont les arythmies ventriculaires létales et les sidérations myocardiques. Il est maintenant reconnu que l’angiotensine II (ANGII) est l'un des principaux médiateurs de l’IC. Ses effets délétères découlent de l’activation du récepteur de type 1 de l’ANGII (AT1) et entraînent le développement d’hypertrophie. Toutefois, son rôle dans la genèse d’arythmies demeure incompris. De ce fait, l'étude des mécanismes électriques et contractiles sous-jacents aux effets pathologiques de l’ANGII s’avère essentielle afin de mieux comprendre et soigner cette pathologie. Il est souvent perçu que les femmes sont protégées envers les maladies cardiovasculaires. Cependant, le nombre total de femmes décédant d’IC est plus grand que le nombre d’hommes. Également, l’impact des facteurs de risque diffère entre chaque sexe. Ces différences existent, mais les mécanismes sous-jacents sont encore peu connus. De plus, les femmes reçoivent fréquemment un diagnostic ou un traitement inapproprié en raison d’un manque d’information sur les différences entre les sexes dans la manifestation d’une pathologie. Ce manque de données peut découler du fait que les sujets de sexe féminin sont souvent sous-représentés dans les essais cliniques ou la recherche fondamentale ce qui a grandement limité l’avancement de nos connaissances sur ~50 % de la population. Ainsi, il semble plus que nécessaire d’approfondir notre compréhension des différences entre les sexes, notamment dans la progression de l’IC. L’utilisation d’un modèle de souris transgénique surexprimant le récepteur AT1 (souris AT1R) a permis d’étudier les changements électriques, structurels et contractiles avant et après le développement d’hypertrophie. Premièrement, chez les souris AT1R mâles, un ralentissement de la conduction ventriculaire a été observé indépendamment de l’hypertrophie. Ce résultat était expliqué par une réduction de la densité du courant Na+, mais pas de l’expression du canal. Ensuite, le rôle des protéines kinases C (PKC) dans la régulation du canal Na+ par l’ANGII a été exploré. Les évidences ont suggéré que la PKCα était responsable de la modulation de la diminution du courant Na+ chez les souris AT1R mâles et dans les cardiomyocytes humains dérivés de cellules souches induites pluripotentes (hiPSC-CM) en réponse à un traitement chronique à l’ANGII. Ensuite, les différences entre les sexes ont été comparées chez la souris AT1R. Une plus grande mortalité a été constatée chez les femelles AT1R suggérant qu’elles sont plus sensibles à la surexpression de AT1R. Le remodelage électrique ventriculaire a donc été comparé entre les souris AT1R des deux sexes. Les courants ioniques étaient altérés de façon similaire entre les sexes excluant ainsi leur implication dans la mortalité plus élevée chez les femelles. Ensuite, l’homéostasie calcique et la fonction cardiaque ont été étudiées. Il a été démontré que les femelles développaient une hypertrophie et une dilatation ventriculaire plus sévère que les mâles. De plus, les femelles AT1R avaient de petits transitoires calciques, une extrusion du Ca2+ plus lente ainsi qu’une augmentation de la fréquence des étincelles Ca2+ pouvant participer à des troubles contractiles et à la venue de post-dépolarisations précoces. En conclusion, l’ANGII est impliquée dans le remodelage électrique, structurel et calcique associé à l'émergence de l’IC. De surcroît, ces altérations affectent plus sévèrement les femelles soulignant la présence de différences entre les sexes dans le développement de l’IC.

Les mécanismes sous-jacents aux effets pathologiques cardiaques de l’angiotensine II dans le remodelage électrique et contractile entre les sexes

L’insuffisance cardiaque (IC) est associée à un taux de mortalité et d’hospitalisations élevé causant un fardeau économique important. Les deux causes majeures de décès de l’IC sont les arythmies ventriculaires létales et les sidérations myocardiques. Il est maintenant reconnu que l’angiotensine II (ANGII) est l'un des principaux médiateurs de l’IC. Ses effets délétères découlent de l’activation du récepteur de type 1 de l’ANGII (AT1) et entraînent le développement d’hypertrophie. Toutefois, son rôle dans la genèse d’arythmies demeure incompris. De ce fait, l'étude des mécanismes électriques et contractiles sous-jacents aux effets pathologiques de l’ANGII s’avère essentielle afin de mieux comprendre et soigner cette pathologie. Il est souvent perçu que les femmes sont protégées envers les maladies cardiovasculaires. Cependant, le nombre total de femmes décédant d’IC est plus grand que le nombre d’hommes. Également, l’impact des facteurs de risque diffère entre chaque sexe. Ces différences existent, mais les mécanismes sous-jacents sont encore peu connus. De plus, les femmes reçoivent fréquemment un diagnostic ou un traitement inapproprié en raison d’un manque d’information sur les différences entre les sexes dans la manifestation d’une pathologie. Ce manque de données peut découler du fait que les sujets de sexe féminin sont souvent sous-représentés dans les essais cliniques ou la recherche fondamentale ce qui a grandement limité l’avancement de nos connaissances sur ~50 % de la population. Ainsi, il semble plus que nécessaire d’approfondir notre compréhension des différences entre les sexes, notamment dans la progression de l’IC. L’utilisation d’un modèle de souris transgénique surexprimant le récepteur AT1 (souris AT1R) a permis d’étudier les changements électriques, structurels et contractiles avant et après le développement d’hypertrophie. Premièrement, chez les souris AT1R mâles, un ralentissement de la conduction ventriculaire a été observé indépendamment de l’hypertrophie. Ce résultat était expliqué par une réduction de la densité du courant Na+, mais pas de l’expression du canal. Ensuite, le rôle des protéines kinases C (PKC) dans la régulation du canal Na+ par l’ANGII a été exploré. Les évidences ont suggéré que la PKCα était responsable de la modulation de la diminution du courant Na+ chez les souris AT1R mâles et dans les cardiomyocytes humains dérivés de cellules souches induites pluripotentes (hiPSC-CM) en réponse à un traitement chronique à l’ANGII. Ensuite, les différences entre les sexes ont été comparées chez la souris AT1R. Une plus grande mortalité a été constatée chez les femelles AT1R suggérant qu’elles sont plus sensibles à la surexpression de AT1R. Le remodelage électrique ventriculaire a donc été comparé entre les souris AT1R des deux sexes. Les courants ioniques étaient altérés de façon similaire entre les sexes excluant ainsi leur implication dans la mortalité plus élevée chez les femelles. Ensuite, l’homéostasie calcique et la fonction cardiaque ont été étudiées. Il a été démontré que les femelles développaient une hypertrophie et une dilatation ventriculaire plus sévère que les mâles. De plus, les femelles AT1R avaient de petits transitoires calciques, une extrusion du Ca2+ plus lente ainsi qu’une augmentation de la fréquence des étincelles Ca2+ pouvant participer à des troubles contractiles et à la venue de post-dépolarisations précoces. En conclusion, l’ANGII est impliquée dans le remodelage électrique, structurel et calcique associé à l'émergence de l’IC. De surcroît, ces altérations affectent plus sévèrement les femelles soulignant la présence de différences entre les sexes dans le développement de l’IC.

La concentración de calcio iónico se asocia a la presencia de fibrilación auricular post operatoria en pacientes llevados a revascularización miocardica

Objetivo. Determinar en un grupo de pacientes llevados a revascularización miocárdica si existió asociación entre la presencia de niveles de calcio iónico inferiores a 1,1 en las 24 horas del post operatorio y la ocurrencia de fibrilación auricular post operatoria. Metodología. Estudio observacional, analítico de casos y controles, en donde de manera consecutiva se incluyeron 110 sujetos (57 en el grupo de casos con presencia de fibrilación auricular post operatoria y 54 en el grupo de controles sin evidencia de fibrilación auricular) estos sujetos fueron llevados a revascularización miocárdica en la Fundación Cardioinfantil en los años 2010 a 2015. Resultados. Hubo 13 casos de fibrilación auricular post operatoria en pacientes con niveles de calcio iónico inferiores a 1,1 mmol/l en las primeras 24 horas del post operatorio OR: 0,5, IC (0,2-1,2) p: 0,1. Sin determinarse asociación por limitaciones del estudio, sin embargo un 29% de los pacientes con fibrilación auricular tuvieron niveles de calcio inferiores a 1,1 mmol/l en las primeras 24 horas del post operatorio, este valor aumenta a 31% cuando se analizan por separado los valores de calcio obtenidos a las 12 horas. Conclusiones. Aunque no se logró determinar asociación entre la fibrilación auricular post operatoria y las concentraciones de calcio iónico, de manera exploratoria se pudo establecer que un 29% de los pacientes con fibrilación auricular tuvieron concentraciones de calcio iónico inferiores a 1,1 mmol/l, este valor aumenta a 31% cuando se analizan los niveles de calcio iónico por separado.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulurn Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PR,L) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to nonpregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (U). The expression of the sarcoendoplasmic reticulum Ca2+-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in P-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of PI 9 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

Exercício e restrição alimentar aumentam o RNAm de proteínas do trânsito de Ca2+ miocárdico em ratos

FUNDAMENTO: Treinamento físico (TF) aumenta a sensibilidade dos hormônios tireoidianos (HT) e a expressão gênica de estruturas moleculares envolvidas no movimento intracelular de cálcio do miocárdio, enquanto a restrição alimentar (RIA) promove efeitos contrários ao TF. OBJETIVO: Avaliar os efeitos da associação TF e RIA sobre os níveis plasmáticos dos HT e a produção de mRNA dos receptores HT e estruturas moleculares do movimento de cálcio do miocárdio de ratos. MÉTODOS: Utilizaram-se ratos Wistar Kyoto divididos em: controle (C, n = 7), RIA (R50, n = 7), exercício físico (EX, n = 7) e exercício físico + RIA (EX50, n = 7). A RIA foi de 50% e o TF foi natação (1 hora/dia, cinco sessões/semana, 12 semanas consecutivas). Avaliaram-se as concentrações séricas de triiodotironina (T3), tiroxina (T4) e hormônio tireotrófico (TSH). O mRNA da bomba de cálcio do retículo sarcoplasmático (SERCA2a), fosfolamban (PLB), trocador Na+/Ca+2 (NCX), canal lento de cálcio (canal-L), rianodina (RYR), calsequestrina (CQS) e receptor de HT (TRα1 e TRβ1) do miocárdio foram avaliados por reação em cadeia da polimerase (PCR) em tempo real. RESULTADOS: RIA reduziu o T4, TSH e mRNA do TRα1 e aumentou a expressão da PLB, NCX e canal-L. TF aumentou a expressão do TRβ1, canal-L e NCX. A associação TF e RIA reduziu T4 e TSH e aumentou o mRNA do TRβ1, SERCA2a, NCX, PLB e correlação do TRβ1 com a CQS e NCX. CONCLUSÃO: Associação TF e RIA aumentou o mRNA das estruturas moleculares cálcio transiente, porém o eixo HT-receptor não parece participar da transcrição gênica dessas estruturas.

Effect of arginine and oscillatory calcium ion (II) on vascular response mediated via nitric oxide signaling in normal and salt sensitive hypertensive rat mesenteric arterioles

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca 2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via &agr;-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 ± μM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.

Effect of Arginine and Oscillatory Ca2+ on Vascular Response Mediated Via Nitric Oxide Signaling in Normal and Salt Sensitive Hypertensive Rat Mesenteric Arterioles

Hypertension, a major risk factor in the cardiovascular system, is characterized by an increase in the arterial blood pressure. High dietary sodium is linked to multiple cardiovascular disorders including hypertension. Salt sensitivity, a measure of how the blood pressure responds to salt intake is observed in more than 50% of the hypertension cases. Nitric Oxide (NO), as an endogenous vasodilator serves many important biological roles in the cardiovascular physiology including blood pressure regulation. The physiological concentrations for NO bioactivity are reported to be in 0-500 nM range. Notably, the vascular response to NO is highly regulated within a small concentration spectrum. Hence, much uncertainty surrounds how NO modulates diverse signaling mechanisms to initiate vascular relaxation and alleviate hypertension. Regulating the availability of NO in the vasculature has demonstrated vasoprotective effects. In addition, modulating the NO release by different means has proved to restore endothelial function. In this study we addressed parameters that regulated NO release in the vasculature, in physiology and pathophysiology such as salt sensitive hypertension. We showed that, in the rat mesenteric arterioles, Ca2+ induced rapid relaxation (time constants 20.8 ± 2.2 sec) followed with a much slower constriction after subsequent removal of the stimulus (time constants 104.8 ± 10.0 sec). An interesting observation was that a fourfold increase in the Ca2+ frequency improved the efficacy of arteriolar relaxation by 61.1%. Our results suggested that, Ca2+ frequency-dependent transient release of NO from the endothelium carried encoded information; which could be translated into different steady state vascular tone. Further, Agmatine, a metabolite of L-arginine, as a ligand, was observed to relax the mesenteric arterioles. These relaxations were NO-dependent and occurred via α-2 receptor activity. The observed potency of agmatine (EC50, 138.7 ± 12.1 µM; n=22), was 40 fold higher than L-arginine itself (EC50, 18.3 ± 1.3 mM; n = 5). This suggested us to propose alternative parallel mechanism for L-arginine mediated vascular relaxation via arginine decarboxylase activity. In addition, the biomechanics of rat mesentery is important in regulation of vascular tone. We developed 2D finite element models that described the vascular mechanics of rat mesentery. With an inverse estimation approach, we identified the elasticity parameters characterizing alterations in normotensive and hypertensive Dahl rats. Our efforts were towards guiding current studies that optimized cardiovascular intervention and assisted in the development of new therapeutic strategies. These observations may have significant implications towards alternatives to present methods for NO delivery as a therapeutic target. Our work shall prove to be beneficial in assisting the delivery of NO in the vasculature thus minimizing the cardiovascular risk in handling abnormalities, such as hypertension.

Human atrial beta1L-adrenoceptor but not beta3-adrenoceptor activation increases force and Ca2+ current at physiological temperature

BACKGROUND AND PURPOSE It has been proposed that BRL37344, SR58611 and CGP12177 activate b3-adrenoceptors in human atrium to increase contractility and L-type Ca2+ current (ICa-L). b3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the b1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective b-adrenoceptor subtype antagonists to clarify cardiostimulant b-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on ICa-L. Contractile effects were determined on right atrial trabeculae. KEY RESULTS BRL37344 increased force which was antagonized by blockade of b1- and b2-adrenoceptors but not by blockade of b3-adrenoceptors with b3-adrenoceptor-selective L-748,337 (1 mM). The b3-adrenoceptor agonist SR58611 (1 nM–10 mM) did not affect atrial force. BRL37344 and SR58611 did not increase ICa-L at 37°C, but did at 24°C which was prevented by L-748,337. (-)-CGP12177 increased force and ICa-L at both 24°C and 37°C which was prevented by (-)-bupranolol (1–10 mM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS We conclude that the inotropic responses to BRL37344 are mediated through b1- and b2-adrenoceptors. The inotropic and ICa-L responses to (-)-CGP12177 are mediated through the low affinity site b1L-adrenoceptor of the b1-adrenoceptor. b3-adrenoceptor-mediated increases in ICa-L are restricted to low temperatures. Human atrial b3-adrenoceptors do not change contractility and ICa-L at physiological temperature.

A raman and infrared spectroscopic study of Ca2+ dominant members of the mixite group from the Czech Republic

Raman microscopy of two mixite minerals BiCu6(AsO4)3(OH)6.3H2O from Jáchymov and from Smrkovec (both Czech Republic) has been used to study their molecular structure, which is interpreted and the presence of (AsO4)3-, (AsO3OH)2-, (PO4)3- and (PO3OH)2- units, molecular water and hydroxyl ions were inferred. O-H…O hydrogen bond lengths were calculated from the Raman and infrared spectra using Libowitzky’s empirical relation. Small differences in the Raman spectra between both samples were observed and attributed to compositional and hydrogen bonding network differences.