Angiotensina II: modulación de la contractilidad y la relajación miocárdica

La Angiotensina II (Ang II) es la molécula efectora del sistema renina- angiotenesina, y se ha visto implicada en varios procesos fisiológicos y fisiopatológicos. Su efecto vasoconstrictor es el más conocido; sin embargo, en los últimos años ha adquirido importancia la modulación que la Ang II ejerce sobre el acoplamiento éxcito-contráctil y la contractilidad en el músculo cardíaco. El conocimiento de los mecanismos de acción de la Ang II sobre el miocardio en condiciones fisiológicas, favorecerá la interpretación de la fisiopatologia de aquellas enfermedades que la involucren. Para una mejor comprensión de los mecanismos subcelulares por medio de los cuales la Ang II modula la contractilidad cardíaca resulta fundamental hacer una revisión de los eventos que participan en el acoplamiento éxcito-contráctil en el miocardio y de su regulación.Facultad de Ciencias Médica

Unbalance between sarcoplasmic reticulum Ca2 + uptake and release: A first step toward Ca2 + triggered arrhythmias and cardiac damage

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners’ proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.Fil: Federico, Marilén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Valverde, Carlos Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Mattiazzi, Ramona Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Palomeque, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina. Universidad Abierta Interamericana. Secretaría de Investigación. Centro de Altos Estudios En Ciencias Humanas y de la Salud - Sede Buenos Aires; Argentin

Apoptosis cardíaca temprana en diferentes modelos de hiperactividad del sistema renina-angiotensina-aldosterona inducida por activación de CaMKII

La angiotensina II (AngII) induce apoptosis in vitro y ex vivo a través de la activación de la kinasa dependiente de Ca2+-Calmodulinaδ (CaMKIIδ). Sin embargo, aun no ha sido determinado si la producción endógena de AngII in vivo induce apoptosis cardíaca a través de la CaMKIIδ.Facultad de Ciencias Médica

Early apoptosis in different models of cardiac hypertrophy induced by high renin-angiotensin system activity involves CaMKII

The objective of this study was to establish whether 1) hyperactivity of renin-angiotensin-aldosterone system (RAAS) produces apoptosis in early stages of cardiac disease; and 2) Ca2+-calmodulin-dependent protein kinase II (CaMKII) is involved in these apoptotic events. Two models of hypertrophy were used at an early stage of cardiac disease: spontaneously hypertensive rats (SHR) and isoproterenol-treated rats (Iso-rats). At 4 mo, SHR showed blood pressure, aldosterone serum levels, used as RAAS activity index, and left ventricular mass index, used as hypertrophy index, above control values by 84.2 6 2.6 mmHg, 211.2 6 25.8%, and 8.6 6 1.1 mg/mm, respectively. There was also an increase in apoptotis (Bax-to-Bcl-2 ratio and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling positive cells) associated with an enhancement of CaMKII activity with respect to age-matched controls (phosphorylated-CaMKII, 98.7 6 14.1 above control). Similar results were observed in 4-mo-old Isorats. Cardiac function studied by echocardiography remained unaltered in all groups. Enalapril treatment significantly prevented hypertrophy, apoptosis, and CaMKII activity. Moreover, intracellular Ca2+ handling in isolated myocytes was similar between SHR, Iso-rats, and their aged-matched controls. However, SHR and Iso-rats showed a significant increase in superoxide anion generation (lucigenin) and lipid peroxidation (thiobarbituric acid reactive substance). In transgenic mice with targeted cardiomyocyte expression of a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C), Iso treatment increased thiobarbituric acid reactive substance in both strains, whereas it increased CaMKII activity and apoptosis only in AC3-C mice. Endogenous increases in RAAS activity induce ROS and CaMKII-dependent apoptosis in vivo. CaMKII activation could not be associated with intracellular Ca2+ increments and was directly related to the increase in oxidative stress.Centro de Investigaciones Cardiovasculare

Na+-Ca2+ exchange function underlying contraction frequency inotropy in the rat myocardium

In most mammalian species, an increase in stimulation frequency (ISF) produces an increase in contractility (treppe phenomenon), which results from larger Ca2+ transients at higher frequencies, due to an increase in sarcoplasmic reticulum Ca2+ load and release. The present study attempts to elucidate the contribution of the Na+-Ca2+ exchanger (NCX) to this phenomenon. Isolated cat ventricular myocytes, loaded with [Ca2+]i- and [Na+]i-sensitive probes, were used to determine whether the contribution of the NCX to the positive inotropic effect of ISF is due to an increase in Ca2+ influx (reverse mode) and/or a decrease in Ca2+ efflux (forward mode) via the NCX, due to frequency-induced [Na+]i elevation, or whether it was due to the reduced time for the NCX to extrude Ca2+. The results showed that the positive intropic effect produced by ISF was temporally dissociated from the increase in [Na+]i and was not modified by KB-R7943 (1 or 5 μM), a specific blocker of the reverse mode of the NCX. Whereas the ISF from 10 to 30 beats min-1 (bpm) did not affect the forward mode of the NCX (assessed by the time to half-relaxation of the caffeine-induced Ca2+ transient), the ISF to 50 bpm produced a significant reduction of the activity of the forward mode of the NCX, which occurred in association with an increase in [Na+]i (from 4.33 ± 0.40 to 7.25 ± 0.50 mM). However, both changes became significant well after the maximal positive inotropic effect had been reached. In contrast, the positive inotropic effect produced by ISF from 10 to 50 bpm was associated with an increase in diastolic [Ca2+]i, which occurred in spite of a significant increase in the relaxation rate and at a time at which no increases in [Na+]i were detected. The contribution of the NCX to stimulus frequency inotropy would therefore depend on a decrease in NCX-mediated Ca2+ efflux due to the reduced diastolic interval between beats and not on [Na+]i-dependent mechanisms.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Mayor comunicación entre el retículo sarcoplasmático y la mitocondria favorecen la apoptosis corazones pre diabéticos

La interacción entre el retículo sarcoplasmático (RS) y la mitocondria juega un rol importante tanto en situaciones fisiológicas como patológicas. Varias proteínas, como Mfn-2 (Mitofusina-2), GRP75 (del inglés; glucose regulated protein 75) y VDAC (del inglés; voltage-dependent anion channel) se encuentran involucradas en esta relación y participan además, en el tráfico de calcio entre ambas organelas. En trabajos previos demostramos que la fosforilación del receptor de rianodina del RS (RyR2) por CaMKII (calcio-calmodulina kinasa II), la perdida de Ca+2 por el RS y la despolarización de la membrana mitocondrial son críticas en la cascada apoptótica que ocurre en los corazones pre diabéticos. Por esto, nuestra hipótesis es que la apoptosis en los corazones pre-diabéticos, es en parte debida al incremento de la interacción entre la mitocondria y el RS. Objetivo: evaluar en los animales tratados con una dieta rica en fructosa (DRF) respecto de los animales tratados con una dieta control (DC), si hay cambios en la relación entre las mitocondrias y el RS que pudieran impactar en la cascada apoptótica de los animales pre-diabéticos.Facultad de Ciencias Médica

Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species

Rationale: Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. Objective: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. Methods and results: Ang II (1 μmol/L) reduced cat/rat myocytes viability by ≈40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca2+/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H2O2, even in the presence of the Ca 2+chelator BAPTA-AM, in myocytes and in EGTA-Ca2-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. Conclusions: (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca2+concentrations, suggesting a new mechanism by which ROS reset the Ca2+dependence of CaMKII to extremely low Ca2+levels.Facultad de Ciencias Médica

Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species

Rationale: Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. Objective: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. Methods and results: Ang II (1 μmol/L) reduced cat/rat myocytes viability by ≈40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca2+/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H2O2, even in the presence of the Ca 2+chelator BAPTA-AM, in myocytes and in EGTA-Ca2-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. Conclusions: (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca2+concentrations, suggesting a new mechanism by which ROS reset the Ca2+dependence of CaMKII to extremely low Ca2+levels.Facultad de Ciencias Médica

CaMKII activation in early diabetic hearts induces altered sarcoplasmic reticulum-mitochondria signaling

Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca2+ leak and arrhythmias due to increased activity of the Ca2+/calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.Fil: Federico, Marilén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Zavala, Maite Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Vico, Tamara Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: López, Sofía. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Portiansky, Enrique Leo. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarez, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Villa-Abrille, María Celeste. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Palomeque, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentin

Corazón prediabético: señalización intracelular en la apoptosis cardíaca inducida por una dieta rica en fructosa

Los objetivos del presente trabajo son verificar la presencia de apoptosis en el corazón con TGA y definir las vías de señalización intracelular que conducen a ella, apuntando a esclarecer el rol de la CaMKII en esta cascada de señales.Facultad de Ciencias Médica